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Publication numberCN101793826 A
Publication typeApplication
Application numberCN 200910246173
Publication date4 Aug 2010
Filing date7 Jun 2005
Priority date7 Jun 2004
Also published asCN100583434C, CN101002322A, CN101793826B, CN102680440A, CN103884698A, CN103884698B, EP1754257A2, EP1754257A4, EP1754257B1, EP2701194A2, EP2701194A3, EP2752874A1, EP2933630A2, EP2933630A3, US7307802, US7588672, US7906072, US8048378, US8512640, US8721968, US8926905, US9234237, US9663821, US20060006067, US20080088952, US20090294703, US20100320364, US20120035080, US20120264226, US20140011204, US20140024559, US20150203896, US20160194685, WO2005121864A2, WO2005121864A3, WO2005121864A9
Publication number200910246173.0, CN 101793826 A, CN 101793826A, CN 200910246173, CN-A-101793826, CN101793826 A, CN101793826A, CN200910246173, CN200910246173.0
InventorsB克拉克森, CG西泽, GR费瑟, MA厄金, N斯威兹
Applicant先锋生物科技股份有限公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Optical lens system and method for microfluidic devices
CN 101793826 A
Abstract
An apparatus for imaging one or more selected fluorescence indications from a microfluidic device (205). The apparatus includes an imaging path coupled to least one chamber in at least one microfluidic device (205). The imaging path provides for transmission of one or more fluorescent emission signals derived from one or more samples in the at least one chamber of the at least one microfluidic device (205). The chamber has a chamber size, the chamber size being characterized by an actual spatial dimension normal to the imaging path. The apparatus also includes an optical lens system (210, 212) coupled to the imaging path. The optical lens system is adapted to transmit the one or more fluorescent signals associated with the chamber.
Claims(49)  translated from Chinese
  1. 一种给微流体器件成像的方法,所述方法包括:传输微流体器件的空间区域的像,所述空间区域与不止96个室有关;以及用与图像检测设备有关的图像检测空间区域来拍摄所述空间区域的图像,所述图像检测空间区域具有和所述微流体器件的空间区域大约相等的尺寸。 A microfluidic device for imaging, the method comprising: transmitting a spatial region like a microfluidic device, the chamber 96 is more than the relevant spatial region; and image detection spatial region associated with the image detection device to capture the image, the image detection spatial region and a space region having a spatial region of the microfluidic device of approximately equal size.
  2. 2. 如权利要求1所述的方法,其特征在于,所述空间区域除以所述图像检测空间区域大约等于一(1)。 2. The method of claim 1, wherein the spatial region divided by the image detection spatial region is about equal to one (1).
  3. 3. 如权利要求1所述的方法,其特征在于,所述空间区域除以所述图像检测空间区域大约等于O. 99或更小。 The method according to claim, characterized in that the spatial region divided by the image detection spatial region is about equal to O. 99 or less.
  4. 4. 如权利要求3所述的方法,其特征在于,所述空间区域除以所述图像检测空间区域大约等于O. 88或更小。 4. The method according to claim 3, characterized in that the spatial region divided by the image detection spatial region is about equal to O. 88 or less.
  5. 5. 如权利要求1所述的方法,其特征在于,所述图像检测空间区域大约是30. 7mmX30. 7mm或更大。 5. The method according to claim 1, characterized in that the image detection spatial region is about 30. 7mmX30. 7mm or more.
  6. 6. 如权利要求1所述的方法,其特征在于,所述图像检测空间区域大约是27. 6mmX27. 6mm或更大。 6. The method according to claim 1, characterized in that the image detection spatial region is about 27. 6mmX27. 6mm or more.
  7. 7. 如权利要求l所述的方法,其特征在于,所述图像检测空间区域包括大约2048X2048个像素区域。 7. The method of claim l, wherein said image detection spatial region comprises a region of approximately 2048X2048 pixels.
  8. 8. 如权利要求1所述的方法,其特征在于,所述图像检测空间区域包括大约2048 X 2048个像素区域,每一个像素区域具有约为15 y mX 15 ym或更大的空间维度。 8. The method according to claim 1, characterized in that the image detection spatial region comprises about 2048 X 2048 pixel areas, each pixel area having about 15 y mX 15 ym or more spatial dimensions.
  9. 9. 如权利要求8所述的方法,其特征在于,至少两个像素区域与至少一个室相关。 9. The method according to claim 8, characterized in that at least two pixel regions associated with the at least one chamber.
  10. 10. 如权利要求9所述的方法,其特征在于,至少九个像素区域与至少一个室相关。 10. The method according to claim 9, characterized in that at least nine pixel region associated with the at least one chamber.
  11. 11. 如权利要求10所述的方法,其特征在于,不止20个像素区域与至少一个室相关。 11. The method according to claim 10, characterized in that more than 20 pixel region associated with the at least one chamber.
  12. 12. —种给微流体器件成像的方法,所述方法包括:在小于1分钟的时间帧内,通过使用图像检测空间区域,给和微流体器件中至少确定数目的室相关的空间区域拍摄图像,在哪儿拍摄所述空间区域的图像基本上不受缝合和/ 或扫描过程的影响。 12. - seed to the method of imaging microfluidic devices, the method comprising: at a time frame of less than one minute, by using the image detection spatial region, and microfluidic devices to determine at least the number of the spatial region of the captured image associated chamber , where the captured image of the spatial region is substantially free from influence stitching and / or scanning process.
  13. 13. 如权利要求12所述的方法,还包括:在大于10毫秒的时间帧内,通过使用图像检测空间区域,给和所述微流体器件中至少确定数目的室相关的空间区域拍摄图像。 13. The method of claim 12, further comprising: greater than 10 ms time frame, by using the image detection spatial region, and the microfluidic device for determining the number of chambers at least the spatial region associated with the captured image.
  14. 14. 如权利要求12所述的方法,其特征在于,拍摄所述空间区域的图像是在小于30秒的时间帧内发生的。 14. The method according to claim 12, characterized in that the spatial region of the image captured in less than 30 seconds occurred within the frames.
  15. 15. 如权利要求12所述的方法,其特征在于,拍摄所述空间区域的图像是在小于15秒的时间帧内发生的。 15. The method according to claim 12, characterized in that the spatial region of the image captured in the time frame of less than 15 seconds occurs.
  16. 16. 如权利要求12所述的方法,其特征在于,在所述小于1分钟的时间帧内,对所述确定数目的室进行同步拍摄。 16. The method according to claim 12, characterized in that, in the time frame of less than one minute, for the determined number of chambers is synchronized to shoot.
  17. 17. 如权利要求12所述的方法,其特征在于,在使所述确定数目的室保持在确定的温度范围中的同时,进行所述拍摄。 17. The method according to claim 12, characterized in that, in determining the number of said chamber is maintained at a determined temperature range is performed while the photographing.
  18. 18. 如权利要求17所述的方法,其特征在于,所述确定的温度范围是2C。 18. The method according to claim 17, characterized in that said determined temperature range is 2 C.
  19. 19. 如权利要求17所述的方法,其特征在于,所述确定的温度是与PCR过程相关的循环温度环境的一部分。 19. The method of claim 17, wherein said determined temperature is part of the PCR process, the cycle temperature environment.
  20. 20. 如权利要求12所述的方法,其特征在于,所述确定数目的室是至少96个或更多。 12 20. The method according to claim, wherein said determining number of chambers is at least 96 or more.
  21. 21. 如权利要求12所述的方法,还包括:在小于1分钟的时间帧内,通过使用图像检测空间区域,给和所述微流体器件中至少确定数目的室相关的空间区域拍摄至少第二张图像,在哪儿拍摄所述空间区域的第二张图像代表了荧光发射。 21. The method of claim 12, further comprising: a time frame of less than one minute, by using the image detection spatial region, and the microfluidic device for determining at least the spatial region associated with the number of photographing at least the first chamber two images, where the shooting of the second picture spatial region represents the fluorescence emission.
  22. 22. —种用于分析弹性微流体器件中的各种过程的方法,所述方法包括:在小于1分钟的时间周期内给至少96个室拍摄图像,其中所述至少96个室中的每一个室都处于和所述至少96个室中的任何其它室流体隔离的状态;以及处理所述图像。 22. - Methods Elastic microfluidic device in various processes, the method comprising for species: in less than one minute time period to the chamber 96 at least the captured image, wherein the at least 96 chambers in each of the are in a chamber and the chamber in the at least 96 chambers fluidly isolated from any other state; and processing the image.
  23. 23. 如权利要求22所述的方法,还包括:在小于1分钟的时间周期内,给所述至少96个室拍摄第二张图像;以及处理所述第二张图像。 23. The method of claim 22, further comprising: in less than one minute time period, the chamber is at least 96 to capture a second image; and processing said second image.
  24. 24. 如权利要求22所述的方法,其特征在于,通过使用其数值孔径大于或等于0. 23且其放大率小于或等于2的光学系统,来执行图像拍摄。 24. The method according to claim 22, characterized in that, through the use of a numerical aperture greater than or equal to 0.23 and less than or equal to its magnification of the optical system 2, to perform image capturing.
  25. 25. 如权利要求24所述的方法,其特征在于,所述光学系统具有大于或等于0. 23的数值孔径。 25. The method of claim 24, wherein said optical system has a numerical aperture of greater than or equal to 0.23.
  26. 26. 如权利要求22所述的方法,其特征在于,所述至少96个室中的每一个室都包括约100纳升或更小的体积。 26. The method according to claim 22, characterized in that said at least chambers 96 each chamber comprises from about 100 nanoliters or smaller volume.
  27. 27. 如权利要求26所述的方法,其特征在于,所述至少96个室中的每一个室都包括约IO纳升或更小的体积。 27. The method according to claim 26, characterized in that said at least chambers 96 each chamber comprises from about IO nanoliter or smaller volumes.
  28. 28. 如权利要求27所述的方法,其特征在于,所述至少96个室中的每一个室都包括约100皮升或更小的体积。 28. The method according to claim 27, characterized in that said at least chambers 96 each chamber comprises from about 100 picoliters or less volume.
  29. 29. 如权利要求22所述的方法,其特征在于,所述至少96个室的密度大约是每平方厘米有100个室或更大。 29. The method of claim 22, wherein the density of said chamber at least about 96 per square centimeter chamber 100 or greater.
  30. 30. 如权利要求29所述的方法,其特征在于,所述至少96个室的密度大约是每平方厘米有250个室或更大。 29 30. The method according to claim, characterized in that the at least chamber 96 is approximately the density per square centimeter chamber 250 or greater.
  31. 31. 如权利要求30所述的方法,其特征在于,所述至少96个室的密度大约是每平方厘米有1000个室或更大。 30 31. The method according to claim, characterized in that the at least chamber 96 is approximately the density per square centimeter chamber 1000 or greater.
  32. 32. —种给包括多个处理位点的微流体器件成像的装置,所述多个处理位点包含从M 种样品中选出的至少一种样品以及从N种试剂中选出的至少一种试剂,所述装置包括:照明系统,耦合到所述微流体器件并适用于用电磁辐射来照射所述微流体器件; 成像系统,耦合到所述微流体器件并适用于接收从所述多个处理位点那儿发出的电磁辐射;以及耦合到所述成像系统的检测器。 32. - kind of microfluidic device to the image forming apparatus includes a plurality of processing sites, the plurality of processing site comprises at least one sample selected from M kinds of sample and reagent selected from at least one of the N reagents, the apparatus comprising: an illumination system coupled to the microfluidic device and adapted to be irradiated with electromagnetic radiation of the microfluidic device; imaging system, coupled to the microfluidic device and adapted to receive from the multi- a treatment site where the electromagnetic radiation emitted; and a detector coupled to the imaging system.
  33. 33. 如权利要求32所述的装置,其特征在于,所述微流体器件是一种弹性微流体器件。 33. The apparatus of claim 32, wherein the microfluidic device is an elastomeric microfluidic device.
  34. 34. 如权利要求32所述的装置,其特征在于,所述处理位点是封闭的反应室。 32 34. The apparatus according to claim, characterized in that the processing site is a closed reaction chamber.
  35. 35. 如权利要求34所述的装置,其特征在于,所述多个封闭的反应室包括每平方厘米有大约250个流体隔离的反应室或更大的反应室密度。 34 35. The apparatus according to claim, wherein said reaction chamber comprises a plurality of closed per square centimeter about 250 fluidly isolated from the reaction chamber of the reaction chamber or greater density.
  36. 36. 如权利要求32所述的装置,其特征在于,从所述多个处理位点中发出的电磁辐射是荧光信号。 36. The apparatus according to claim 32, characterized in that the electromagnetic radiation emitted from the plurality of processing sites is a fluorescent signal.
  37. 37. 如权利要求36所述的装置,还包括耦合到所述微流体器件的热控制器。 37. The apparatus according to claim 36, further comprising the microfluidic device coupled to a thermal controller.
  38. 38. 如权利要求37所述的装置,其特征在于,所述多个处理位点以大约100纳升或更小的体积为特征。 38. The apparatus of claim 37, wherein said plurality of processing sites to about 100 nanoliters or smaller volume characteristics.
  39. 39. 如权利要求38所述的装置,其特征在于,所述多个处理位点以大约IO纳升或更小的体积为特征。 39. The apparatus of claim 38, wherein said plurality of processing sites to about IO nanoliters or smaller volume characteristics.
  40. 40. 如权利要求38所述的装置,其特征在于,所述多个处理位点以每平方厘米有250个位点或更大的阵列密度为特征。 40. The apparatus of claim 38, wherein said plurality of processing sites per square centimeter 250 sites or larger array density characteristics.
  41. 41. 如权利要求38所述的装置,其特征在于,所述多个处理位点以每平方厘米有1000 个位点或更大的阵列密度为特征。 41. The apparatus of claim 38, wherein said plurality of processing sites per square centimeter 1,000 sites or larger array density characteristics.
  42. 42. 如权利要求32所述的装置,其特征在于,所述多个处理位点适用于支持蛋白质结晶过程。 42. The apparatus of claim 32, wherein said plurality of processing sites adapted to support a protein crystallization process.
  43. 43. 如权利要求32所述的装置,其特征在于,所述电磁辐射是光辐射。 43. The apparatus of claim 32, wherein said electromagnetic radiation is light radiation.
  44. 44. 一种光学成像系统,所述系统包括: 计算机;所述弹性微流体阵列设备包括由多层构成的弹性模块,其中至少一层具有形成于其中的至少一个凹陷,所述凹陷具有至少一个可弯曲的膜,所述膜与具有所述凹陷的层构成一体;以及光学检测系统。 44. An optical imaging system, said system comprising: a computer; said elastomeric microfluidic array device includes an elastic module consists of a plurality of layers, wherein at least one layer having formed therein at least one recess, said recess having at least one flexible film, the film layer is formed integrally with the recess; and an optical detection system.
  45. 45. 如权利要求44所述的光学成像系统,其特征在于,所述至少1536个反应室都以小于100纳升的体积为特征。 The optical imaging system 44 according to claim 45., characterized in that said at least 1,536 reaction chambers are in a volume less than 100 nanoliters characterized.
  46. 46. 如权利要求44所述的光学成像系统,其特征在于,所述至少1536个反应室按照每平方厘米大于或等于250个室的阵列密度来进行空间排列。 The optical imaging system 44 according to claim 46., characterized in that said at least 1536 per square centimeter in accordance with the reaction chamber is greater than or equal to the density of the array chamber 250 to carry out spatial arrangement.
  47. 47. 如权利要求44所述的光学成像系统,其特征在于,M种样品和N种试剂分布在所述至少1536个反应室中。 The optical imaging system 44 according to claim 47., wherein, M N kinds of samples and reagents in the distribution of at least 1536 in the reaction chamber.
  48. 48. 如权利要求44所述的光学成像系统,其特征在于,所述光学照明系统从所述至少1536个反应室的子集中激发出一个或多个荧光信号的发射。 The optical imaging system 44 according to claim 48., characterized in that the optical illumination system from at least 1,536 reaction chambers sub said centralized stimulate one or more fluorescent emission signals.
  49. 49. 如权利要求44所述的光学成像系统,其特征在于,所述光学检测系统适用于提供与所述至少1536个反应室的子集相关的蛋白质结晶过程的一个或多个图像。 49. The optical imaging system according to claim 44, characterized in that the optical detection system is adapted to provide at least 1,536 reaction chambers with an associated subset of said protein crystallization process or a plurality of images.
Description  translated from Chinese

用于微流体器件的光学透镜系统和方法 An optical lens system and method for microfluidic devices

[0001] 本发明专利申请是申请日为2005年6月7日、申请号为200580025595. 5、名称为 [0001] The present invention is a patent application filed on June 7, 2005, Application No. 200,580,025,595.5 named

"用于微流体器件的光学透镜系统和方法"的发明专利申请的分案申请。 Divisional application "System and method for optical lenses for microfluidic devices," the patent application. [0002] 絲申i青,营照、 [0002] i wire Shen Qing, camp photo,

[0003] 本申请要求2004年6月7日提交的美国临时申请60/578, 106的优先权。 [0003] This application claims the United States June 7, 2004 filed provisional application priority 60/578, 106. 技术领域 FIELD

[0004] 本发明一般涉及微流体技术。 [0004] The present invention relates generally to microfluidic technology. 特别是,本发明提供用于给微流体器件的室内的(例如悬浮在流体体积中的) 一个或多个实体成像的方法和系统。 In particular, the present invention is to provide a chamber for microfluidic devices (e.g., suspended in a fluid volume of) one or more entities imaging methods and systems. 更具体地讲,这种成像方法和系统利用了与微流体器件中的一个或多个实体有关的荧光信号指示。 More specifically, this image forming method and system for use with the microfluidic device of one or more entities associated fluorescence signal indicates. 仅作为示例,通过使用耦合到微流体器件的荧光、化学发光和生物发光读出器,来应用微流体方法和系统技术,但是将会认识到本发明具有更广的应用范围。 Of example only, by using coupled to a fluorescent, chemiluminescent and biological microfluidic device emitting the reader, to apply microfluidic methods and systems technology, it will be recognized that the invention has a broader range of applications.

[0005] 经共同努力已开发并制造出各种微流体系统,来执行化学和生物化学的各项分析与综合。 [0005] have been developed through the joint efforts and create a variety of microfluidic systems to perform various chemical and biochemical analysis and synthesis. 这些系统已被开发成用于各项制备和分析应用。 These systems have been developed as for the preparation and analysis applications. 制造这种微尺寸器件的目标源自常规大尺度分析与综合(这通常很笨重且不太有效)的小型化过程所实现的诸多重要益处。 This micro-sized target devices manufactured from conventional large-scale analysis and synthesis of a number of important benefits (which are usually very cumbersome and less effective) miniaturization of process implementation. 通过使用这些微流体系统,便实现了时间大幅减少、成本更低、空间分配更有效等诸多益处。 By using these microfluidic systems, will significantly reduce the time to achieve a lower cost, more efficient space allocation, and many other benefits. 对于使用这些微流体器件的自动化系统而言,附带的好处可以包括人工操作更少。 For these microfluidic devices using automated systems, the benefits may include manual that came with less. 自动化系统还减少了操作人员的过失以及其它和操作人员有关的局限。 Automated system also reduces mistakes and other limitations associated with the operator of the operator. 已经提出,微流体器件可用于各种应用,其中包括毛细电泳、气体层析和细胞分离。 Has been proposed, the microfluidic device can be used for a variety of applications, including capillary electrophoresis, gas chromatography and cell separation.

[0006] 适于进行核酸扩增过程的微流体器件有可能有广泛的应用。 [0006] adapted to perform a nucleic acid amplification process of the microfluidic devices have a wide range of possible applications. 例如,这种器件作为一种分析工具,可以用于确定在样品中是否存在特定的靶核酸。 For example, such a device as an analytical tool that can be used to determine whether there is a specific target nucleic acid in the sample. 利用微流体器件作为分析工具的示例包括: The microfluidic device as an example of the use of analysis tools including:

[0007] •鉴定过程(例如,亲子鉴定和法医学应用); [0007] • identification process (eg, paternity testing and forensic applications);

[0008] •检测和表征与特定疾病或遗传病相关联的特定核酸; [0008] • detection and characterization of specific nucleic acid with a specific disease or genetic disease associated;

[0009] *检测与特定药物行为相关联的基因表达分布型/序列(例如药物遗传学,即选择 [0009] * Detection and specific behaviors associated with drug distribution patterns of gene expression / sequence (such as pharmacogenetics, choose

对特定遗传概况相容的/特别有效的/无害的药物);以及 Of particular genetic profile compatible / particularly effective / harmless drug); and

[0010] •进行基因型分析和基因表达分析(例如,差别基因表达研究)。 [0010] • genotyping and gene expression analysis (for example, differences in gene expression studies) conducted.

[0011] 或者,该器件可以按制备方式用于核酸扩增,从而产生足以进行进一步分析的水 [0011] Alternatively, the device can be prepared by methods for nucleic acid amplification, thereby producing water sufficient for further analysis

平的扩增产物。 Flat amplification product. 这些分析方法的示例包括扩增产物的测序、细胞分型、DNA指纹鉴定等。 Examples of these analysis methods include sequencing of the amplification product, cell typing, DNA fingerprinting and the like. Expand

增产物也可用于各种遗传工程应用。 Stimulation may also be used for a variety of genetic engineering applications. 这些遗传工程应用包括(但不限于)生产所需蛋白质 These genetic engineering applications include (but are not limited to) the production of a desired protein

产物,这是通过将扩增产物插入载体,然后用该载体转化细胞使其产生所需蛋白质产物完成的。 The product, which is inserted into the vector by amplification product, then the cells transformed with the vector to produce the desired protein product is completed.

[0012] 尽管有这些潜在的应用,适用于从这种微流体器件中收集并处理成像数据(例 [0012] Despite these potential applications, suitable for collecting and processing the imaging data (from which a microfluidic device

如,荧光数据)的成像系统(也被称为读出器)具有各种缺点。 E.g., fluorescence data) of the imaging system (also called a reader) have various disadvantages. 一些常规的读出器按扫描模式工作,其中激光束在微流体器件上进行光栅扫描。 Some conventional reader works as the scan mode, wherein the laser beam on the microfluidic device is raster scanned. 在其它这样的系统中,器件被平移,或 In other such systems, the device is translated, or

者激光和器件同时平移。 By laser and devices simultaneously pan. 这些扫描器按照和激光源/器件的光栅扫描相关联的有序方式, 从微流体器件中存在的反应室中收集荧光数据。 These scanners raster scanning in an orderly manner and associated laser source / device, the presence of a microfluidic device from the reaction chamber to collect fluorescence data. 其它常规的扫描器按缝合模式工作,按顺序地给小区域(例如,尺寸小于1mm2的区域)成像,并且将这些小图像缝合到一起,从而形成测试中的微流体器件的图像。 Other conventional scanners work by stitch pattern, sequentially to a small area (e.g., an area size of less than 1mm2) imaging, and these small images stitched together, to form an image of the microfluidic test device.

[0013] 扫描和缝合模式都有缺点。 [0013] scan and stitch patterns have drawbacks. 例如,两种类型的系统都工作在相对低的系统频率下, 而系统频率作为时间的函数正比于成像面积。 For example, both types of systems work at relatively low system frequency, and the system frequency as a function of time proportional to the imaging area. 常规系统的工作频率在每分钟l-20cm2的量级。 Conventional system frequency in the order of l-20cm2 per minute. 对于某些感兴趣的试验,比如蛋白质热量测定和核酸扩增,通常需要大于约每分钟l-20cm2的系统频率,以便对微流体器件的反应器皿中发生的荧光过程进行成像。 For some tests of interest, such as proteins and nucleic acid amplification calorimetry, typically require more than about l-20cm2 per minute of system frequency, so that the process of the fluorescence reaction vessel in the microfluidic device for imaging. 常规的扫描和缝合系统无法满足这些性能目标。 Conventional scanning and suturing system can not meet these performance goals. 除了使系统通量放缓以外,这些扫描和缝合系统还会限制利用某些试验(例如,实时PCR的性能)的可能性。 In addition to slowing down the system outside of flux, these scanning and stitching system will restrict the use of certain tests (for example, the performance of real-time PCR) possibilities.

[0014] 因此,本领域需要改进的方法和系统,来对微流体器件的反应室中的流体体积中悬浮的一个或更多的实体进行成像。 [0014] Accordingly, the art requires an improved method and system for imaging one or more entities of the reaction chamber in the microfluidic device suspended in the fluid volume.

发明内容 SUMMARY

[0015] 根据本发明,提供了用于微流体系统的技术。 [0015] The present invention provides a technique for microfluidic systems. 特别是,本发明提供了用于给微流体器件的反应室中的流体体积中悬浮的一个或多个实体成像的方法和系统。 In particular, the present invention provides for a reaction chamber in the microfluidic device to the fluid volume in the suspension method and system for imaging one or more entities. 更具体地讲,本发明的成像方法和系统利用了与微流体器件中的一个或多个实体相关联的荧光信号指示。 More specifically, the image forming method and system of the present invention utilizes microfluidic device of one or more entities associated with the fluorescent signal indicates. 仅仅作为示例,通过使用和微流体器件相耦合的荧光、化学发光和生物发光读出器,来应用微流体方法和系统技术,但是将会认识到本发明具有更广的应用范围。 Example only, and by using the microfluidic device coupled to a fluorescent, chemiluminescent and bioluminescent reader, to apply microfluidic methods and systems technology, it will be recognized that the invention has a broader range of applications.

[0016] 在特定的实施例中,本发明提供了一种给微流体器件中的一个或多个选出的荧光指示成像的装置。 [0016] In a particular embodiment, the present invention provides a microfluidic device to one or more selected fluorescence indicates imaging apparatus. 该装置包括成像通道,耦合到至少一个微流体器件中的至少一个反应室。 The apparatus includes an imaging path coupled to the at least one microfluidic device at least one reaction chamber. 该成像通道用于传输从上述至少一个微流体器件的至少一个反应室中的一个或多个样品那儿获得的一个或多个荧光发射信号。 The imaging channel is used to transmit one or more of fluorescence from the at least one of said at least one reaction chamber in the microfluidic device of one or more sample obtained where the transmission signal. 该反应室具有一定的大小,其实际空间维度与成像通道正交。 The reaction chamber has a certain size, the actual spatial dimension orthogonal to the imaging channel. 该装置还包括耦合到成像通道的光学透镜系统。 The apparatus further includes an optical lens system coupled to the imaging path. 该光学透镜系统用于传输与该反应室有关的一个或多个荧光信号。 The optical lens system for transmitting one or more fluorescent signals associated with the reaction chamber.

[0017] 在另一个特定的实施例中,提供了一种用于给微流体器件的至少一个反应室中的一个或多个选出的荧光指示成像的方法。 [0017] In another particular embodiment, there is provided at least one reaction chamber to a microfluidic device used in the one or more selected fluorescence indicates imaging method. 该方法包括:沿耦合到至少一个反应室的成像通道,传输从至少一个微流体器件的至少一个反应室内的一个或多个样品那儿获得的一个或多个荧光发射信号。 The method comprising: edge coupled to the at least one reaction chamber of the imaging channel, transmitted from one or more fluorescent emission signals at least one of the at least one reaction chamber one or more sample to get there, the microfluidic device. 上述至少一个反应室具有一定的大小,其实际空间维度与成像通道正交。 Said at least one reaction chamber having a certain size, the actual spatial dimension orthogonal to the imaging channel. 该方法还包括:通过耦合到成像通道的光学透镜系统,传输与该反应室相关联的一个或多个荧光发射信号。 The method further comprising: transmitting a signal through an optical lens system coupled to the imaging channel, the transmission associated with the reaction chamber or a plurality of fluorescent. 该光学透镜系统用于将实际空间尺寸的大小减小到确定的水平。 The optical lens system is used to reduce the size of the actual spatial dimension to a determined level. [0018] 在本发明的另一个特定实施例中,提供了一种用于给微流体器件的一个或多个反应室内的一个或多个指示成像的系统。 [0018] In another particular embodiment of the present invention, there is provided a microfluidic device is used for the one or more reaction chamber one or more indications of the imaging system. 该系统包括光学通道,该光学通道能够传输微流体器件的空间区域的一部分的一个或多个图像。 The system includes an optical channel, the optical channel can transmit a part of a spatial region of a microfluidic device or a plurality of images. 在一个实施例中,上述微流体器件的空间区域的那部分具有第一尺寸。 In one embodiment, that part of the spatial region of the microfluidic device having a first dimension. 该系统还包括第一透镜系统,它耦合到上述光学通道的第一部分。 The system further includes a first lens system, which is coupled to a first portion of said optical path. 该第一透镜系统具有第一光学特性。 The first lens system having a first optical characteristic. 该系统还包括第二透镜系统,它耦合到上述光学通道的第二部分。 The system further includes a second lens system, which is coupled to a second portion of said optical path. 第二透镜系统具有第二光学特性。 A second lens system having a second optical characteristic. 该系统另外包括检测器,该检测器耦合到上述光学通道的第三部分。 The system additionally includes a detector, the detector is coupled to the third portion of the optical channel. 该检测器可用于给上述空间区域的那部分拍摄一个或多个图像。 That portion of the detector may be used to give the above-mentioned space areas taken one or more images. 此外,该检测器适用于拍摄一个或多个图像。 In addition, the detector is suitable for photographing one or more images. 在该检测器处,上述一个或多个图像具有确定的尺寸,约为第一尺寸或更小。 At the detector, the one or more images having a determined size, approximately the first size or smaller.

[0019] 在可选实施例中,提供了一种给微流体器件的一个或多个反应室的一个或多个指示成像的方法。 [0019] In an alternative embodiment, there is provided a microfluidic device to one or a plurality of reaction chambers or method of imaging a plurality of directions. 该方法包括:沿光学通道,传输微流体器件的空间区域的一部分的一个或多个图像。 The method comprising: an optical path along a portion of the spatial region of the microfluidic device or transmitted plurality of images. 上述微流体器件的空间区域的那部分具有第一尺寸。 That part of the spatial region of the microfluidic device having a first dimension. 该方法还包括:将第一透镜系统耦合到光学通道的第一部分。 The method further comprising: a first lens system coupled to the first portion of the optical channel. 第一透镜系统具有第一光学特性。 A first lens system having a first optical characteristic. 该方法另外包括:将第二透镜系统耦合到光学通道的第二部分。 The method further comprises: a second lens system coupled to a second portion of the optical channel. 第二透镜系统具有第二光学特性。 A second lens system having a second optical characteristic. 此外,该方法包括:用检测器给上述空间区域的那部分拍摄一个或多个图像。 Moreover, the method comprising: one or more images captured by the detector to that portion of said space region. 该检测器耦合到光学通道的第三部分,并且在检测器处, 一个或多个图像具有确定的尺寸,约为第一尺寸或更小。 The detector is coupled to a third portion of the optical path, and at the detector, one or more images having a determined size, approximately the first size or smaller. [0020] 在另一个可选实施例中,提供了一种给微流体器件成像的方法。 [0020] In another alternative embodiment, there is provided a microfluidic device to the image forming method. 该方法包括:在小于一分钟的时间范围内,通过使用图像检测空间区域,给和至少确定数目的微流体器件反应室相关联的空间区域拍摄图像。 The method comprising: in less than a minute by using the image detection spatial region, and to determine at least a spatial region of the microfluidic device number associated with the reaction chamber captured image. 在特定的实施例中,上述空间区域的图像拍摄基本上不受缝合和/或扫描过程的影响。 In a particular embodiment, the image-capturing area of said space substantially unaffected by impact of the suture and / or scanning process.

[0021] 在另一个可选实施例中,提供了一种给微流体器件的一个或多个选出的荧光指示成像的装置。 [0021] In another alternative embodiment, there is provided a to one or more microfluidic devices selected fluorescence indicates imaging apparatus. 该装置包括成像通道,该成像通道耦合到至少一个微流体器件的至少一个反应室。 The apparatus includes an imaging channel, the imaging channel coupled to at least one of the at least one reaction chamber of the microfluidic device. 该成像通道用于传输从上述至少一个微流体器件的至少一个反应室内的一个或多个样品那儿获得的一个或多个荧光发射信号。 The imaging channels for transmission from said one or more fluorescent emission signals of at least one microfluidic device at least one reaction chamber one or more samples where obtained. 该装置还包括滤光器件,它耦合到成像通道 The apparatus further comprises a filter device, which is coupled to the imaging channel

(用于传输一个或多个发射信号)的第一空间部分。 (Used to transmit one or more transmit signals) of the first space part. 滤光器件用于传输一个或多个荧光发射信号的选定光谱带宽,还用于将与一个或多个荧光发射信号相关联的一种或多种色差处理到确定的程度。 Optical device for transmitting a selected one or a plurality of fluorescent spectral bandwidth of the transmitted signal, but also for the degree of the one or more fluorescent emission signals associated with one or more of the processing to determine the color difference.

[0022] 在特定的实施例中,提供了一种在弹性微流体器件中分析各种处理的方法。 [0022] In a particular embodiment, there is provided a method of analyzing various processes in elastomeric microfluidic devices. 该方法包括:在不到一分钟的时间周期内,拍摄至少96个反应室的图像。 The method comprising: in less than one minute time period, the captured image, at least 96 of the reaction chamber. 在一个实施例中,这至少96个反应室处于彼此之间的流体隔离状态。 In one embodiment, at least 96 reaction chambers which are in fluid isolation between each other. 该方法还包括处理该图像。 The method also includes processing the image. [0023] 在另一个特定实施例中,提供了一种用于给包括多个处理位置的微流体器件成像的装置。 [0023] In another particular embodiment, there is provided an apparatus for comprising a plurality of processing positions to a microfluidic device for imaging. 上述多个处理位置包含选自M个样品的至少一个样品以及选自N种试剂的至少一种试剂。 At least one sample is selected from the plurality of processing positions comprising M samples and a reagent selected from the N kinds of at least one agent. 该装置包括照明系统,该照明系统耦合到微流体器件并用电磁辐射照亮该微流体器件。 The apparatus includes an illumination system, the illumination system is coupled to the microfluidic device and the microfluidic device with electromagnetic radiation to illuminate. 该装置还包括成像系统,该成像系统耦合到微流体器件并用于接收从多个处理位置发射过来的电磁辐射。 The apparatus further comprises an imaging system, the imaging system is coupled to the microfluidic device and for receiving emitted from the plurality of processing positions over the electromagnetic radiation. 该装置另外包括耦合到成像系统的检测器。 The apparatus additionally includes an imaging system coupled to the detector.

[0024] 在另一个特定实施例中,提供了一种光学成像系统。 [0024] In another particular embodiment, there is provided an optical imaging system. 该光学成像系统包括计算机和光学照明系统,该光学照明系统用于照射弹性微流体阵列器件,这种器件至少包括1536 个处于流体隔离状态中的反应室。 The optical imaging system includes a computer and an optical illumination system, the optical illumination system for illuminating the elastic microfluidic array device, which device comprises at least 1536 in fluid isolation of the reaction chamber. 该弹性微流体阵列器件包括由多个层构成的弹性块。 The elastomeric microfluidic array device comprising an elastic block is composed of a plurality of layers. 在这多个层中,至少一层具有形成于其中的至少一个凹陷。 In the plurality of layers, at least one layer having formed therein at least one recess. 该凹陷具有至少一个可弯曲的膜, 该膜被整合到凹陷的层上。 The recess has at least one flexible film which is integrated into the recessed layer. 光学成像系统还包括光学检测系统。 The optical imaging system further comprises an optical detection system.

[0025] 在另一个可选的特定实施例中,提供了一种给微流体器件的多个反应室内的一个或多个选出的荧光指示成像的方法。 [0025] In another alternative particular embodiment, is provided to one or more of a microfluidic device of the plurality of reaction chamber indicates the selected method of imaging fluorescence. 该方法包括:沿成像通道,传输一个或多个荧光发射信号,所述一个或多个荧光发射信号是从微流体器件的多个反应室中的至少一个反应室内的一个或多个样品那儿获得的。 The method comprising: an imaging channel, the transmission of one or more fluorescent emission signals along said one or more fluorescent emission signals from a plurality of reaction chambers in the microfluidic device of at least one reaction chamber one or more sample to get there a. 该方法还包括:通过使用滤光器件,沿成像通道,选择性地传输上述一个或多个荧光发射信号的子集,该滤光器件用于使预定光谱带宽内的荧光发射信号通过,并且将与上述一个或多个荧光发射信号相关联的一种或多种色差处理到预定的程度。 The method further comprising: using a filter device, along the imaging channel, selectively transmitting said one or more subset of the fluorescence emission signal, the optical device for causing a fluorescence emission signal by a predetermined spectral bandwidth, and the with one or more fluorescent emission signals associated with one of said one or more color processing to a predetermined extent.

[0026] 在特定的实施例中,该方法还包括:在检测器处,读取上述一个或多个荧光发射信号的子集的一部分;拍摄上述一个或多个荧光发射信号,这些信号是从微流体器件的多个 [0026] In a particular embodiment, the method further comprising: at the detector, to read a part of the subset of one or more fluorescent emission signals; shooting said one or a plurality of fluorescent emission signals, which are from a plurality of microfluidic devices

7反应室中的至少一个反应室内的一个或多个样品那儿获得的;照亮上述至少一个微流体器件中的至少96个反应室,其中在多于48个反应室构成的一组中的每一个反应室与多于48 个反应室构成的另一组中的每一个反应室都处于流体隔离状态,并且使上述至少一个微流体器件在确定的时间维持预定的温度,其中预定时间处的预定温度是多步热循环分布中的一部分。 7 in the reaction chamber at least one reaction chamber one or more samples obtained there; a group of at least one of each of said illuminating microfluidic device, at least 96 reaction chambers, wherein more than 48 in the reaction chamber consisting of more than one reaction chamber and the other group consisting of the reaction chamber 48 of each reaction chamber are in fluid isolation, and the said at least one microfluidic device to maintain a predetermined temperature at a determined time, wherein the predetermined time at a predetermined temperature is the multi-step thermal cycling profile part. 在另一个特定的实施例中,处理与上述一个或多个荧光发射信号相关联的一种或多种色差的过程包括:使上述一种或多种色差减小到预定的程度。 In another particular embodiment, treatment with one or more of one or more of the color of the fluorescent emission signals associated with the above-described process comprising: one or more of the color difference is reduced to a predetermined extent. 该预定的程度以预定的焦点移动为特征,而该预定的焦点移动又与具有第一颜色的第一光线和具有第二颜色的第二光线相关联。 The predetermined degree of focus movement for a predetermined characteristic, and the focus movement and a predetermined first light and second light having a first color is associated with a second color. 在另一个特定的实施例中,滤光器件包括多个零屈光度双透镜和多个光谱滤光片。 In another particular embodiment, the filter device includes a plurality of pairs of zero-diopter lens and a plurality of spectral filters.

[0027] 在本发明的一个实施例中,使用光学成像系统来收集和微流体器件有关的信息。 [0027] In one embodiment of the present invention, the use of an optical imaging system to collect and microfluidic device related information. 在特定的实施例中,成像系统的使用包括一种由不止63个反应室构成的微流体器件。 In a particular embodiment, the imaging system used by more than 63 comprising a reaction chamber consisting of a microfluidic device. 在另一个特定的实施例中,成像系统的使用包括一种具有弹性微流体器件的微流体器件。 In another particular embodiment, using an imaging system includes a microfluidic device having a microfluidic device of the elastic. 在可选的实施例中,成像系统的使用包括由不止95个反应室构成的微流体器件。 In an alternative embodiment, using an imaging system comprising more than 95 from the reaction chamber consisting of a microfluidic device. 在另一个可选的实施例中,成像系统的使用包括一种由不止383个反应室构成的微流体器件。 In another alternative embodiment, using an imaging system includes a reaction chamber by more than 383 constituting microfluidic device. 在另一个可选实施例中,成像系统的使用包括一种由不止511个反应室构成的微流体器件。 In another alternative embodiment of the microfluidic device using an imaging system comprising more than 511 from the reaction chamber constructed embodiment. 在另外的实施例中,成像系统的使用包括一种由2304个或更多个反应室构成的微流体器件。 In a further embodiment, using an imaging system includes a by 2304 or more reaction chambers of the microfluidic device configured. 在另一个附加实施例中,成像系统的使用包括一种由9216个或更多个反应室构成的微流体器件。 In another additional embodiment, using an imaging system includes a by 9216 or more reaction chambers of the microfluidic device configured. 在另一个附加实施例中,成像系统的使用包括一种由100000个或更多个反应室构成的微流体器件。 In another additional embodiment, using an imaging system includes a by 100,000 or more reaction chambers of the microfluidic device configured.

[0028] 根据本发明的另一个实施例,使用光学成像系统来收集和微流体器件有关的信息。 [0028] According to another embodiment of the present invention, using an optical imaging system to collect and microfluidic device related information. 光学系统的使用包括一种弹性微流体器件,这种弹性微流体器件适合于在特定的实施例中执行蛋白质结晶化处理。 Using an optical system comprising an elastomeric microfluidic device, such elastic microfluidic device adapted to perform protein crystallization process in a particular embodiment. 在另一个实施例中,成像系统的使用包括一种弹性微流体器件,这种弹性微流体器件适合于执行有关处理。 In another embodiment, using an imaging system includes an elastomeric microfluidic device, such elastic microfluidic device adapted to perform the relevant processing. 在另一个实施例中,成像系统的使用包括一种弹性微流体器件,这种弹性微流体器件适合于在选定的温度或温度范围中进行各种反应。 In another embodiment, using an imaging system includes an elastomeric microfluidic device, such elastic microfluidic device is adapted to carry out various reactions at the selected temperature or temperature range. 在另一个特定的实施例中,光学成像系统被用于收集和PCR反应有关的信息。 In another particular embodiment, the optical imaging system is used for information relating to the collection and PCR reactions. 在另一个特定的实施例中,成像系统的使用包括一种微流体器件,这种微流体器件包括处于微流体隔离状态中的封闭式反应室。 In another particular embodiment, using an imaging system includes a microfluidic device, the microfluidic device comprises a microfluidic isolated state is in a closed reaction chamber. 在其它实施例中,成像系统的使用包括耦合到该微流体器件的热控制器。 In other embodiments, using an imaging system includes a controller coupled to the heat of the microfluidic device.

[0029] 在可选的实施例中,使用光学成像系统来给微流体器件成像。 [0029] In an alternative embodiment, an optical imaging system used to image a microfluidic device. 在这种可选实施例中,光学成像系统具有多个双透镜,适合于将色差的值减小到预定的程度。 In this alternative embodiment, an optical imaging system having a plurality of lenticular lens, adapted to the value of the color difference is reduced to a predetermined extent. 在一个实施例中,光学成像系统具有大于0. 23的NA。 In one embodiment, the optical imaging system having greater than 0.23 of NA. 在另一个实施例中,光学成像系统具有大于或等于0. 36的NA。 In another embodiment, an optical imaging system having greater than or equal to 0.36 of NA. 在另一个实施例中,光学成像系统具有大于0. 2的NA。 In another embodiment, the optical imaging system has greater than 0.2 of NA. 在特定的实施例中,光学成像系统包括:第一光谱滤光片,耦合到第一双透镜;以及第二光谱滤光片,耦合到第二双透镜。 In certain embodiments, the optical imaging system comprising: a first spectral filter, coupled to the first pair of lens; and a second spectral filter, coupled to the second pair of lenses. 此外,在另外的实施例中,第一双透镜使第一光谱滤光片所透射的第一波长处的色差值减小到第一预定程度,而第二双透镜使第二光谱滤光片所透射的第二波长处的色差值减小到第二预定程度。 Further, in another embodiment, the first pair of lens color difference value of the first spectral filter the transmitted first wavelength is decreased to a first predetermined level, and the second two-lens of the second spectral filter the transmitted color value sheet second wavelength is decreased to a second predetermined level. 在某些实施例中,第一双透镜和第二双透镜是零屈光度双透镜。 In certain embodiments, the first and second pair of two-lens is a zero diopter lens lenticular lens. 在其它实施例中,第一光谱滤光片、第一双透镜、第二光谱滤光片和第二双透镜是作为滤光轮来排列的。 In other embodiments, the first spectral filter, a first pair of lenses, the second spectral filter and the second lenticular lens is arranged as a filter wheel.

[0030] 在本发明的另一个实施例中,使用荧光成像系统从微流体器件那儿获取多个图像,其中包括一个或多个荧光发射信号。 [0030] In another embodiment of the present invention, using the fluorescence imaging system to obtain a plurality of images from the microfluidic device there, which includes one or more fluorescent emission signals. 荧光成像系统的使用包括一种具有不止63个反应室的微流体器件,这些反应室都处于流体隔离状态中。 Using the fluorescence imaging system comprises more than one reaction chamber 63 having a microfluidic device, these chambers are in fluid isolation state. 在一个实施例中,荧光成像系统的使用包括一种具有图像微流体器件的微流体器件。 In one embodiment, using the fluorescence imaging system includes a microfluidic device having a microfluidic device of the image. 在另一个实施例中,荧光成像系统的使用包括多个其体积小于10nl的反应室。 Example, using a fluorescent imaging system comprising a plurality of its volume is less than 10nl In another embodiment of the reaction chamber. 在另一个实施例中,荧光成像系统的使用包括一种弹性微流体器件,这种弹性微流体器件具有每平方厘米大于或等于100个反应室的阵列密度。 In another embodiment, using the fluorescence imaging system includes an elastomeric microfluidic device, the microfluidic device having such an elastic array density per square centimeter is greater than or equal to 100 of the reaction chamber. 在可选的实施例中,荧光成像系统包括一种滤光器件,该滤光器件适合于使上述一个或多个荧光发射信号中选定的光谱带宽透射,并且还适合于将与上述一个或多个荧光发射信号相关联的一种或多种色差处理到确定的程度。 In an alternative embodiment, the fluorescence imaging system comprises a filter device, the filter device is adapted to cause said one or more fluorescent emission signals selected spectral bandwidth transmission, and is further adapted to move with said one or one or more of the color difference signals a plurality of fluorescent emission associated with the processing to the extent determined.

[0031] 在本发明的一个实施例中,提供了一种对微流体系统中的样品进行询问的光学系统。 [0031] In one embodiment of the present invention, there is provided a microfluidic system inquiry sample optical system. 在本实施例所提供的光学系统中,检测器由一个阵列(例如,CCD阵列)构成,其尺寸等于或大于要被成像到其上的微流体系统。 In the present embodiment of the optical system provided in the embodiment, by a detector array (e.g., CCD array) form, its size is equal to or greater than the microfluidic system to be imaged thereon. 在另一个实施例中,检测器通过使用至少一个透镜,以光学方式耦合到微流体器件。 In another embodiment, the detector by using at least one lens, optically coupled to the microfluidic device. 在另一个实施例中,检测器并不与微流体器件保形接触。 In another embodiment, the detector is not in contact with the microfluidic device conformal. 在特定的实施例中,检测器与微流体器件保形接触。 In a particular embodiment, the microfluidic device and the detector in conformal contact. 在另一个特定的实施例中,在光学系统中使用了在整个说明书中都讨论到的色差校正系统。 In another particular embodiment, in the optical system used throughout this specification are discussed chromatic aberration correction system.

[0032] 在另一个实施例中,提供了一种对微流体器件进行询问的光学系统。 [0032] In another embodiment, there is provided a microfluidic device an inquiry for an optical system. 光学系统具有放大率M > NA。 An optical system having a magnification M> NA. /NAdet,其中NA。 / NAdet, which NA. 是物方/样品那一侧的NA,而NAdet是在因检测器表面反射所造成的损耗超过阈值之前可允许到达该检测器的最大的NA。 NA is the object-side / sample side, and NAdet before the detector surface due to reflection loss caused by exceeding a threshold maximum NA of the detector can be allowed to reach. 在特定的实施例中,检测器是CCD,并且NAdet = 0. 36。 In a particular embodiment, the detector is a CCD, and NAdet = 0. 36. 在另一个实施例中,检测器是CCD,并且O. 23《NAdet《0. 5。 In another embodiment, the detector is a CCD, and O. 23 "NAdet" 0. 5. [0033] 在附加的实施例中,提供了一种对微流体系统中的样品进行询问的光学系统。 [0033] In an additional embodiment, there is provided a microfluidic system inquiry sample optical system. 检测器包括一种阵列(例如,CCD阵列),其大小等于包含多个反应室的微流体器件的面积的M倍。 Detector comprises an array (e.g., CCD array), which is equal to the size of the reaction chamber comprising a plurality of microfluidic device area of M times. 在一个实施例中,微流体器件的面积包括多个要被询问的样品室。 In one embodiment, the microfluidic device comprises a plurality of areas to be asked of the sample chamber. 在该实施例中,M =脆。 In this embodiment, M = brittle. /脆^+/-10%,其中NA。 / Brittle ^ + / - 10%, including NA. 是由焦深等种种考虑所确定的可接受的最大NA,而焦深等种种考虑则涉及反应室之间可接受的串扰、它们的XY间隔和尺寸以及它们沿Z轴的宽度,并且NAdet使得《40X的入射光因反射、晕映等原因而在检测器表面处损失了。 NA is the maximum acceptable depth of focus considerations by the determined, and depth of focus considerations involves an acceptable cross-talk between the reaction chamber, and their size and their XY spacing along the Z-axis width, and so NAdet "40X incident light due to reflection, vignetting and other reasons, the loss at the surface of the detector. 在另一个特定的实施例中,NAdet使得《20%的入射光因反射、晕映等原因而在检测器表面处损失了。 In another particular embodiment, NAdet such that "20% of the incident light due to reflection, vignetting and other reasons, the loss at the surface of the detector. 在另一个特定的实施例中,NA^t使得《10%的入射光因反射、晕映等原因而在检测器表面处损失了。 In another particular embodiment, NA ^ t such that "10% of incident light due to reflection, vignetting and other reasons, the loss at the surface of the detector. 在可选的实施例中,M^ 1。 In an alternative embodiment, M ^ 1. 在另一个可选的实施例中,M = 1。 In another alternative embodiment, M = 1. [0034] 和常规技术相比,通过使用本发明,实现了大量的益处。 [0034] Compared to conventional techniques, through the use of the present invention, to achieve a large number of benefits. 一些实施例提供了光学成像系统,以产生并检测来自微流体器件的荧光。 Some embodiments provide an optical imaging system, to generate and detect fluorescence from the microfluidic device. 另外,本发明的各实施例提供了一些光学系统,这些光学系统通过使用耦合到光谱滤光片的零屈光度消色差双透镜来减小色差。 Further, embodiments of the present invention provides an optical system, the optical system through the use of these spectral filter coupled to a zero diopter achromatic doublet lens to reduce chromatic aberration. [0035] 本发明的各实施例能够在单个平台上执行许多应用。 Embodiments [0035] The present invention can perform many of the applications on a single platform. 例如,通过使用本发明的各实施例,来执行各种基因组应用,其中包括基因表达和基因型鉴定。 For example, by using the various embodiments of the present invention, to perform a variety of genomic applications, including gene expression and genotyping. 此外,数字隔离与检测(DID)应用是可行的。 In addition, the digital detection and isolation (DID) is feasible. 作为一个示例,提供了许多涉及癌症检测、单细胞大分子检测和量化等应用。 As an example, provides a number of cancers involving detection, detection and quantification of cellular macromolecules single applications. 此外,通过本发明的各实施例,提供了许多蛋白质组学的应用,其中包括蛋白质配体结合以及免疫测定处理。 Further, embodiments of the present invention, there is provided a number of proteomics applications, including ligand binding and protein immunoassay processing.

[0036] 另外,根据本发明的各实施例,可以实现某些益处和/或优点,以克服其它技术的某些局限。 [0036] Further, according to various embodiments of the invention, certain benefits can be realized and / or advantages to overcome some of the limitations of other techniques. 下面的内容都是基于计算而确定的: The following contents were determined based on the calculation:

[0037] —些扫描系统具有期望的分辨率10微米。 [0037] - Some scanning system having the desired resolution of 10 microns. 在这些系统中,总共有3000x3000 (= 约1067)个"点",期望针对这些"点"来使荧光量化。 In these systems, a total of 3000x3000 (= about 1067) a "point", it is desirable for these "points" to the fluorescent quantification. 例如,为了在IO秒内实现上述这一点,每一个点处的停留时间是〜1微秒。 For example, in order to achieve the above-mentioned point IO seconds, the residence time at each point is ~ 1 microsecond. 相应地,在3毫秒内将完成线扫描(3000个点)。 Accordingly, within 3 milliseconds to complete the scan line (3000 dots). 为了获得和使用本发明各实施例而实现的信噪比等价的信噪比,该"点"处的光强应该比本发明的成像系统通过在1秒周期内获得一个信号从而实现的光强要亮〜106倍。 In order to obtain equivalent SNR SNR and use embodiments of the present invention achieved, light from the "point" should be stronger than at the imaging system of the present invention by obtaining a signal in a one second period in order to achieve light Strong ~106 times brighter. 为了在3毫秒内越过3厘米扫描一个点,1000次/秒在机械方面常常是很有挑战性的。 In order to scan across a point 3 cm within 3 ms, 1000 times / sec are often mechanically challenging. 另外,期望高效地收集发射信号。 Further, it is desirable to efficiently collect the transmitted signal. 这通常需要很大的场光学镜片或NA很高的浮动头(例如,以>=10m/s的速度移动)。 This usually requires a large field high NA optical lens or floating head (e.g., at a rate of> = 10m / s the movement). 在这种情况下,和器件左上角处收集的数据相比,器件右下角处收集的数据将晚IO秒,这在某些应用中是不期望出现的。 In this case, upper left corner of the device, and data collected as compared to the data collected at the lower right corner of the device will be late IO seconds, which in certain applications is undesirable arise.

[0038] —些缝合系统获取多个图像,并且将它们缝合到一起从而形成复合图像。 [0038] - some suturing system acquiring a plurality of images, and the stitching them together to form a composite image. 在一个系统中,为了用多个3mmx3mm的图像缝合出一个图像,利用了100个图像。 In a system, in order to use a plurality of image stitching a 3mmx3mm image, use 100 images. 获取这些单张图像所用的总时间将是100毫秒。 Get the total time used by a single image will be 100 ms. 假设大约一半的时间用在移动上面,另一半时间用在获取图像上面,则图像时间将大约是50毫秒。 Assumptions about half of their time moving above the other half the time in obtaining the above image, the image of time would be approximately 50 milliseconds. 该系统通常需要比本发明各实施例在l秒周期内获取1个信号的过程中所用的光强要强20倍的光强,和/或还需要NA更高的物镜。 The system typically requires 20 times stronger intensity light intensity in Example 1 to obtain a signal in the second period within l used in the process of, and / or a higher NA objective lens required ratio of various embodiments of the present invention. 另外, 这些系统通常需要快速的镜台_能够移动3毫米并且能够在50毫秒内完全结束。 In addition, these systems typically require a fast Mirror _ movable 3 mm and can be fully completed within 50 msec. 另外,这些系统通常需要对移动和光强进行仔细的校准,以消除缝合过程中的伪像。 In addition, these systems typically require the mobile and light intensity carefully calibrated to eliminate artifacts during suturing. 和左上角处收集的数据相比,右下角处收集的数据将晚IO秒,这在某些应用中是不期望出现的。 And compared to the data collected at the upper left corner, lower right corner of the data collected at the late IO seconds, which in certain applications is undesirable arise. [0039] 本发明的一些实施例提供了能够使微流体器件所在区域一次全部成像的成像系统。 Some embodiments [0039] The present invention provides a microfluidic device capable region where the imaging system for imaging all at once. 在每个图像可以有10秒的一个实施例中,可以有若干秒的整合时间。 In each image can have a 10-second embodiment, there may be several seconds integration time. 同步化问题减小了或消除了,因为数据是同时从所有的像素中收集的。 Synchronization problem is reduced or eliminated, because the data is collected simultaneously from all pixels. 此外,本发明的各实施例通常同时照亮整个微流体器件(〜31mmx31mm的区域),并且使用具有高NA和大视场的透镜系统。 In addition, embodiments of the present invention generally simultaneously illuminate the entire microfluidic device (~31mmx31mm region), and having a high NA and a large field of view lens system. 如本说明书中所描述的,本发明的各实施例提供了比常规系统简单且更有用的方法和系统。 As described in this specification, embodiments of the present invention provide a simpler and more useful than conventional systems methods and systems. 根据各实施例,本发明各实施例的方法和系统可以克服上述这些局限中的一种或多种。 The method and system according to various embodiments, various embodiments of the present invention can overcome these limitations in one or more of. 在本说明书中,尤其是在下文中,可以找到本发明的细节。 In the present specification, particularly in the following, the details of the invention can be found. 根据各实施例,可能存在这些益处中的一种或多种。 According to various embodiments, these benefits may be present in one or more of. 在本说明书中,尤其是在下文中,会描述这些和其它益处。 In the present specification, particularly in the following, these and other benefits will be described. [0040] 参照下面的详细描述和附图,可以更全面地理解本发明的各种其它的目的、特征和优点。 [0040] reference to the following detailed description and drawings, may be more fully understood the present invention various other objects, features and advantages.

附图说明 Brief Description

[0041] 图1A是示出了本发明一实施例的光学成像系统的简化示意图; [0041] FIG 1A is a diagram showing the present invention is a simplified schematic diagram of an optical imaging system according to an embodiment;

[0042] 图1B描绘了本发明一可选实施例的典型成像系统的概观; [0042] Figure 1B depicts an alternative exemplary of the present invention is an imaging system of an embodiment of an overview;

[0043] 图1C是示出了本发明一实施例的热控制器件的简化示意图; [0043] Figure 1C is a simplified schematic diagram illustrating a thermal control device of an embodiment of the present invention;

[0044] 图2A-2C是示出了本发明一实施例的透镜组件的简化示意图; [0044] Figures 2A-2C is a simplified schematic diagram illustrating a lens assembly according to an embodiment of the present invention;

[0045] 图3是根据本发明一实施例在微流体器件的许多反应室中通过反应而产生的以 [0045] Figure 3 is in accordance with an embodiment of the invention in a number of reaction chambers in the microfluidic devices produced by the reaction of

第一波长为中心的荧光发射的照片; The first fluorescence emission wavelength of the center of the photo;

[0046] 图4是根据本发明一实施例在微流体器件的许多反应室中通过反应而产生的以第二波长为中心的荧光发射的照片; [0046] FIG. 4 is transmitted according to a second center wavelength of fluorescence by an embodiment of the invention produced by the reaction in a number of reaction chambers in the microfluidic device of the photo;

[0047] 图5-7是使用本发明一实施例而产生的选定波长的光点图; [0047] FIG. 5-7 is a selected wavelength of light spot diagram using an embodiment of the present invention is produced;

[0048] 图8是使用本发明一实施例而产生的照明图,该照明图示出了相对均匀性和位置的函数关系图; [0048] Figure 8 is an embodiment of the present invention produced illumination pattern, the illumination diagram illustrating relative uniformity as a function of position and;

[0049] 图9-11是本发明若干实施例的正方块块内能量图; [0049] Figures 9-11 are within the present invention, a number of square Kuaikuai energy diagram of an embodiment;

10[0050] 图12是示出了本发明一实施例所提供的光学系统的场曲和畸变的图; 10 [0050] FIG. 12 is a diagram illustrating an embodiment of the present invention, curvature of field and distortion in Example provided by the optical system;

[0051] 图13是示出了本发明一实施例的系统所产生的"双波长对焦点"的图 [0051] FIG. 13 is a diagram showing an embodiment of the present invention is produced by the system embodiment of "dual wavelength focus" FIG

[0052] 图14-16是使用本发明一实施例而产生的选定波长的光点图 [0052] Figures 14-16 are embodiments of the present invention a selected wavelength generated by a light spot FIG.

[0053] 图17-19是使用本发明一实施例而产生的选定波长的光点图 [0053] Figures 17-19 are embodiments of the present invention a selected wavelength generated by a light spot FIG.

[0054] 图20-22是使用本发明一实施例而产生的选定波长的光点图 [0054] Figure 20-22 is an embodiment of the present invention to produce a selected wavelength of light dot plot

[0055] 图23-25是使用本发明一实施例而产生的选定波长的光点图 [0055] Figures 23-25 are embodiments of the present invention a selected wavelength generated by a light spot FIG.

[0056] 图26-28是本发明若干实施例的正方块块内能量图。 [0056] FIGS. 26-28 are within the present invention, a number of square Kuaikuai energy diagram of an embodiment.

具体实施方式 DETAILED DESCRIPTION

[0057] 根据本发明,提供了用于微流体系统的多种技术。 [0057] The present invention provides a variety of techniques for microfluidic systems. 特别是,本发明提供了一种给微流体器件的反应室内的流体体积中悬浮的一个或多个实体成像的方法和系统。 In particular, the present invention provides a method and system to the fluid volume of the reaction chamber in the microfluidic device of one or more entities suspended imaging. 更具体地讲,这种成像方法和系统利用了与微流体器件中的一个或多个实体相关联的荧光信号的指示。 More specifically, this image forming method and system utilizing a microfluidic device indicating one or a plurality of fluorescent signal associated with the entity. 仅仅作为示例,通过使用和微流体器件相耦合的荧光、化学发光和生物发光读出器,来应用微流体方法和系统技术,但是将会认识到本发明具有更广的应用范围。 Example only, and by using the microfluidic device coupled to a fluorescent, chemiluminescent and bioluminescent reader, to apply microfluidic methods and systems technology, it will be recognized that the invention has a broader range of applications. [0058] 在本申请中,提到了微流体器件中的某些类型的"反应"室。 [0058] In the present application, referred to the microfluidic device of some type of "reaction" chamber. 通常,这些"反应室" 包括处理位置、处理室、和/或反应位置、它们的任意组合等等。 Typically, these "reaction chamber" includes a processing position, the process chamber, and / or reaction site, any combination thereof and the like. 这些室室可以是闭合的、部分闭合的、打开的、部分打开的、密封的、或它们的组合等等,其中包括涉及这些状态中的任何状态的任何暂时或瞬时条件。 These chambers chambers can be closed, partially closed, open, partially open, closed, or a combination thereof and the like, including reference to any of these states of any temporary or transient conditions. 在一些实施例中,这些室室是密封的、能够密封的、可闭合的、隔离的、能够隔离的、和它们的组合、以及涉及这些状态中的任何状态的任何暂时或瞬时条件中的任何组合或单个条件。 In some embodiments, these chambers are sealed chambers, sealable, closeable, isolated, can be isolated, and combinations thereof, and any temporary or transient conditions involving any of these states of any or a combination of a single condition. 因此,术语"反应室"的使用并不旨在限制本发明,而是包括这些其它结构。 Thus, the term "reaction chamber" is used not intended to limit the present invention but are to include these other structures. 另外,术语"室"并不旨在限制本发明,而是应该按其普通的意思来使用,除非已经引用和该室相关联的特定特征。 Further, the term "room" is not intended to limit the invention, but should be used according to their ordinary meaning, unless a particular reference feature associated with the chamber. 当然,可以有其它变化、修改和替换。 Of course, there can be other variations, modifications and substitutions. [0059] 此外,在本申请中,提到了来自微流体器件的荧光指示。 [0059] Further, in the present application, referred to the fluorescence of an instruction from the microfluidic device. 本发明的范围内不仅包括荧光指示,还包括发光指示,其中包括化学发光、电致发光、电化学发光、磷发光、生物发光和其它发光过程、或包括可用检测器件检测的任何其它类型的指示的任何其它过程。 Included within the scope of the present invention is not fluorescent indicator, including flares, including chemiluminescence, electroluminescence, ECL, phosphorus, bioluminescence and other emission process, or include any other type of indicator can be used to detect the device detection any other process. 对于本领域的技术人员,很明显,可用于检测并分析这些荧光和发光指示的方法和系统可以从一种指示转移到另一种指示。 Skilled in the art, it is clear that the method can be used to detect and analyze the fluorescence and luminescence and systems of these instructions can be transferred from one indication to another indication. 另外,尽管本发明的一些实施例利用光谱滤光片作为光学元件,但是本发明并没有要求这一点。 In addition, although some embodiments of the present invention utilizing spectral filter as an optical element, but the present invention does not require this. 一些荧光和发光应用并不在光激发通道、光发射通道或二者中使用光谱滤光片。 Some applications are not fluorescent and luminescent light excitation channel, or both of the light emission path using spectral filter. 如本文所述,其它实施方式采用光谱滤光片。 As described herein, other embodiments using spectral filter. 本领域的技术人员会理解与特定应用相关联的诸多差异。 Those skilled in the art will appreciate that many differences associated with specific applications.

[0060] 在一些实施方式中,使用了用于进行微流体分析的各种器件和方法,其中包括可用于进行热循环反应如核酸扩增反应的器件。 [0060] In some embodiments, using a variety of devices and methods for micro-fluid analysis, including thermal cycling can be used for reactions such as nucleic acid amplification reaction device. 这些器件和常规微流体器件相比不同之处在于:它们包括了弹性组件;在某些情况下,该器件大部分或全部都是由弹性材料构成的。 These devices are different compared to the conventional microfluidic devices in that: they include elastomeric components; in some cases, most or all of the device are made of an elastic material. 例如,扩增反应可以是线性扩增(用一种引物扩增)以及指数扩增(即用正向和反向组进行扩增)。 For example, amplification reactions can be linear amplification (using a primer), and exponential amplification (i.e., the group with the forward and reverse amplification).

[0061] 本发明的一些实施方式所提供的方法和系统在执行核酸扩增反应的过程中利用了盲端通道类型的器件。 [0061] Some embodiments of the method and system of the present invention there is provided the use of the blind end of the channel type of the device during the execution of a nucleic acid amplification reaction. 在这些器件中,通常沉积在反应位点内的试剂是进行所需类型的扩增反应所必需的那些试剂。 In these devices, the reagent is typically deposited within the reaction sites are those desired type of amplification reaction reagents necessary to carry out. 通常,这意味着沉积以下某些或全部试剂:引物、聚合酶、核苷酸、金属离子、缓冲液和辅因子。 Typically, this means that some or all of the following deposition of reagents: primers, polymerase, nucleotides, metal ions, buffer, and cofactors. 在这种情况下,引入反应位点的样品是核酸模板。 In this case, the introduction of samples of the reaction sites is a nucleic acid template. 然而,可 However, you can

11沉积该模板,并且使扩增试剂流入反应位点。 Depositing the template 11, and the amplification reagents into the reaction sites. 如本发明中详细讨论的那样,当使用矩阵器件进行扩增反应时,含核酸模板的样品流过垂直的流动通道,而扩增试剂则流过水平的流动通道,反之亦然。 As discussed in detail in the present invention, when performing an amplification reaction using a matrix device, a sample containing nucleic acid template to flow through the vertical flow channels, and amplification reagents flowing through the horizontal flow channel, and vice versa.

[0062] PCR或许是已知最好的扩增技术。 [0062] PCR is perhaps the best known amplification techniques. 本发明各实施例所使用的器件并不限于进行PCR扩增。 Device used in Example embodiments of the present invention is not limited to PCR amplification. 可以进行的其它类型的扩增反应包括但不限于:(i)连接酶链反应(LCR)(参照Wu和Wallace, Genomics 4:560(1989)以及Landegren等人,Science 241:1077(1988)); (ii)转录扩增(参照Kwoh等人,Proc. Natl. Acad. Sci. USA 86:1173(1989)) ;(iii)自动维持序列复制(参照Guatelli等人,Proc. Nat. Acad. Sci. USA, 87 :1874 (1990));以及(iv)基于核酸的序列扩增(NASBA)(参照Sooknanan, R.和Malek, L. , BioTechnology 13 : 563-65(1995))。 Other types of amplification reactions can be carried out include, but are not limited to: (i) ligase chain reaction (LCR) (Wu and reference Wallace, Genomics 4: 560 (1989) and Landegren et al., Science 241: 1077 (1988)) ; (ii) transcription amplification (see Kwoh et al., Proc Natl Acad Sci USA 86:.... 1173 (1989)); (iii) automatically maintains sequence replication (see Guatelli et al., Proc Nat Acad Sci... . USA, 87: 1874 (1990)); and (iv) a nucleic acid sequence-based amplification (NASBA) (refer Sooknanan, R. and Malek, L., BioTechnology 13: 563-65 (1995)). 上述各种参考文献以其全部内容引用在此。 The above cited reference in its entirety herein.

[0063] 此外,设计某些器件,以采用包括一个或多个弹性阀的器件进行热循环反应(如PCR),所述弹性阀用来调控流过该器件的溶液。 [0063] In addition, some devices designed to use the valve comprises one or more elastic devices thermal cycling reactions (e.g., PCR), the resilient valve for the regulation of solution flow through the device. 因此,也提供了用这种设计的器件来进行扩增反应的方法。 Therefore, this design also provides a device to carry out the amplification reaction.

[0064] 不管是被隔离在反应室内还是在随后的任何时刻,都可以用检测核酸的常规方法来检测并区分扩增产物(扩增子)。 [0064] The reaction is isolated either indoors or at any subsequent time, can be detected by a conventional method to detect and distinguish between the nucleic acid amplification product (amplicon). 使用嵌入染料如SYBRTM、Pico Green(俄勒冈州Eugene市Molecular Probes有限公司)、溴化乙锭等(参照Zhu等人,1994, Anal. Chem. 66 : 1941-48)和/或凝胶电泳,可以检测包含双链DNA的扩增子。 Using embedded dye as SYBRTM, Pico Green (Molecular Probes Eugene, Oregon City, Ltd.), ethidium bromide (see Zhu et al., 1994, Anal Chem 66:.. 1941-48) and / or gel electrophoresis, can Detection of double-stranded DNA containing the amplicon. 更常见的是,使用序列特异性检测方法(即基于核苷酸序列检测扩增子)。 More commonly, the use of sequence-specific detection method (i.e., detection based on the nucleotide sequence of the amplicon). 检测方法的示例包括:固定的寡核苷酸或多核苷酸的阵列杂交;以及使用基于差别标记分子信标的检测系统,或者其它"荧光共振能量转移"(FRET)检测系统。 Examples of detection methods include: a fixed array hybridization of oligonucleotide or polynucleotide; and using the difference labeled molecular beacons based detection system, or other "Fluorescence resonance energy transfer" (FRET) detection system. 根据本发明的一些实施方式,FRET检测是较佳的检测方法。 According to some embodiments of the invention, FRET detection is the preferred detection method. 在FRET试验中,检测供体/受体荧光团对中供体(报道物)和/或受体(淬光剂)荧光团的荧光变化。 In FRET experiments, testing the donor / acceptor fluorophore on the body (the reporter) and / or acceptor (quencher) fluorophore for fluorescence changes. 选择供体和受体荧光团,使得供体的发射光谱与受体的激发光谱交迭。 Select the donor and acceptor fluorophores so that the excitation spectrum overlaps the emission spectrum of the donor and acceptor. 因此,当使这对荧光团彼此靠得足够近时,会发生从供体到受体的能量转移,并且可以检测到这种能量转移。 Therefore, when the fluorophores which are close enough to each other occurs when energy is transferred from the donor to the acceptor, and can detect this energy transfer. 已知各种试验,包括但不限于:模板延伸反应、定量RT-PCR、分子信标(Molecular Beacons)和侵入检测(Invader assay),下面对它们进行简述。 Various tests are known, including, but not limited to: the template extension reactions, quantitative RT-PCR, molecular beacons (Molecular Beacons) and intrusion detection (Invader assay), below them briefly.

[0065] FRET和模板延伸反应使用:用供体/受体对中的一个成员标记的引物,以及用此供体/受体对中的另一成员标记的核苷酸。 [0065] FRET and template extension reactions using: using donor / acceptor pair a member labeled primers and nucleotides with this donor / acceptor pair in another member mark. 在模板依赖性延伸反应期间将标记核苷酸掺入引物中之前,先将供体和受体分隔得足够远,以使能量转移无法发生。 During the template-dependent extension reaction in the labeled nucleotide incorporated into the primer before, first the donor and acceptor are separated far enough, so that energy transfer can not occur. 然而,如果标记核苷酸掺入引物并且间隔足够近,则会发生能量转移并且可以检测到这种能量转移。 However, if the labeled nucleotide incorporated into the primer and spaced sufficiently close, then energy transfer occurs and can detect this energy transfer. 这些方法在检测单核苷酸多态性的单碱基对延伸反应中特别有用,参见美国专利5, 945, 283和PCT 公开W0 97/22719。 These methods of detecting single nucleotide polymorphism is particularly useful for single base extension reactions, see U.S. Patent No. 5, 945, 283, and PCT Publication W0 97/22719. 通过使用本说明书中所述的温度控制方法和装置,可以任选地对这些反应进行热循环,以增大信号。 By using the temperature control method and apparatus according to the present specification, these reactions may optionally be carried out on thermal cycling, to increase the signal.

[0066] 也可以使用各种所谓的"实时扩增"方法或"实时定量PCR"方法,通过测量扩增过 [0066] You can also use a variety of so-called "real-time amplification" methods or "real-time quantitative PCR" method, by measuring the amplification is too

程期间或之后所形成的扩增产物的量,来确定样品中存在的靶核酸量。 Amount of amplification product formed during or after the process, to determine the amount of target nucleic acid present in the sample. 发荧光的核酸酶试 Fluorescing nuclease test

验是实时定量方法的一个特定示例,实时定量方法可以和本文所述器件一起成功地使用。 Test is a specific example of real-time quantitative methods, real-time quantitative methods and the device can be used with success in this article.

这种监控扩增产物的形成的方法包括:用双标记的荧光寡核苷酸探针连续测量PCR产物累 This method of monitoring the formation of amplification product comprises: a fluorescent-labeled oligonucleotide probe double tired continuous measurement of PCR product

积,此方法在文献中常被称为"TaqMan"法。 Product, this method is often referred to in the literature "TaqMan" method. 参见例如,美国专利5, 723, 591。 See, e.g., U.S. Patent No. 5, 723, 591.

[0067] 在使用分子信标的情况下,当它杂交到扩增产物的互补区域时探针构象的变化产 [0067] In the case where the molecular beacon when it hybridizes to a complementary region of the amplification product produced probe conformational change

生了可检测的信号。 Gave birth to a detectable signal. 探针自身包括两部分:5'端的一部分,以及3'端的另一部分。 Probe itself consists of two parts: the 5 'portion of the end, and the 3' end of the other portion. 这些部分侧接于退火到探针结合位点的探针部分,并且彼此互补。 These sections flank the probe annealing to the portion of the probe binding site, and complementary to each other. 一端部分通常连接于报道染料, 而另一端部分通常连接到淬光染料。 Is commonly connected to one end portion of the reporter dye, while the other end portion is typically connected to the quencher dye. 在溶液中,这两个端部分可以彼此杂交形成发夹环。 In solution, the two end portions may hybridize to each other forming a hairpin loop. 在这种构象中,报道物和淬光剂靠得足够近,使得报道物的荧光被淬光染料有效地淬灭。 In this conformation, the reporter and quencher close enough so that the fluorescent reporter dye is quenched efficiently quench the light. 相反,杂交探针产生线性化构象,其中淬灭程度降低。 In contrast, hybridization probes produce linearized conformation, which reduces the degree of quenching. 由此,通过监测这两种染料的发射光变化,有可能间接地监测扩增产物的形成。 Thus, by monitoring the change in light emission of the two dyes, it is possible to indirectly monitor the formation of amplification product. 这种类型的探针及其使用方法在下面的文献中有进一步的描述:如Piatek等人,1998年,Nat. Biotechnol. 16 :359-63 ;Tyagi和Kramer, 1996年,Nat. Biotechnology 14 :303-308 ;以及Tyagi等人,1998年,Nat. Biotechnol. 16 : 49-53(1998)。 This type of probe and method of use there is in the following further description of the literature: e.g. Piatek et al., 1998, Nat Biotechnol 16: 359-63; Tyagi and Kramer, 1996 年, Nat Biotechnology 14...: . 303-308; and Tyagi et al., 1998, Nat Biotechnol 16: 49-53 (1998)..

[0068] 下列文献中描述了Scorpion检测法:Thelwell等人,2000年,Nucleic AcidsResearch,28 :3752-3761 ;以及Solinas等人,2001年,"SNP分析和FRET应用中的双链体Scorpion弓|物"(Duplex Scorpion primers in SNP analysis and FRETapplications) ,Nucleic Acids Research 29:20。 [0068] The following documents describe the Scorpion assays: Thelwell et al., 2000, Nucleic AcidsResearch, 28: 3752-3761; and Solinas et al., 2001, "SNP analysis and FRET applications duplexes Scorpion Bow | matter "(Duplex Scorpion primers in SNP analysis and FRETapplications), Nucleic Acids Research 29:20. Scorpion引物是荧光PCR引物,其中探针元件通过PCR终止物连接到5'端。 Scorpion primer is a fluorescent PCR primers, wherein the probe element is connected to the 5 'end by PCR Terminator. 它们用于均一溶液中PCR产物的实时扩增子特异性检测。 They are used in a homogeneous solution of PCR products in real-time amplicon-specific detection. 可能有两种不同的形式,即"茎-环"形式和"双链体"形式。 There may be two different forms, i.e., "stem - loop" form and the "duplex" form. 在这两种情况下, 探测机制都是作用于分子内。 In both cases, detection mechanisms are acting on the molecule. 所有形式的Scorpion基本元件是:(i)PCR引物;(ii)PCR终止物,用于防止探针元件的PCR通读(read-through) ;(iii)特定探针序列;以及(iv)荧光检测系统,该系统包含至少一个荧光团和淬光剂。 Scorpion basic element of all forms are: (i) PCR primers; (ii) PCR was terminated, to prevent PCR read (read-through) a probe element; (iii) a specific probe sequence; and (iv) fluorescence detection system, which contains at least one fluorophore and quencher. 在Scorpion引物的PCR延伸之后,所得扩增子包含与探针互补的序列,在各PCR循环的变性阶段将其分解成单链。 After PCR extension of the Scorpion primer, the resultant amplified promoter contains sequences complementary to the probe, the denaturation step in each PCR cycle will be broken down into single strands. 在冷却时,该探针可自由地结合该互补序列,从而增加荧光,因为淬光剂不再位于荧光团附近了。 Upon cooling, the probe can be freely combined with the complementary sequence, and thus an increase in fluorescence, since the quencher is no longer in the vicinity of the fluorophore. PCR终止物防止T叫DNA聚合酶进行不期望的探针通读。 PCR terminator T called DNA polymerase prevent undesirable probe read.

[0069] 侵入检测(Third Wave Technologies公司,Madison市,威斯康星州)被特别地用于SNP基因型分析,它利用与靶核酸(DNA或RNA)或多态性位点互补的寡核苷酸,称为信号探针。 [0069] Intrusion Detection (Third Wave Technologies Corporation, Madison City, Wisconsin) are particularly useful for SNP genotyping, which uses the target nucleic acid (DNA or RNA) or polymorphism site complementary oligonucleotides, called the signal probe. 第二寡核苷酸(称为侵入寡核苷酸)包含相同的5'核苷酸序列,但3'核苷酸序列包含核苷酸多态性(位点)。 Second oligonucleotide (referred invasive oligonucleotide) contains the same 5 'nucleotide sequence, but the 3' nucleotide sequence comprises nucleotide polymorphism (loci). 侵入核苷酸干扰信号探针与靶核酸的结合,使得信号探针的5'端在包含多态性(位点)的核苷酸处形成"下垂物"。 Nucleotide binding interference signal invasive probe and a target nucleic acid, so that the signal of the probe 5 'end to form a "sag was" at nucleotides containing polymorphisms (sites). 该复合物被一种称为Cleavase 酶的结构特异性内切核酸酶识别。 Endonuclease recognition within the structural specificity of the complex is called Cleavase enzyme. Cleavase酶切下核苷酸的5'下垂物。 Cleavase enzyme under nucleotides 5 'sagging thereof. 所释放的下垂物与携带FRET标签的第三探针结合,由此形成另一种由Cleavase酶识别的双链体结构。 Sag was released and a third carrying FRET label probe binding, thereby forming another duplex structure recognized by the Cleavase enzyme. 此时,Cleavase酶从淬光剂中切下荧光团,产生荧光信号。 At this point, Cleavase enzyme cut from the quencher fluorophore, resulting in a fluorescent signal. 对于SNP基因型分析,设计信号探针,使其与参比(野生型)等位基因或变异(突变)的等位基因杂交。 For SNP genotyping, the signal probe is designed so that the reference (wild type) allele or the variant (mutant) allele hybridization. 不像PCR,有一种线性的信号扩增,其中没有核酸扩增。 Unlike PCR, there is a linear amplification of the signal, wherein there is no nucleic acid amplification. 足以指导本领域普通技术人员的诸多细节参见:Neri, BP等人,Advances in Nucleic Acid and Protein Analysis3826 :117-125, 2000年;以及美国专利6, 706,471。 Sufficient to guide the many details of ordinary skill in the art, see: Neri, BP, et al., Advances in Nucleic Acid and Protein Analysis3826: 117-125, 2000 years; and US Patent 6, 706,471.

[0070] 各种多重扩增系统可与本发明联用。 [0070] various multiplex amplification system associated with the present invention can be used. 在一种类型中,可使用多个标记不同的探针(每一个探针被设计成只杂交到特定的目标)同时检测若干个不同的靶标。 In one type may be used a plurality of different labeled probes (each probe is designed to hybridize only to a specific target) simultaneously detect a plurality of different targets. 因为各探针具有不同的标记,所以根据荧光信号检测与各靶标的结合。 Because each probe has a different label, so that binding with the target of the fluorescence signal detection. 通过明智地选择所使用的不同标记,可进行在单次反应中以不同波长激发和/或检测不同标记的各种分析。 By judicious choice of the different markers used, can be carried out in a single reaction to a different wavelength of the excitation and / or detection of a variety of different markers analyzed. 例如,可以参照:《荧光光谱学》(FluorescenceSpectroscopy) (Pesce等人著)Marcel Dekker, New York, (1971) ;White等人著,《荧光分析:实践方法》(Fluorescence Analysis :A Practical Approach) , MarcelDekker, New York, (1970) ;Berlman著,《芳族分子的荧光光谱手册》 For example, you can refer: "Fluorescence Spectroscopy" (FluorescenceSpectroscopy) (Pesce et al.) Marcel Dekker, New York, (1971); White et al., "Fluorescence analysis: practices" (Fluorescence Analysis: A Practical Approach), MarcelDekker, New York, (1970); Berlman forward, "aromatic molecule fluorescence spectroscopy Handbook"

13(Handbookof Fluorescence Spectra of Aromatic Molecules),第二片反,Academic Press, NewYork, (1971) ;Griffiths著,《有机分子的颜色和构造》(Colour and Constitutionof Organic Molecules) , Academic Press, New York, (1976);《指不物》(Indicators) (Bishop 编),Pergamon Press, Oxford, 19723 ;以及Haugland著,《荧光探针和研究用化学物质手册》(Handbook of Fluorescent Probes and ResearchChemicals) , Molecular Probes, Eugene市(1992)。 13 (Handbookof Fluorescence Spectra of Aromatic Molecules), the second sheet trans, Academic Press, NewYork, (1971); Griffiths a, "the color and structure of organic molecules" (Colour and Constitutionof Organic Molecules), Academic Press, New York, ( 1976); "means do not matter" (Indicators) (Bishop edited), Pergamon Press, Oxford, 19723; and Haugland with, "fluorescent probes and research with chemicals Handbook" (Handbook of Fluorescent Probes and ResearchChemicals), Molecular Probes, Eugene City (1992).

[0071] 许多与基因组修饰有关的疾病,无论是宿主有机体的抑或感染有机体的疾病,是少量核苷酸发生变化(常常包括单个核苷酸改变)的结果。 [0071] Many diseases associated with genomic modification, either the host organism or organisms infected with the disease, is the result of a small number of nucleotide changes (often including single nucleotide changes) of. 这种单核苷酸变化称为单核苷酸多态性(简称为SNP),并且发生SNP的位点通常称为多态性位点。 Such single nucleotide change is called single nucleotide polymorphism (abbreviated as SNP), and the occurrence of the SNP site is usually referred to as a polymorphic site. 本文所述的器件可用于确定这种多态性位点处存在的核苷酸的身份。 The device described herein may be used to determine the identity of the presence of this polymorphic site at nucleotide. 作为这种能力的扩展,这些器件可用于基因型分析。 As an extension of this capability, these devices can be used for genotyping. 基因型分析包括:确定二倍体生物体(即各基因有两个拷贝的生物体)是否包含两个拷贝的参比等位基因(参比型纯合子),或者包含参比和变异等位基因各一个拷贝(即杂合子),抑或包含两个拷贝的变异等位基因(即变异型纯合子)。 Genotype analysis includes: determining diploid organisms (ie, there are two copies of each gene in the organism) contain two copies of the reference allele (homozygous reference), or contains, such as reference and mutations a copy of each gene (i.e., heterozygous), or contains two copies of the variant allele (i.e., variant homozygous). 当进行基因型分析时,可用本发明方法询问单个变异位点。 When genotype analysis, the method of the present invention can be used to ask a single mutation sites. 然而,如下文关于多道传输那部分所进一步描述的,这些方法也可用于确定许多不同的DNA位置(loci)(相同基因上的DNA位置、不同基因上的DNA位置或二者的组合)中单个(基因)的基因型。 However, as discussed with regard to that portion of multi-channel transmission further described, these methods may also be used to determine the number of different DNA positions (loci) (DNA position on the same gene, or a combination of different genes on the DNA position) in single (gene) genotypes.

[0072] 用于进行基因型分析的器件被设计成采用大小合适的反应位点,以在统计学上确保该反应位点中存在可行DNA浓度的二倍体的两种等位基因各一拷贝。 A copy of each device [0072] for performing genotype analysis is designed to use suitably sized reaction site, in order to ensure the presence of viable DNA concentration of the reaction sites in the statistically diploid two alleles . 否则,分析可能产生表明杂合子就是纯合子的结果,仅仅因为该反应位点处不存在第二种等位基因的拷贝。 Otherwise, the analysis may yield results indicate that heterozygous homozygous, simply because the reaction at the site of the second copy of the allele is not present. 下表1指出本文所述器件可利用的各种示范性DNA浓度下1纳升反应体积中存在的基因组的拷贝数。 The following table 1 indicates the copy number of various exemplary DNA concentrations of the device described herein can be used in a nanoliter reaction volume present in the genome.

[0073] 表格1 :在所示DNA浓度下1纳升体积中存在的基因组拷贝数。 [0073] Table 1: Number of genomic DNA copies shown at a concentration of 1 nanoliter volume exist. [0074] [0074]

<table>table see original document page 14</column></row> <table>[0075] —般说来,因样品的随机配比,扩增反应开始之前存在的拷贝数决定了测量中可能的误差。 <Table> table see original document page 14 </ column> </ row> <table> [0075] - In general, the result of a random sample of the ratio, the number of copies of the amplification reaction came into existence before the decision of the measurements possible error. 通常用DNA浓度约为0. lOi! g/y L的样品来进行使用某些器件的基因型分析, 尽管本发明人已经在每一反应位点有一个基因组的浓度下成功地运行了T叫Man反应。 Usually DNA concentration of about 0. lOi! G / y L samples for genotyping using certain devices, although the present invention have a genome in a concentration in each reaction site under the successful operation of the T called Man reactions. [0076] 基因型分析可以使用各种不同的方法来进行。 [0076] Genotypic analysis can use a variety of methods. 在这些方法中,通常足以获得"是" 或"否"的结果,即检测过程只需要能够回答是否存在给定等位基因的问题。 In these processes, is usually sufficient to get "yes" or "no" outcome, i.e., the detection process only needs to be able to answer whether a given allele problem exists. 因此,只有使用用来检测可能位于多态性位点处的一种等位基因的存在所必需的引物或核苷酸,才能够进行各种分析。 Therefore, only the use may be located to detect the presence of an allele at a polymorphic site necessary primers or nucleotides, to be able to carry out various analyzes. 然而,更常见的是,包括了用于检测多态性位点处可能存在各等位基因的引物和核苷酸。 However, more commonly, comprising detecting polymorphic sites may exist for each allele primers and nucleotides.

[0077] 单碱基对延伸(SBPE)反应是一种专门针对基因型分析而开发的技术。 [0077] single base pair extension (SBPE) reaction is a specially developed for genotyping technology. 尽管已经开发出许多SPBE试验,但是一般方法相当相似。 Although many SPBE tests have been developed, but generally the method is quite similar. 通常,这些试验包括杂交一种与靶核酸互补的引物,使得该引物的3'端恰恰是变异位点的5'端或与其相邻。 Generally, these tests include hybridization with a primer complementary to the target nucleic acid such that the primer 3 'end of variable sites precisely 5' end or adjacent thereto. 在一种或多种标记的不可延伸的核苷酸和聚合酶的存在下进行延伸,这种核苷酸与占据变异位点的核苷酸互补。 In the presence of one or more labeled non-extendible nucleotide and extends polymerase, nucleotides and nucleotide occupying such variable sites complementary. 不可延伸的核苷酸是一种核苷酸类似物,一旦将该核苷酸类似物掺入引物中便可以防止聚合酶的进一步延伸。 Non-extendable nucleotide is a nucleotide analog, once the nucleotide analogues can be incorporated into the primer in the polymerase to prevent further extension. 如果所添加的不可延伸的核苷酸与变异位点的核苷酸互补,则标记的不可延伸的核苷酸被掺入该引物的3'端,从而产生标记的延伸产物。 If the added non-extendible nucleotide is a nucleotide complementary to the mutation site, the non-extendible nucleotide is incorporated into a labeled 3 'end of the primer, resulting in extension products labeled. 因此,延伸引物可以表示哪一种核苷酸存在于靶核酸的变异位点处。 Thus, extension primer which can represent one nucleotide present in the polymorphic sites of the target nucleic acid. 这些方法和相关的方法在下列文献中进行了讨论:美国专利5, 846, 710、6, 004, 744、5, 888, 819、5, 856, 092、5, 710, 028以及W0 92/16657。 These methods and related methods are discussed in the following documents: U.S. Patent No. 5, 846, 710,6, 004, 744,5, 888, 819,5, 856, 092,5, 710, 028 and W0 92/16657 .

[0078] 也可用定量PCR方法进行基因型分析。 [0078] can also be used quantitative PCR genotyping methods. 在这种情况下,与各等位基因形式互补的区别标记探针与引物、核苷酸和聚合酶一起被包括成为试剂。 In this case, with the difference between the allelic forms of a labeled probe complementary to the primer, nucleotide and polymerase are included together into a reagent. 然而,可以只用一种探针进行反应,尽管这会产生不确定性,即没有信号是因为没有特定的等位基因还是简单地因为反应失效。 However, only one probe can be reacted, although this would create uncertainty, i.e., no signal because there is no particular allele or simply because the reaction failed. 对于一个多态性位点有两个等位基因的典型双等位基因情况,试剂混合物中通常包含两个区别标记的探针(各自与等位基因之一完美互补),以及扩增引物、核苷酸和聚合酶。 For a polymorphic locus has two alleles of a typical two-allele case, the reagent mixture typically contains two probes diacritical marks (each with one allele perfect complement), as well as primers, nucleotides and polymerase. 将包含靶DNA的样品引入反应位点。 The sample containing the target DNA is introduced into the reaction site. 如果靶DNA中存在与探针互补的等位基因,则进行扩增,由此产生在上述检测中所描述的可检测信号。 If the presence of the allele probe is complementary to the target DNA, the amplification is performed, thereby generating a detectable signal in the detection described. 根据所获的差别信号,可以确定多态性位点处的核苷酸身份。 According to the obtained difference signal, you can determine the identity of the nucleotide at the polymorphic site. 如果两个信号都被检测到,则两个等位基因都存在。 If the two signals are detected, then both alleles are present. 如上述温度控制部分所述,进行反应期间的热循环。 As the above-described temperature control section, a heat cycle during the reaction.

[0079] 基因表达分析包括:测定在特定细胞中表达一种或多种基因的水平。 [0079] Gene expression analysis comprising: determining the expression levels of one or more genes in a particular cell. 这种测定可以是定性的,但通常是定量的。 Such assays may be qualitative, but generally is quantitative. 在差别基因表达分析中,将一种细胞(例如受试细胞)中的基因水平与另一种细胞(对照细胞)中相同基因的表达水平进行比较。 In differential gene expression analysis, the expression level of a cell (e.g., a test cell) level of the gene with another cell (control cell) are compared in the same gene. 可以做出非常多的这种比较。 Can make a lot of this comparison. 示例包括但不限于:健康细胞和病变细胞之间的比较;用一种药物治疗的个体的细胞和未治疗个体的细胞之间的比较;接触和未接触具体毒剂的细胞之间的比较,等等。 Examples include, but are not limited to: comparison between healthy cells and diseased cells; the cells of the individual comparison between the individual cells with a drug treatment and untreated; Comparative contact and non-contact between the cell specific agents, etc. and so on. 在受试和对照细胞中表达水平不同的那些基因可以用作治疗的标记物和/或靶点。 Those different levels of gene expression in the test and control cells can be used as a marker of treatment and / or targets. 例如, 如果在病变细胞而非健康细胞中发现某组基因被上调,则这些基因可以用作疾病的标记物并且有可能用作诊断测试的基础。 For example, if a group of genes found to be up-regulated in diseased cells rather than healthy cells, these genes can be used as a marker of the disease and may be used as basis for diagnostic tests. 这些基因也可以是靶点。 These genes may also be targets. 治疗该疾病的方案可包括导致这些上调基因表达降低的步骤。 Treatment of the disease programs may include steps to reduce these lead to increase gene expression.

[0080] 本发明实施方式中所使用的微流体器件的设计有利于各种基因表达分析。 [0080] The microfluidic device design embodiment of the present invention is used in a variety of beneficial gene expression analysis. 因为这些器件包含大量的反应位点,所以可同时测定大量的基因和/或样品。 Because these devices contain a large number of reactive sites, it can be determined simultaneously a large number of genes and / or samples. 采用盲端流体通道器件,可以同时测定成百上千种基因的表达水平。 Blind-side fluid-channel device, can simultaneously measured the expression levels of hundreds of genes. 这些器件也有利于差别基因表达分析。 These devices are also conducive to differential gene expression analysis. 在使用矩阵设计的情况下,获自健康细胞的样品可以在一条流体通道中进行测试,而来自病变细胞的样品则在紧邻的通道中运行。 In the case of using the matrix design, the sample obtained from a healthy cell can be tested in one flow channel, while the sample from the diseased cells run in adjacent channels. 这种特征使检测变得容易,并且提高了结果的精确度,因为两个样品同时在同一器件上运行并且处于相同的条件下。 This feature makes the detection becomes easy, and improves the accuracy of the results because the two samples run on the same device and in the same conditions.

[0081] 根据本发明的各实施方式,也使用各种矩阵器件或以阵列为基础的器件。 [0081] According to embodiments of the present invention, the device also uses a variety of matrix or array-based devices. 某些这样的器件包括:(i)第一类多个流体通道,形成于弹性基板中;(ii)第二类多个流体通道, 形成于弹性基板中并与第一类多个流体通道相交从而限定了反应位点的阵列;(iii)多个隔离阀,设置在第一类和第二类多个流体通道内,可用于将各反应位点内的溶液与其它反应位点处的溶液隔离开;以及(iv)多个保护通道,它们覆盖在一条或多条流体通道和/或一个或多个反应位点上,以防止溶液蒸发。 Some of these devices include: (i) a plurality of first type fluid passage formed in the elastic substrate; (ii) a second plurality of fluid channel type, formed in the elastic substrate with a plurality of fluid channels intersecting the first type thereby defining an array of reactive sites; (iii) a plurality of isolation valve located in the first class and second class plurality of fluid channels for the solution in the solution of the reaction sites and other reactions at the site isolated; and (iv) a plurality of guard channels, they are overlaid on one or more fluid channels and / or one or more reaction sites, in order to prevent the solution was evaporated. 上述器件可用于许多不同类型的反应,包括那些涉及温度调节的反应(例如,核酸分析的热循环)。 The devices can be used in many different types of reactions, including those involving temperature regulation of the reaction (e.g., thermal cycling nucleic acid analysis).

[0082] —些微流体器件使用一种常被称为"盲端通道"或"盲端填充"的设计,这种设计具有多个盲端通道,盲端通道是具有一个死端或隔离端使得溶液只能从该盲端通道的一个端进出的这样一种流体通道(即盲端通道没有单独分离的入口和出口)。 [0082] - the device uses a slightly fluid often called "blind-side channel" or "blind side fill" the design, which has a plurality of blind-ended channels, blind-ended channel having a dead-end or end the isolation makes Such a solution can only be a fluid passage from one end of the blind end of the tunnel and out (ie, dead-end channel separation is no separate entrance and exit). 这些器件只需要给每一个盲端通道配单个阀,以便隔离盲端通道的区域从而形成隔离的反应位点。 These devices just give each channel with a blind-side single valve to isolate the blind end of the channel region to form isolated reaction sites. 在这种类型的器件的制造过程中,用于进行分析的一种或多种试剂被沉积在反应位点处,由此使输入和输出的次数显著减小。 In the manufacturing process of this type of device, for analysis of one or more reagents are deposited at the reaction site, whereby the number of input and output is significantly reduced. 另外,这些盲端通道连接到互连的通道网络,使得可以从单个或有限个样品输入填充所有的反应位点。 In addition, these blind-side channel is connected to the interconnected network of channels, making it possible to fill all of the reaction sites from a single or a finite number of input samples. 因为输入和输出复杂性减小了并且只使用单个阀来隔离每一个反应位点,所以可用于反应位点的空间增大了。 Because the input and output complexity is reduced and only use a single valve to isolate each reaction site, so that the space available for reaction sites is increased. 由此,这些器件的特征意味着, Thereby, characteristics of these devices means,

每一个器件可以包括大量的反应位点(例如,多达上万个),并且可以实现高反应位点密度(例如,每平方厘米超过1, 000-4, 000个反应位点)。 Each device may include a large number of reactive sites (e.g., up to tens of thousands) and can achieve high reaction site densities (e.g., per square centimeter than 1, 000-4, 000 reaction site). 和常规的微流体器件相比,上述这些特征也直接地使器件的尺寸显著减小。 And compared to the conventional microfluidic device, these characteristics also make the size of the device directly reduced significantly.

[0083] 本文所揭示的其它微流体器件使用矩阵设计。 [0083] Other microfluidic devices disclosed herein using the matrix design. 通常,这种类型的微流体器件使用多个相交的水平和垂直流体通道,以便在相交的点处定义反应位点的阵列。 Typically, this type of microfluidic device using a plurality of intersecting horizontal and vertical flow channels to define an array of reaction sites at the point of intersection of the. 由此,这种设计的器件也具有阵列或反应位点;然而,在使用的这种设计时,仍然有大量的样品输入和相应的样品输出来供给大量的样品。 Thus, devices of this design also have an array or reaction sites; however, when used in this design, there are still a large number of sample inputs and corresponding output sample is supplied to a large number of samples. 被称为可切换式流体阵列构架的阀系统能够使溶液选择性地流过水平或流体通道,由此允许对矩阵中的各种流体通道进行可切换的隔离。 Is called switchable fluid valve array system architecture enables solution to flow through the horizontal or selective fluid passage, thereby allowing the matrix to isolate various fluid passages switchable. 因此,尽管盲端通道被设计成在不同的条件下用有限数目的样品来进行大量的分析,但是矩阵器件被构建成在有限的条件下分析大量的样品。 Thus, although the blind end of the passage is designed to use a limited number of samples for a large number of analyzes under different conditions, but the matrix devices are constructed such that, under conditions of limited analysis of large numbers of samples. 其它器件都是这两个通用设计类型的混合。 Other devices are mixing these two types of universal design. [0084] 其它微流体器件是整体分隔器件(DID),在下列文献中对此进行了描述:PCT公报W0 2004/089810 ;美国专利申请10/819088,作为US 20050019792予以公布;2006年6月 [0084] Other microfluidic devices is a whole separate device (DID), in the following documents This is described: PCT Gazette W0 2004/089810; US patent application 10/819088, to be published as US 20050019792; June 2006

2日提交的共同待批共同转让的专利申请No._且题为〃 Analysis using Commonly assigned co-pending patent application filed on the 2nd and entitled 〃 Analysis using No._

microfluidic partitioning devices"(代理人档案标号020174-012400),这些文献全部引用在此作为参考。通过使用整体分隔器件,样品可以被分隔到多个隔离的反应室中,并且在每一个室内同时执行多个反应。 microfluidic partitioning devices "(Attorney Docket numeral 020174-012400), these documents are fully incorporated herein by reference. By using the whole separating device, the sample can be separated into a plurality of isolated reaction chambers, and in each indoor perform multiple simultaneous reactions.

[0085] 本文所描述的微流体器件进一步还具有下列部分特征:使用由弹性材料制成的各种组件,比如流体通道、控制通道、阀和/或泵。 [0085] The microfluidic device described herein is further characterized in further comprising the following parts: various components of an elastic material, such as fluid channels, control channels, valves and / or pumps. 在一些情况下, In some cases,

[0086] 基本上整个器件都是由弹性材料制成的。 [0086] substantially the entire device is made of an elastic material. 结果,和用塑料或以硅为主的材料构成的常规微流体器件相比,上述器件在形状和功能方面具有显著的不同。 As a result, and as compared with a conventional plastic microfluidic device, or silicon-based material, said device having significantly different in shape and function. 根据本发明的各实施例,所提供的反应室的数目可以有所不同。 According to various embodiments of the present invention, the number of the provided reaction chamber may vary.

[0087] 这些器件的设计能够使它们与许多不同的加热系统相结合地使用。 [0087] These devices are designed so that they can be used with many different heating systems used in combination. 由此,这些器件可用于进行需要温度控制的各种不同的分析。 Thus, these devices can be used for various analyzes requiring temperature control. 另外,适合于用在加热应用中的微流体器件可以包括另一种设计特征,以便使反应位点处样品的蒸发最小化。 Further, suitable for use in heating applications of microfluidic device may comprise another design feature, so that the evaporation of the reaction at the site of the sample is minimized. 这种类型的器件通常包括许多形成于弹性器件内的保护通道和/或储液囊或室,通过它们可以使水流入从而增大构成该器件的弹性材料内的水蒸气气压,由此减小样品材料在反应位点处的蒸发。 This type of device generally comprises a plurality of resilient means formed within the protection channel and / or reservoir capsule or chamber, which allows water to flow through so as to increase the water vapor pressure of an elastic material within the device, thereby reducing evaporation of sample material in the reaction at the site. [0088] 在另一个实施方式中,可用温度循环器件来控制微流体器件的温度。 [0088] In another embodiment, a temperature cycling device can be used to control the temperature of the microfluidic device. 较佳地,微流 Preferably, the microfluidic

16体器件将用于与微流体器件热接触。 16 in contact with the body of the device will be used to heat the microfluidic device. 在基板材料支撑微流体器件的情况下,如载玻片或载板(如塑料托架)底部,可以在载板或载玻片的区域中形成窗口,使得微流体器件(最好是具有弹性模块的器件)可以直接接触温度循环器件的加热/冷却模块。 In the microfluidic device substrate material supporting the case, such as a glass slide or a carrier plate (such as a plastic bracket) at the bottom, the window may be formed in a region of the carrier plate or glass slide, so that the microfluidic device (preferably with an elastic device module) temperature cycling device can be in direct contact with heating / cooling module. 在较佳的实施方式中,加热/冷却模块具有形成于其中的槽,这些槽和真空源相连,用于对该微流体器件施加吸入力,最好对发生反应的位点邻接的部分施加吸入力。 In a preferred embodiment, the heating / cooling module has a groove formed therein, the slots and connected to a vacuum source, for applying a suction force to the microfluidic device, preferably the reaction occurs on the part adjacent to the site of applying suction into force. 或者,可以将刚性导热盘接合到微流体器件,然后与加热和冷却模块配对以便有效地进行热传导。 Alternatively, a rigid disk may be thermally bonded to the microfluidic device, and then paired with the heating and cooling modules for efficient heat conduction.

[0089] 某些器件的阵列格式意味着这些器件可以实现高通量。 [0089] Certain devices array format means that these devices can achieve high throughput. 总的说来,高通量和温度控制能力使这些器件可用于进行大量核酸扩增(例如,聚合酶链反应(PCR))。 In general, high throughput and temperature control capabilities make these devices can be used for a large number of nucleic acid amplification (e.g., polymerase chain reaction (PCR)). 本文将详细讨论这种反应,以说明该器件的实用性,尤其是它们在需要温度控制的任何反应中的应用。 This article will discuss the details of this reaction, in order to illustrate the usefulness of the device, particularly any response they require temperature control application. 然而,应该理解,这些器件并不限于这些特定应用。 However, it should be understood that these devices are not limited to these particular applications. 这些器件可用于各种其它类型的分析或反应之中。 These devices can be used in various other types of analyzes or reactions being. 这些示例包括:分析蛋白质-配体相互作用;以及分析细胞与各种化合物之间的相互作用。 These examples include: analysis of protein - ligand interactions; and analyzing the interaction between the cells with various compounds. 在本说明书中提供了更多的示例。 Provides more examples in this specification.

[0090] 本文所揭示的微流体器件通常至少部分地由弹性材料构成,并且由单层和多层软光刻(MSL)技术和/或牺牲层包裹方法(例如,参照Unger等人,(2000) Science 288 : 113-116 ;以及PCT公报W0 01/01025,它们全部引用在此作为参考)。 [0090] herein disclosed microfluidic device typically is at least partially made of an elastic material, and a single layer and multilayer soft lithography (MSL) techniques and / or sacrificial layer coating process (for example, see Unger et al., (2000 ) Science 288: 113-116; and PCT Publication W0 01/01025, all of which are hereby incorporated by reference). 通过使用这些方法, 微流体器件可以被设计成,溶液流过该器件的流体通道的过程至少部分程度受到控制,其中用弹性膜或区段使一个或多个控制通道和流体通道分隔开。 By using these methods, microfluidic devices can be designed so that the solution flows through the fluid passage of the device of the process is controlled at least partially, wherein an elastic membrane or zone one or more control channels and fluid channels are separated. 通过向控制通道施加作用力,该膜或区段可以弯曲到和控制通道相关联的流体通道中或从该流体通道中縮回来。 By applying a force to the control channels, or section of the film to the fluid passage may be curved and the control channel associated to or retracted from the fluid passageway. 通过控制上述膜弯曲到流体通道中的程度或者控制上述膜从该流体通道中縮回来的程度,在流体通道中的溶液流动可以放缓或完全被阻挡。 By controlling the film is bent into the fluid passage for controlling the extent or degree of the film is retracted from the fluid passage, and the solution flow in the fluid passage can be slowed down or completely blocked. 通过使用这种类型的控制通道和流体通道的组合,便可以制备各种不同类型的阀和泵以便调节溶液流动,在下列文献中对此有更多的细节描述:Unger等人,(2000) Science 288 :113-116 ;以及PCT公报WO/02/43615和W0 01/01025。 By using the control channel and the fluid passage of this type of combination, can be prepared in a variety of different types of valves and pumps for regulating solution flow, in the following documents for more details on this description: Unger et al., (2000) Science 288: 113-116; and PCT Gazette WO / 02/43615 and W0 01/01025.

[0091] 本文所提供的器件包括这种泵和/或阀,以便选择性地隔离允许试剂发生反应的那个反应位点。 [0091] The device provided herein includes such pumps and / or valves for selectively isolating the reaction site allowing the reagents to react. 或者,使用不带泵和/或阀的器件,这些器件使用压力驱动流或聚合过程来关闭合适的通道,并由此选择性地隔离反应位点。 Alternatively, without using a pump and / or valve devices, these devices use a pressure-driven flow or polymerization process to close the appropriate channel, and thereby selectively isolating the reaction sites. 反应位点可以被定位到该器件内大量不同位置中的任何位置。 The reaction sites can be targeted to a number of different positions within the device anywhere. 例如,在一些矩阵型器件中,反应位点被定位到一组流体通道的相交处。 For example, in some matrix-type devices, the reaction site is positioned at the intersection of a set of fluid channels. 在盲端通道器件中,反应位点被定位到盲端通道的一端。 In the blind-side-channel device, the reaction site is located to one side of the blind side of the channel.

[0092] 如果该器件用于温度控制反应(例如,热循环反应),则如下文更详细的描述,弹性器件通常被固定到托架(例如载玻片)上。 A more detailed description [0092] If the device is used to control the reaction temperature (e.g., thermal cycling reaction), then below, the elastic devices are usually carriage (e.g. a glass slide) is fixed to the upper. 所得的结构可以接着被放到温度控制板上, 以便控制各个反应位点处的温度。 The resulting structure may then be placed in the temperature control panel, to control the temperature of each reactor at the site. 在热循环反应的情况下,该器件可以被放到许多热循环板上。 In the case of thermal cycling reactions, the device can be placed in a number of thermal cycling plate.

[0093] 因为这些器件均由相对而言具有光学透明性的弹性材料制成,所以在微流体器件上基本上任何位置处,通过使用各种不同的检测系统,都可以很容易地监控各种反应。 [0093] Because these devices are made by relatively resilient material having optical transparency, so the microfluidic device at substantially any location, by using a variety of detection systems, can easily monitor the various reaction. 然而,通常,都是在反应位点自身处进行检测(例如,在包括流体通道交点的区域或在流体通道的盲端)。 However, in general, are detected (e.g., in the region of intersection including a fluid passage or the fluid passage in the blind side) at the reaction site itself. 上述器件由基本上透明的材料制成这一事实也意味着,某些检测系统可以和目前尚不能与常规硅基微流体器件一起使用的那些器件一起使用。 Said device consists of a substantially transparent material, the fact also means that certain detection systems can not work with those devices currently conventional silicon-based microfluidic device for use in conjunction used together. 通过使用被嵌入上述器件中的检测器,或者通过使用与上述器件分离但与待测器件区域对准的检测器,便可以实现检测。 Is embedded in the above device by using a detector, or separate but aligned with the region of the device under test by using the aforementioned detector device, it can realize the detection. [0094] 使用矩阵设计的器件通常具有多个垂直的和水平的流体通道,这些流体通道彼此 [0094] Matrix devices are typically designed with a plurality of vertical and horizontal fluid passage, the fluid passage to each other

相交以便形成多个结的阵列。 Intersect to form an array of a plurality of junctions. 因为不同的样品和试剂(或试剂组)可以被引入每一个流体 Because of different sample and reagent (or set of reagents) can be introduced into each of the fluid

通道,所以可以按高通量形式在相对大量的反应条件下测试大量的样品。 Channel, it can be a high throughput format at a relatively large number of reaction conditions for testing large numbers of samples. 由此,如果不同的 Thus, if different

样品被引入M个不同的垂直流体通道中的每一个通道之内并且不同的试剂(或试剂组)被 The sample is introduced into the M different vertical flow channels and each channel of a different reagent (or set of reagents) is

引入N个水平的流体通道的每一个通道之内,则可以同时进行MxN个不同的反应。 Within each lane the introduction of the N levels of the fluid passage, it can be MxN different reactions simultaneously. 通常,矩 Typically, the moment

阵器件包括阀,允许对垂直和水平的流体通道进行可切换的隔离。 Array device comprises a valve, allowing for vertical and horizontal fluid passage isolated switchable. 这些阀定位成允许选择 These valves are positioned to allow selection of

性地只流过垂直的流体通道,或只流过水平的流体通道。 Resistance to flow only through the vertical fluid passage, or only the level of the fluid flowing through the passage. 因为该类型的器件具有和样品类 Because this type of device has a sample class

型和数目选择、试剂数目和类型选择等有关的灵活性,所以这些器件可很好地适用于进行 For flexibility type and number of selections, the number and type of agent selection, these devices is well adapted to carry out

许多特定的分析,在这些特定的分析中,人们想要根据相对大量的反应条件来筛选大量的 Many particular analysis, in these particular analysis, according to a relatively large number of people want to screen a large number of reaction conditions

样品。 Samples. 矩阵器件可以选择性地包括保护通道,以便防止样品和试剂的蒸发。 Matrix devices can optionally include the protection channel in order to prevent evaporation of samples and reagents.

[0095] —些高密度矩阵设计利用了微流体器件各层之间的流体连通孔,以便使整个器件 [0095] - some of the high-density matrix designs utilizing microfluidic device in fluid communication hole between the layers, so that the entire device

中的控制线和流体线交织起来。 The control lines and fluid lines intertwined together. 例如,通过使双层弹性模块的每一层中都具有流体线,密度 For example, by double elastic module of each layer, having a fluid line, density

更高的反应室排布是可行的。 Higher reaction chamber arrangement is feasible. 对于本领域的技术人员而言,很明显,多层器件允许流体线彼 Skilled in the art it will be obvious, multilayer device permits fluid line Piguet

此在上或在下相交,同时不处于流体传递的状态中。 This on or next intersection, while not in fluid communication state. 例如,在特定的设计中,第一层中的试 For example, in a particular design, the first layer of the test

剂流体通道通过一个孔连接到第二层中的试剂流体通道,而第二层中还具有样品通道,样 Agent fluid passage connected to the second layer through a hole in a reagent fluid channel, while the second layer further has a sample channel, like

品通道和试剂通道分别终结于样品和试剂室。 Product channels and reagent channel at the end of each sample and reagent chamber. 样品室和试剂室通过一个连接通道而进行流 The sample chamber and the reagent chamber through a connecting passage for traffic

体传递,该连接通道具有相关的连接阀,以便控制反应室的每一个室之间的流体传递。 Are passed, the connecting channel has an associated valve connection, in order to control fluid passes each chamber between the reaction chamber. 在使 In so

用过程中,该连接首先闭合,然后从试剂入口将试剂引入试剂通道中,并且从样品入口将样 During use, the first connection is closed, then reagent is introduced from the reagent inlet reagent channel and the sample from the sample inlet

品引入样品通道中。 Product sample introduction channel. 然后,密封阀闭合,以便使反应室和其它室隔离开。 Then, the sealing valve is closed, so that the reaction chamber and the other chamber isolated. 一旦反应室被隔离, Once the reaction chamber is isolated,

则打开连接阀以便使样品室和试剂室进行流体传递,从而可以发生想要的反应。 Connection valve is opened to allow the sample chamber and the reagent chamber in fluid transfer, whereby the desired reaction can occur. 本领域的 In the art

技术人员将认识到许多变体、修改和替换。 In the art will recognize many variations, modifications and substitutions.

[0096] 相应地,用于微流体器件的特定设计提供了一种微流体器件,它适用于使M种不同的样品和N种不同的试剂进行反应,该器件包括:多个反应室,每一个反应室包括样品室和试剂室,样品室和试剂室通过连接通道进行流体传递,该连接通道具有相关的连接阀,用于控制样品室和试剂室之间的流体传递;多个样品入口,都和样品室相通;多个试剂入口, 都和试剂室相通;其中样品入口或试剂入口之一通过小孔分别与样品室之一或试剂室之一进行流体传递。 [0096] Accordingly, a particular design for microfluidic devices is provided a microfluidic device, so that it applies to different kinds of samples M and N different reagent reaction, the device comprising: a plurality of reaction chambers, each a reaction chamber including a sample chamber and a reagent chamber, the sample chamber and the reagent chamber in fluid passing through the connecting passage, the connecting passage having a relevant connection valve for controlling the fluid sample chamber and the reagent chamber between the transfer; a plurality of sample inlet, are interlinked and the sample chamber; a plurality of reagent inlet, and the reagent chamber are interlinked; one in which the sample inlet or reagent inlet holes respectively by one or the sample chamber with one reagent chamber fluid delivery. 某些实施例包括:在多层结合所构成的弹性模块中形成多个反应室,并且连接阀是可弯曲的膜;使样品入口通过样品通道和样品室相通,使试剂入口通过试剂通道和试剂室相通,样品通道的一部分和试剂通道的一部分定向成大约彼此平行,并且各自具有相关联的密封阀,用于控制所通过的流体传递过程;使与样品通道相关联的阀和与试剂通道相关联的阀通过共用的密封控制通道彼此相通;使密封共用控制通道沿着大约和样品通道或试剂通道正交的线定位。 Some embodiments include: forming the elasticity module in a multi-layer combination consisting of a plurality of reaction chambers, and the connection valve is flexible membrane; sample channel through the sample inlet and the sample chamber communicated through the reagent inlet reagent channel and reagents chamber communicating, portion of the sample and reagent channel portion of the channel is oriented approximately parallel to each other, and which each have an associated sealing valve for controlling fluid transfer process adopted; valve so that associated with the sample channel and associated with the reagent channel linked by a common seal valve control channel communication with each other; the seal around a common control channel along the channel or reagent and sample lines orthogonal channel positioning.

[0097] 本发明各实施例中所使用的微流体器件可以进一步被集成到一种托架器件中, 这在共同待批、共同拥有的由Unger filed于2004年3月29日提交的美国专利申请60/557,715中进行了描述,该专利申请引用在此作为参考。 The microfluidic device used in the examples the embodiments [0097] The present invention may further be integrated into a carriage device, which in co-pending, commonly owned by Unger filed on March 29, 2004 filed U.S. Patent Application 60 / 557,715 are described in the patent application incorporated herein by reference. Unger的托架提供了工作中的连续流体压力,以便保持阀闭合状态远离流体压力源(例如,房间空气压力)。 Unger brackets provide a working pressure in the continuous fluid, in order to maintain the valve closed state away from the fluid pressure source (e.g., room air pressure). 如上述专利申请中所述的,Unger进一步提供了一种自动系统,用于充注并启动本发明的阀。 As described in the aforementioned patent application, Unger further provides an automatic system for filling and start the valve of the present invention. 在另一个较佳实施例中,用于充注蓄能器并启动阀的自动系统使用了一种具有滚筒的器件,该滚筒顶住微流体器件的一个或多个表面,其中该滚筒具有至少两个或多个与受控真空或压力源相通的端口,并且可以包括用于操纵微流体器件多个部分的机械部分,例如但不限于单向止回阀。 In another preferred embodiment, the accumulator for charging and start the automatic valve system uses a cylinder device having one or more of the drum against the surface of the microfluidic device, wherein the cylinder has at least Two or more controlled vacuum or pressure source communicating port, and may include a plurality of portions for the mechanical part of the microfluidic device to manipulate, such as, but not limited to a one-way check valve.

[0098] 本发明各实施例中所使用的另一种器件提供了一种被用作基板的托架,用于使弹性模块稳定。 [0098] Another device used in the examples is provided a bracket to be used as the substrate, so that the elasticity module for stability in all embodiments of the present invention. 较佳地,该托架具有一个或多个下面的特征:一个井或储液囊,它通过形成于托架中或与托架一起形成的至少一个通道而与弹性模块相通;蓄能器,它通过形成于托架中或与托架一起形成的至少一个通道而与弹性模块相通;以及流体端口,与弹性模块相通, 其中该流体端口最好可以接入自动真空或压力源(比如上述自动系统),其中自动源还包括一种具有端口的滚筒,它与上述流体端口配对,以便在用于将流体压力或真空施加到弹性模块的自动系统之间形成隔离的流体连接。 Preferably, the bracket has one or more of the following features: a well or reservoir bag, which is formed by at least one channel in the bracket or the bracket is formed together with the elasticity module in communication; accumulator, It is formed by at least one channel bracket or the bracket is formed together with the elasticity module communication; and a fluid port in communication with the elastic module, wherein the fluid port is preferably vacuum or pressure can automatically access the source (such as the automatic System), which further comprises an automatic source having a cylinder port, with said fluid port pair, so for the fluid pressure or vacuum is applied to the isolation of a fluid connection is formed between the elastic modules of the automated system. 在某些实施例所用的器件中,自动源也可以与一个或多个与托架相关联的蓄能器相通,以便充注并释放蓄能器中所维持的压力。 In certain embodiments of the device used, the automatic source may also be one or more of the accumulator associated with the bracket connected to the filling and release of the accumulator pressure maintained. 在某些实施例中,该托架可以进一步包括定位于该托架范围中接触微流体器件的一个区域,其中该区域由与该托架另一个部分不相同的材料制成,该区域材料的选择是为了改善热传导和热分布特性,这些与该托架的其它部分不相同。 In certain embodiments, the carrier may further comprise the carrier positioned in a region of the contact range of the microfluidic device, wherein the region made of another portion of the carrier is not the same material, the material in the region chosen to improve the thermal conductivity and heat distribution characteristics, these other portions of the carrier are not the same. 用于改善热传导和热分布的较佳材料包括但不限于硅,最好是高度抛光的硅,比如半导体领域中可获得的抛光的晶片或从晶片中切割的一部分即芯片。 For improving the heat conduction and heat distribution preferred materials include, but are not limited to silicon, preferably silicon highly polished, such as semiconductor wafers polished available in the art or cleavage from a wafer that is part of the chip.

[0099] 如下文更全面地描述,本发明的各实施例使用一种热源,但不限于PCR热循环装置,它可能已从其原始制造状态中获得修改。 [0099] As described more fully below, embodiments of the present invention uses a heat source, but not limited to PCR thermal cycle apparatus, it may be manufactured from its original state to obtain modified. 通常,该热源具有热调节部分,该部分与托架的一部分(最好是托架的热传导和热分布部分)配对,以便通过该托架的热传导和热分布部分向弹性模块提供热控制。 Typically, the heat source having a heat-regulating portion, a portion of the bracket portion (preferably the bracket and the heat distribution of the heat conduction portion) pair, in order to provide thermal control to the elastomeric block through the thermal conduction and heat distribution portion of the carrier. 在较佳的实施例中,通过将真空源施加到热源的热调节部分内所形成的一个或多个通道,便改善了热接触,其中形成多个通道以便接触该托架的热传导和热分布部分的表面,从而施加吸力并维持该托架的热传导和热分布部分的位置。 In the preferred embodiment, by the vacuum source applied to one or more channels formed in the heat source heat regulating portion, they improve the thermal contact, wherein the plurality of channels are formed so as to contact the heat conduction and heat distribution of the carrier surface portion, so as to apply suction position and maintain thermal conduction and heat distribution portion of the carrier. 在较佳的实施例中,通过将热传导和热分布部分只附着于弹性模块上并且在热传导和热分布部分的边缘周围留下空隙以便减小由该托架造成的寄生热效应,该托架的热传导和热分布部分并不与该托架的其余部分进行物理接触,而是与该托架的其余部分和弹性模块相关联。 In the preferred embodiment, by the heat conduction and heat distribution section is only attached to the elastic module and leaving a void in the heat conduction and heat distribution around the edge portion in order to reduce parasitic thermal effects caused by the carrier, the carrier of the heat conduction and heat distribution portions are not in physical contact with the rest of the carrier, but is associated with the remainder of the carrier and elasticity module. 应该理解,在本文所描述的本发明的许多方面,较佳的弹性模块可以用本文没有描述但本领域已知的任何微流体器件来替代,例如由美国加州Santa Clara的Affymetrix (R)公司或美国加州Mountain View的Caliper公司所生产的器件GeneChip(R)。 It should be understood that in many aspects of the invention described herein, the elasticity module may be preferred not described herein but known in the art to substitute any microfluidic device, e.g., of Santa Clara, California, by Affymetrix (R) Corporation or Mountain View, California, the company produced devices Caliper GeneChip (R). 授权给Soane、 Parce、Fodor、Wilding、Ekstrom、Quake或Unger的美国专利描述了可以被替代的微流体器件或中尺度流体器件,因为本发明的弹性模块利用了上述温度方面的优点和改进,例如吸入定位,减少到流体器件的其它区域的寄生热转移,这些在上文使用弹性模块的情况下进行过描述。 Issued to Soane, Parce, Fodor, Wilding, Ekstrom, Quake, or Unger U.S. patent describes an alternative can be a microfluidic device or mesoscale fluidic devices, because the elastic module of the present invention takes advantage of the above-mentioned temperature and improvements, e.g. Inhalation positioning, reducing parasitic thermal transfer to other regions of the fluid device, performed when the elasticity module used in the foregoing been described.

[0100] 通过使用本发明各实施例所提供的系统和方法,用384孔系统提高了通量。 [0100] By using the embodiment of the present invention, various systems and methods provided by the embodiment, the system 384 with improved throughput. 例如, 在一些实施方式中使通量以因数4、6、12、24或更大增加。 For example, in some embodiments, the flux by a factor of 4,6,12,24 manipulation or greater increase. 在产生通量增加的同时,还减小了各种操作的逻辑冲突。 Generating flux increased, also reduces the logical conflict various operations. 此外,本发明各实施方式的系统和方法能够进行多个样品的多次试验。 In addition, the system and method embodiments of the present invention is capable of many tests of multiple samples. 例如,在特定的实施方式中,采用96个样品和96次试验,提供了总共9, 216个数据点。 For example, in certain embodiments, the use of 96 samples and 96 tests, providing a total of 9, 216 data points. 在特定的示例中,这96次试验是T叫Man 5'核酸酶试验的一部分。 In a particular example, the 96 runs were called T part of Man 5 'nuclease experiments. [0101] 此外,本发明的各实施方式减小了反应体积。 [0101] In addition, embodiments of the present invention reduces the reaction volume. 在本发明的各实施例中,所用反应体积为10皮升(10—12升)-100纳升(10—9升)。 In various embodiments of the present invention, the use of the reaction volume of 10 picoliters (10-12 liter) -100 nanoliter (10-9 liter). 在一些实施方式中,使用大于100纳升的反应体积。 In some embodiments, the use of more than 100 nanoliter reaction volumes. 仅作为示例,在一个实施方式中,本发明的方法和系统所用反应体积为10皮升、50 Of example only, in one embodiment, the method and system of the present invention is used in the reaction volume of 10 picoliters, 50

19皮升UOO皮升、250皮升、500皮升和1纳升。 19 picoliter UOO picoliters, 250 picoliters, 500 picoliters and a nanoliter. 在另选的实施方式中,使用2纳升、5纳升、10 纳升、20纳升、30纳升、40纳升、50纳升、75纳升和100纳升的反应体积。 In an alternative embodiment, the use of two nanoliter, 5 nanoliters, 10 nanoliters, 20 nanoliters, 30 nanoliters, 40 nanoliters, nanoliter 50, 75 and 100 nanoliter nanoliter reaction volume. [0102] 根据特定微流体器件的几何形状和该微流体器件的大小以及流体连接通道和处理位点的排列情况,本发明的各实施例提供了范围不同的处理位点(或反应室)密度。 [0102] connection arrangement of channels and according to the treatment site specific geometry of the microfluidic device and the size of the microfluidic device and a fluid, the various embodiments of the present invention provide a range of different processing sites (or reaction chamber) Density . 在一些实施例中,在使用本发明的方法和系统时,反应室密度大约从每平方厘米100个反应室到每平方厘米1百万个反应室。 In some embodiments, when the method and system of the invention, the reaction chamber density of about 100 per square centimeter from the reaction chamber to the reaction chamber 1 million per square centimeter. 仅作为示例,根据本发明的各实施例,使用了每平方厘米有250个、1, 000个、2, 500个、10, 000个、25, 000个、100, 000个和250, 000个反应室的反应室密度。 Of example only, in accordance with embodiments of the present invention, the use of 250 per square centimeter, 1, 000, 2, 500, 10, 000, 25, 000, 100, 000 and 250, 000 the density of the reaction chamber the reaction chamber. 在一些实施例中,使用了每平方厘米超过1, 000, 000个反应室的反应室密度,尽管这不是本发明所要求的。 In some embodiments, the use of more than one per square centimeter, 000, the density of the reaction chamber 000 of the reaction chamber, although this is not required by the present invention.

[0103] 操作具有这种小反应体积的微流体器件可以减小试剂用量和样品用量。 Microfluidic devices [0103] operating with such a small reaction volume can be reduced reagent consumption and sample volume. 此外,本发明的一些实施方式提供了与实时定量PCR联用时可进行实时检测的方法和系统。 In addition, some embodiments of the present invention provides a method and system for real-time quantitative PCR and real-time detection can be associated with time. 使用这些系统和方法,可以为某些应用提供六个数量级的线性动态范围,并且使定量分辨率足够高以致于可以检测IO纳升体积中亚纳摩尔级的荧光团浓度。 Using these systems and methods can provide six orders of magnitude of the linear dynamic range for certain applications, and the quantitative resolution is high enough to be detected IO nanoliter volume of the fluorophore of subnanomolar concentrations. 本领域的技术人员将会认识到许多改变、修改和替换。 Those skilled in the art will recognize that many changes, modifications and substitutions.

[0104] 用某些盲端通道类型的器件实现的方法包括:提供一种微流体器件,该微流体器件包括形成于弹性材料内的流体通道;以及多个盲端流体通道,它们与流体通道流体相连, 各盲端流体通道的末端区域限定了一个反应位点。 [0104] The method of the blind end of the channel with some type of device-implemented method comprising: providing a microfluidic device, the microfluidic device comprising a fluid passage formed in the elastomeric material therein; and a plurality of blind-side fluid passage, the fluid passage they fluid communication with, the end region of each blind end defines a fluid passageway reaction site. 至少一种试剂被引入各反应位点中,然后,在一个或多个反应位点处检测反应。 At least one reagent is introduced into each of the reaction sites, and then detecting the reaction in one or more reaction sites of. 该方法可以任选地包括加热反应位点内的至少一种试剂。 The method can optionally include heating the at least one reagent within the reaction site. 因此,一种方法可以包括:引入用于核酸扩增反应的各种成分;然后使这些成分热循环以形成扩增产物。 Therefore, a method may include: introducing various ingredients used in nucleic acid amplification reaction; and then these components to form a thermal cycle amplification product. 如下文更全面的描述那样,根据本发明各实施方式,提供了一种光学成像系统,它适用于表征某些微流体器件中发生的反应。 As more fully described above, according to embodiments of the present invention, there is provided an optical imaging system, which is suitable for characterization of the reaction occurs in certain microfluidic device.

[0105] 如图1A所示,本发明的一些实施例所提供的光学成像系统包括耦合到热控制模块的荧光成像系统。 [0105] As shown in Figure 1A, some of the optical imaging system provided by the embodiments of the present invention comprises a thermal control module coupled to the fluorescence imaging system. 一些系统适用于收集来自几何形状为NxM的微流体芯片的数据。 Some systems suitable for collection from the geometry of the microfluidic chip for NxM data. 在一些实施例中,N等于M。 In some embodiments, N is equal to M. 例如,本发明的各实施例使用具有48x48个反应室的微流体器件、具有96x96个反应室的微流体器件、以及其它几何形状的微流体器件。 For example, the various embodiments of the present invention using a microfluidic device having a reaction chamber 48x48, 96x96 reaction chambers having a microfluidic device, the microfluidic device and other geometric shapes. 在特定的实施例中,在具有96x96反应室几何形状的微流体器件中,使用了96个样品和96个试剂。 In a particular embodiment, the microfluidic device having a 96x96 reaction chamber geometry, the use of the 96 samples and 96 reagent. 对于本领域的技术人员而言,很明显,本发明各实施例所提供的方法和系统能够使一个平台执行多种应用。 For the skilled artisan, it is clear that the various embodiments of the present invention to provide a method and system according to the execution platform enables a wide variety of applications.

[0106] 图1A是示出了本发明一实施例的光学成像系统的简化示意图。 [0106] Figure 1A is a simplified schematic diagram illustrating an optical imaging system according to an embodiment of the present invention. 如图1A所示,根据本发明各实施例,提供了光源242。 1A, in accordance with various embodiments of the present invention, there is provided a light source 242. 如下文更全面地描述,在本发明的一些实施例中,来自光源242的光线被用来诱导样品中的荧光。 As described more fully below, in some embodiments of the present invention, the light from the light source 242 is used to induce fluorescence in the sample. 在其它实施例中,化学发光被用作一种指示。 In other embodiments, chemiluminescence is used as an indication. 根据该实施例,将添加、除去、或使用各种系统成分,这对本领域的技术人员而言是很明显的。 According to this embodiment, to add, remove, or using various system components, which the skilled artisan is obvious. 在各种实施例中,使用发光二极管(LED)、激光器、弧光灯、白炽灯等光源。 In various embodiments, the use of a light emitting diode (LED), lasers, arc lamps, incandescent light sources. 这些源可以是多色的或单色的。 These sources may be polychromatic or monochromatic. 在特定的实施例中,该光源具有第一光谱带宽。 In a particular embodiment, the light source having a first spectral bandwidth. 在特定的实施例中,该光源是白光源,可产生光谱范围介于约400纳米到约700纳米的光学辐射。 In a particular embodiment, the light source is a white light source, can produce optical radiation spectral range between about 400 nanometers to about 700 nanometers. 仅作为示例,在本发明的一些实施例中,从加州Novato市Sutter Instruments公司那里可以买到的Lambda LS 300W氙弧光灯被用作光源。 Of example only, in some embodiments of the invention, from Novato, California City Sutter Instruments Company where commercially available Lambda LS 300W xenon arc lamp is used as a light source. 对于本领域的技术人员而言,很明显,在可选实施例中能够使用光谱带宽更大或更小的其它光源。 For the skilled person that, obviously, in alternative embodiments can use larger or smaller spectral bandwidth of other light sources.

[0107] 图1A示出了激发滤光轮244。 [0107] Figure 1A shows an excitation filter wheel 244. 在一些实施例中(例如,光源为多色的实施例),使用激发滤光轮244,对光源242发出的光线进行光谱滤光。 In some embodiments (e.g., a light source for polychromatic embodiments), using an excitation filter wheel 244, the light emitted from the light source 242 spectral filtering. 当然,也可以使用多个滤光片。 Of course, you can also use multiple filters. 作为一个示例,在一个实施例中,激发滤光轮提供了许多光谱滤光片,每一个光谱滤光片透射能够从样品中激发出特定荧光的预定波长范围。 As one example, in one embodiment, the excitation filter wheel provides a number of spectral filters, each filter spectral transmittance inspire a predetermined wavelength range specific fluorescence from the sample. 如图1A所示,激发滤光轮244耦合到计算机270,从而提供滤光片的计算机控制。 1A, the excitation filter wheel 244 is coupled to a computer 270, thereby providing a computer controlled filter. 在特定的实施例中,激发滤光轮提供了许多光谱滤光片: In a particular embodiment, the excitation filter wheel provides a number of spectral filter:

[0108] 滤光片1 :其中心波长为485nm,光谱带宽为20nm ; [0109] 滤光片2 :其中心波长为530nm,光谱带宽为20nm ;以及[0110] 滤光片3 :其中心波长为580nm,光谱带宽为20nm。 [0108] filter 1: the center wavelength of 485nm, a spectral bandwidth of 20nm; [0109] Filter 2: center wavelength of 530nm, a spectral bandwidth of 20nm; and [0110] Filter 3: center wavelength is 580nm, the spectral bandwidth of 20nm.

[0111] 对于本领域的技术人员而言,很清楚,本发明的各实施例并不限于这些特定的光谱滤光片,而是将针对特定的样品使用适于荧光处理的光谱滤光片。 [0111] skilled in the art is concerned, it is clear that the various embodiments of the present invention is not limited to these specific spectral filter, but to use the processed fluorescence spectral filter adapted for a particular sample. 此外,尽管上述讨论涉及滤光轮的使用,但是这并不是本发明所要求的。 Further, although the above discussion relates to the use of the filter wheel, but this is not required by the present invention. 在可选的实施例中,光谱滤光片以除轮子以外的更多几何形状来设置。 In an alternative embodiment, the spectral filter with more geometric shapes other than the wheel set. 例如,根据本发明的各实施例,可以包括落入滤光片托架中的光谱滤光片、电光滤光片、由执行器将其置入光路中的滤光片等。 For example, according to various embodiments of the present invention, may include a falling filter bay spectral filter, an electro-optical filter, by the actuator to place it in the optical path filters and the like. 此外,在其它实施例中,光源是可调谐的激光器,可以发射适于激发荧光的预定波长的辐射。 Further, in other embodiments, the light source is a tunable laser, can emit a predetermined wavelength suitable to excite the fluorescence radiation. 本领域的技术人员将会认识到许多变体、修改和替换。 Those skilled in the art will recognize many variations, modifications, and alternatives.

[0112] 如图1A所示,根据本发明的各实施例,提供了激发快门246。 [0112] As shown in Figure 1A, according to various embodiments of the present invention, there is provided an excitation shutter 246. 在一些实施例中,在计算机270的控制下,操作该激发快门246,以便阻挡/透射由光源242产生并由激发滤光轮244进行光谱滤光之后的光信号。 In some embodiments, under the control of the computer 270, operation of the excitation shutter 246, so as to block / transmission optical signal 242 generated by the excitation filter wheel 244 after spectral filtering by the light source. 根据这种应用,在样品被插入系统中或从系统中取出样品时以及在校准操作期间,激发源被阻挡。 According to this application, the sample is inserted into the system or removed and the sample during the calibration operation, the excitation source is blocked from the system. 在一些实施例中,激发快门并不用在使用激光光源的实施例中,使用激光光源的实施例提供可替换的手段来使光源熄灭。 In some embodiments, the excitation shutter is not used in an embodiment using a laser light source, a laser light source used in Example provides a means alternative to the light source is off. [0113] 当激发快门处于打开位置时,光激发信号穿过光纤束248并被引导照射到芯片托架中所设置的微流体器件205上。 [0113] When the excitation shutter is in the open position, the excitation signal passes through the optical fiber bundle 248 and is irradiated to the chip on the guide carriage in the microfluidic device 205 set. 本发明的其它实施例使用石英光导、液体光导、其它扰频系统等,来增大照明均一性。 Other embodiments of the present invention is the use of a quartz light guide, liquid light guide, other scrambling systems, to increase the illumination uniformity. 如图1A所示,引导激发光信号,使其通过光照明装置250的反射、折射或其组合,到达微流体器件205的表面上。 1A, the guided excitation light signal, so that the light reflected by the illumination device 250, a refractive or a combination thereof, to the upper surface 205 of the microfluidic device. 如图1A所示,微流体器件的照明是通过光照明装置250进行的。 1A, the illumination of the microfluidic device 250 through the illumination means carried. 在其它实施例中,通过环形光,或通过收集光学系统的一部分(微流体器件和检测器260之间的光路),照明可以从器件的一个或多个侧面倾斜地耦合到该微流体器件。 In other embodiments, by the ring light, or by collecting part (microfluidic device and the detector 260 between the optical path) of the optical system, the illumination can be tilted from one or more sides of the coupling device to the microfluidic device.

[0114] 在一些实施例中,在样品的二维面积上,用激发源产生的光线对微流体器件进行照明。 [0114] In some embodiments, the two-dimensional area of the sample, the light generated by the excitation source to illuminate the microfluidic device. 在这些实施例中,提供了较大的视场,这能够执行许多涉及到给时间分辨化学过程和反应成像的荧光应用。 In these embodiments, provides a greater field of view, which is capable of performing many relates to chemical processes and to the time-resolved fluorescence imaging applications reactions. 作为一个示例,蛋白质热量测定和核酸控制过程的荧光成像都是可从本发明各实施例中获益的时间分辨过程。 As one example, the protein calorimetry and nucleic acid fluorescence imaging control process are available from the present invention in each example embodiment benefit time-resolved process. 在这些过程中,期望同时激发许多反应室中所提供的多个荧光样品,并且还期望同时收集由这些反应室中正发生的各种反应所产生的荧光信号。 In these processes, it is desirable in many samples simultaneously excite a plurality of fluorescent reaction chamber provided, and also desirable simultaneously collect fluorescence signals generated by these various reactions of the reaction chamber CKS generated. 在其它过程(例如荧光寿命成像)中,在简短的激发脉冲之后,就是荧光信号的检测(和分析),因为荧光信号从其初始水平随时间流逝而衰减。 In other processes (e.g., fluorescence lifetime imaging), after a brief excitation pulse is detected (and analysis) of the fluorescence signal, since the fluorescence signal over time from its initial level of attenuation. 本领域的技术人员将会认识到许多变体、修改和替换。 Those skilled in the art will recognize many variations, modifications, and alternatives.

[0115] 作为一个示例,核酸扩增过程通常包括靶DNA、耐热DNA聚合酶、两种寡核苷酸引物、脱氧核苷酸三磷酸(dNTP)、反应缓冲液和镁盐。 [0115] As one example, a target nucleic acid amplification process typically includes DNA, thermostable DNA polymerase, two kinds of oligonucleotide primers, deoxynucleotide triphosphate (dNTP), reaction buffer, and magnesium salts. 一旦混合后,就将该反应置于热循环器中,该热循环器是一种使该反应在一系列不同温度下保持不同时间的仪器。 Once mixed, the reaction was placed on a thermal cycler, which is a thermal cycler The reaction was held at different times of a series of different temperatures instrument. 这一系列温度和时间调节被称为一个扩增循环。 This series of temperature and time adjustment is called an amplification cycle. 在该反应中,每一个循环从理论上讲能使靶序列(扩增子)的量倍增。 In this reaction, the amount of each cycle allows the target sequences (amplicons) multiplication theory. 10次循环从理论上讲使该扩增子乘以约为1000的因数;在大约几个小时中,20次循环能使该扩增子乘以大于1百万的因数。 10 cycles so that the amplicon theory multiplied by a factor of about 1000; in about a few hours, 20 cycles enables the amplicon is multiplied by a factor greater than 1 million. 在一些应用中,需要在几秒到几分钟的时间内获得很大面积(例如,若干个平方厘米的数量级)的荧光成像数据。 In some applications it is desirable to obtain a large area (e.g., several cm of magnitude) of fluorescence imaging data in a few seconds to several minutes. [0116] 在本发明的一些实施例中,本发明各实施例所提供的方法和系统有利于在预定的时间周期内执行的图像拍摄过程。 [0116] In some embodiments of the present invention, the various embodiments of the present invention is a method and system for image capturing procedure provided in Example favor performed within a predetermined time period. 仅作为一个示例,在本发明的一个实施例中,提供了一种给微流体器件成像的方法。 As just one example, in one embodiment of the present invention, there is provided a microfluidic device to the image forming method. 该方法包括:在不到一分钟的时间帧中,通过使用图像检测空间区域,拍摄与微流体器件的至少确定数目的反应室相关联的空间区域的图像,因此空间区域的图像拍摄基本上不受缝合和/或扫描过程的影响。 The method comprising: in less than a minute in the time frame, by using the image detection spatial region, shoot and microfluidic devices to determine at least the spatial region of the image number associated with the reaction chamber, and therefore the image pickup space region does not substantially Affected stitching and / or scanning process.

[0117] 本发明的各实施例提供了用于图像拍摄的各种时间帧,范围从1毫秒到1分钟。 [0117] Embodiments of the present invention provides a variety of time frames for the image-capturing range from 1 millisecond to 1 minute. 在一些实施例中,用于图像拍摄的时间帧大于1分钟。 In some embodiments, the captured image frame for time greater than 1 minute. 根据与微流体器件的反应室中所执行的各种过程相关联的发光特性,用于图像拍摄的时间帧将发生变化。 The light emitting characteristics of various processes in the reaction chamber in the microfluidic device associated performed, the time frame for the captured image will change. 例如,在一个实施例中,时间帧是10ms、50ms、100ms、250ms、500ms、750ms或1秒。 For example, in one embodiment, a time frame is 10ms, 50ms, 100ms, 250ms, 500ms, 750ms or 1 second. 在其它实施例中,时间帧是2 秒、5秒、10秒、15秒、20秒、30秒、40秒、50秒、或1分钟。 In other embodiments, the time frame is 2 seconds, 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, or 1 minute. 当然,时间帧将取决于特定的应用。 Of course, the time frame will depend on the particular application.

[0118] 在一些实施例中,图像拍摄过程是按同步方式来执行的,从而可以同时拍摄确定数目的反应室的图像。 [0118] In some embodiments, the image capturing process is performed by a synchronized manner, thereby determining the number of simultaneously captured image of the reaction chamber. 作为一个示例,在示例性的PCR过程中,微流体器件维持92t: 15秒。 As one example, in an exemplary PCR process, the microfluidic device to maintain 92t: 15 seconds. 接下来,该微流体器件维持6(TC 60秒。在一分钟的循环周期内重复加热和冷却,并且进行许多次循环。通过使用本发明的各实施方式,同步地获得了微流体器件中存在的确定数目的反应室的图像,同时这些反应室根据位置保持在均匀的温度下。例如,在微流体器件保持在温度6(TC的情况下,使用30秒曝光,便可以获得整个微流体器件的二维图像。和光栅扫描或缝合系统相比,本领域的技术人员应该理解本发明所提供的诸多益处,因为在光栅扫描或缝合系统中微流体器件第一部分(例如,左上象限)中的反应室的图像是先于微流体器件第二部分(例如,右下象限)中的反应室的图像而获得的。 Next, the microfluidic device to maintain 6 (TC 60 seconds repeated heating and cooling in a minute cycle, and subjected to many cycles. By using the embodiments of the present invention, in obtaining synchronization microfluidic device in the presence of The determined number of images of the reaction chamber while the reaction chamber according to the position maintained at a uniform temperature. For example, the microfluidic device is maintained at a temperature of 6 (in the case of TC, using 30 second exposure, can be obtained throughout the microfluidic device The two-dimensional image. and rasterization or suturing system compared to those skilled in the art will appreciate the many benefits provided by the present invention, since the microfluidic device in the system a first portion of the raster scanning or stitching (e.g., upper left quadrant) of The reaction chamber is the first image in the microfluidic device of the second part (e.g., lower right quadrant) of the image obtained by the reaction chamber.

[0119] 在其它实施方式中,在不到一分钟的时间帧内,获得了确定数目的反应室的多张图像。 [0119] In other embodiments, in less than one minute time frame, obtain a certain number of reaction chambers multiple images. 作为这些实施例的一个示例,在一个特定的实施例中获得了与多个荧光团相关联的多张图像。 As an example of these embodiments, in a particular embodiment, a plurality of images obtained with a plurality of fluorophores associated. 在微流体器件保持在温度6(TC下的60秒时间周期内,可以针对三种不同的荧光团(例如,RoxTM、Vic⑧和FamTM),获得使用20秒曝光的三张连续的图像。当然,根据该应用,曝光时间可以更短,甚至短到l秒或更短。通过使用这些图像,可以计算并分析不同的荧光比。当然,根据荧光发射的强度,可以修改各种荧光团的曝光次数,以适应特定的应用。 这样,本发明的各实施例可以在微流体器件保持恒定温度的同时在多个光谱波带中给微流体器件成像。像上述示例所示,恒定的温度可以是包括循环温度处理的PCR过程的一部分。 [0120] 本发明各实施例所提供的方法和系统也适合执行并分析化学发光过程。在这些过程中,各种反应发生在第一时间标度,而化学发光过程的图像则是在第二时间标度获得的。 在特定的过程中,第二时间标度小于第一时间标度。因此,当感兴趣的反应室中的样品已经反应了相等的时间量时,本发明的各实施例适于拍摄化学发光过程的同步图像。在这些过程中,提供了包括样品温度循环的温度控制,而在其它实施例中,反应室保持在恒定的温度下。 In the microfluidic device is maintained at 6 (60 seconds at a temperature of TC time period, can target three different fluorophores (e.g., RoxTM, Vic⑧ and FamTM), obtained using a 20-second exposure of three consecutive images. Of course, According to this application, the exposure time may be shorter, and even as short as l seconds or less. By using these images, can be calculated and analyzed different fluorescence ratio. Of course, depending on the intensity of fluorescence emission, a variety of impressions can be modified fluorophore , to suit a particular application. Thus, the various embodiments of the present invention may be in the microfluidic device while maintaining a constant temperature in a plurality of spectral wave bands to the microfluidic device imaging. Like in the preceding example may include a constant temperature circulating a portion of the PCR process temperature treatment. [0120] Embodiments provide a method and system according to the present invention is also adapted to perform and analyze the chemiluminescence process. In these processes, various reactions occur in the first time scale, while the chemical The emission process is an image at a second time scale obtained. In a particular process, the second time scale is less than the first time scale. Thus, when the reaction of interest in the sample chamber has a reaction time equal to When the amount of, various embodiments of the present invention are suitable for shooting an image synchronization chemiluminescence process. In these processes, there is provided a temperature control comprises temperature cycling of the sample, while in other embodiments, the reaction chamber is maintained at a constant temperature.

[0121] 如图1A所示,根据本发明的各实施例,提供了热控制器240,也被称为温度控制器240。 [0121] As shown in Figure 1A, according to various embodiments of the present invention, there is provided a thermal controller 240, the temperature controller 240 is also known. 改变精密化的许多不同的选择可用于控制微流体器件的选定区域内的温度或整个微流体器件的温度。 Change the precision of a number of different options available for the temperature control of the microfluidic device the temperature within the selected area or the entire microfluidic device. 因此,如本文所使用的那样,术语温度控制器广泛地指代一种器件或元件,它能够调节整个微流体器件的温度或微流体器件的一部分内(例如,在特定的温度区域内或微流体器件的通道矩阵的一个或多个结处)的温度。 Thus, as used herein, the term temperature controller broadly refer to a device or element, it can be adjusted within a portion of the temperature of the entire microfluidic device or microfluidic device (e.g., within a specific temperature range or slightly one or more of the fluid junction device channel matrix) temperature.

[0122] 图1C是示出了本发明一实施例的热控制器件的简化示意图。 [0122] Figure 1C is a simplified schematic diagram illustrating a thermal control device of an embodiment of the present invention. 如图1C所示,微流体器件205包括样品阵列206。 1C, the microfluidic device 205 includes a sample array 206. 对于本领域的技术人员而言,很明显,尽管图中样品阵列206 是一维的,但是本发明各实施例提供三维样品阵列。 For the skilled artisan, it is clear that although the figure is one-dimensional sample array 206, but the embodiments of the present invention provide a three-dimensional array of samples. 作为一个示例,在本发明各实施例所使用的一些微流体器件中,反应室的阵列和流体连接通道延伸到该图所在的平面中。 As one example, in some embodiments of the present microfluidic device used in the invention, the reaction chamber array and a fluid connection passage extending into the plane of the diagram is located. 包括反应室在内的微流体器件的各种元件具有第三维度即深度。 Various elements comprising a reaction chamber, including the microfluidic device has a third dimension i.e. depth. 微流体器件205由托架207支撑, 托架207由托架立柱208支撑。 The microfluidic device 205 supported by bracket 207, the bracket 207 by a bracket 208 supporting columns. 微流体器件或芯片底层209耦合到托架207和集成热传播装置(IHS)241。 The microfluidic device or chip 209 is coupled to the bottom bracket and integrated heat propagation means 207 (IHS) 241. 热滚筒243在图1C中有示出并且在下文中进行更完整地描述。 Heat roll 243 in FIG. 1C have shown and more fully described hereinafter. 在一些实施例中,提供了在微流体器件和HIS/滚筒之间的硬接触。 In some embodiments, there is provided a hard contact between the microfluidic device and HIS / cylinder. 此外,如下文更详细地描述,在一些实施例中使用真空技术,使微流体器件相对于托架定位并固定住。 Furthermore, as described in more detail, using vacuum techniques in some embodiments, the microfluidic device relative to the bracket is positioned and fixed.

[0123] 通常,这些器件被置于热循环板上,以便使该器件热循环。 [0123] Typically, these devices are placed in a heat cycle plate, so that the device heat cycle. 各种这样的板都可以从下列公司买到:ThermoHybaid Px2 (Franklin, MA) ;MJ ResearchPTC-200 (South San Francisco,CA) ;Eppendorf Part#E5331(Westbury,NY) ;Techne Part#205330(Princeton, NJ)。 A variety of such plates are commercially available from the following companies: ThermoHybaid Px2 (Franklin, MA); MJ ResearchPTC-200 (South San Francisco, CA); Eppendorf Part # E5331 (Westbury, NY); Techne Part # 205330 (Princeton, NJ).

[0124] 在一些实施例中,微流体器件与热控制器件接触,使得热控制器件与热控制源热连通,所以作为热控制源的温度变化的结果,至少一个反应室中的反应温度会发生变化。 [0124] In some embodiments, the microfluidic device in contact with the thermal control device, such that the thermal control device in thermal communication with the thermal control source, so as a result of temperature changes in the thermal control source, at least one reaction temperature in the reaction chamber will occur change. 在不同的实施例中,热转移器件可以包括半导体(比如硅),可以包括反射材料,和/或可以包括金属。 In various embodiments, the thermal transfer device may comprise a semiconductor (such as silicon), may include a reflective material, and / or may comprise a metal.

[0125] 热控制器件可以向热转移器件施加力,以便朝着热控制源推动热转移器件。 [0125] thermal control devices can apply a force to the heat transfer device to push toward the heat source control heat transfer devices. 在不同的实施例中,这种力可以包括机械压力、磁力、静电力、或真空力。 In various embodiments, this force may comprise a mechanical pressure, magnetic, electrostatic, or vacuum force. 例如,在一个实施例中, 这种力包括真空力,真空力是通过热控制器件或热转移器件的表面中所形成的通道朝着热转移器件施加的。 For example, in one embodiment, this force comprises a vacuum force, a vacuum force is the surface of the device by thermal or thermal transfer channel devices formed in the thermal transfer device towards the applied control. 可以检测热控制器件的表面和热转移器件的表面(或表面的一部分)之间所实现的真空度。 Can be detected between the surface of the thermal control device (or a part of the surface) and the surface heat transfer device achieved degree of vacuum. 这种检测可以用真空度检测器(它位于沿通道远离真空源的一位置处)来执行。 (It is located at a position along the path away from the vacuum source) which detect the degree of vacuum can be used to perform the detector. 当真空没有超过预设的水平时,可能出现警报,或者可能进行重新对准。 When the vacuum does not exceed a preset level, an alarm may occur, or may be re-aligned. [0126] 通过使用一个或多个机械或电机械定位器件,阵列器件可以与热控制器件接触。 [0126] By using one or more mechanical or electromechanical positioning devices, the array device may be contacted with the thermal control device. 该方法的执行过程可以受到自动地控制和监控。 During the execution of the method may be automatically controlled and monitored. 例如,用自动控制系统可以执行这种自动控制和监控,该自动控制系统与机器人控制系统相连,以便将该阵列器件引入热控制器件或从热控制器件中取出该阵列器件。 For example, the automatic control system can perform this automatic control and monitoring, the automatic control system connected to the robot control system, so the introduction of the thermal control device array device or the array device is removed from the thermal control device. 各种反应的进程也可以受到监控。 Process various reactions may be monitored.

[0127] 可以提供一种包括热控制器件的单元。 [0127] can provide a thermal control device unit included. 可以提供一种包括阵列器件和热控制器件的系统。 May be provided a device comprising an array of thermal control devices and systems. 为了确保热循环步骤的精确度,在某些器件中,包括传感器是很有用的,这些传感器可用于检测该器件各个区域的温度。 To ensure the accuracy of thermal cycling steps, in certain devices, including sensors are very useful, these sensors can be used to detect the temperature of various regions of the device. 一种用于检测温度的结构是热电偶。 A structure for detecting temperature is a thermocouple. 这种热电偶可以作为底层基板材料上图形化的薄膜导线而产生,或者作为被直接引入微制造的弹性材料中的导线而产生。 Such thermocouples can be generated on the underlying substrate material as a patterned thin film conductor, or as an elastic material is introduced directly into the micro-fabrication in the wire to produce.

[0128] 通过电阻变化,也可以检测温度。 [0128] The change in resistance, temperature can be detected. 例如,使用常规技术在底层半导体基板上制造的热敏电阻的阻抗变化可以被校准到给定的温度变化。 For example, conventional techniques using a thermistor on the underlying semiconductor substrate manufacturing of impedance change may be calibrated to a given temperature change. 或者,热敏电阻可以被直接插入微制造的弹性材料中。 Alternatively, the thermistor can be inserted directly into the micro-fabrication of a resilient material. 在Wu等人的文章"MEMS Flow Sensorsfor Nano-fluidic Applications"(Sensors and Actuators A 89152—158, 2001年)中,描述了另一种利用阻抗来检测温度的方法,该文章整体引用在此作为参考。 In Wu et al. Article "MEMS Flow Sensorsfor Nano-fluidic Applications" (Sensors and Actuators A 89152-158, 2001 年), there is described another to detect the temperature of the impedance method, which article is hereby incorporated by reference in their entirety by reference . 该文章描述了将掺杂多晶硅结构应用于控制和检测温度。 This article describes the doped polysilicon structure used to control and detect temperature. 对于多晶硅和其它半导体材料,阻抗的温度系数可以由杂质的类别和量来精确地控制,由此针对给定的应用使传感器的性能最佳化。 For polysilicon and other semiconductor materials, the temperature coefficient of impedance can be accurately controlled by the type and amount of impurities, whereby a given application for the best performance of the sensor.

[0129] 热-色材料(Thermo-chromatic material)是另一种可用于检测扩增器件各区域温度的结构。 [0129] Thermal - coloring material (Thermo-chromatic material) is another structure can be used to detect amplification of each region of the device temperature. 具体来讲,某些材料在经历不同温度时会急剧地且可复现地改变颜色。 Specifically, certain materials when undergoing different temperatures will rapidly and reproducibly change color. 这种材料可以被添加到溶液中,因为它们历经不同的温度。 Such material may be added to the solution, because they experienced different temperatures. 热-色材料可以形成于底层基板上, 或者被包括到弹性材料中。 Thermal - Color material may be formed on the base substrate, or included in the elastomeric material. 或者,热-色材料可以以颗粒的形式被添加到样品溶液中。 Alternatively, heat - color material in particulate form can be added to the sample solution. [0130] 另一种检测温度的方法是使用红外照相机。 [0130] Another method is to use the detected temperature of the infrared camera. 红外照相机和显微镜相结合可以被用来确定整个扩增结构的温度分布。 Infrared cameras and microscopes combination can be used to determine the temperature distribution of the entire amplification structure. 对于合适波长的辐射(例如,热辐射、红外辐射等)而言, 弹性材料的渗透性将有利于这种分析。 For appropriate wavelength radiation (e.g., thermal radiation, infrared radiation, etc.), the permeability of the elastomeric material will facilitate this analysis.

[0131] 温度检测的另一种方法是使用热电传感器。 Another method of [0131] the temperature detection is to use a pyroelectric sensor. 具体来讲,一些结晶材料特别是那些呈现出压电行为的材料可以呈现出热电效应。 Specifically, some crystalline materials, particularly those that exhibit the piezoelectric behavior of a material can exhibit the pyroelectric effect. 该效应描述了一种现象,即材料晶格的极化和材料上的电压高度依赖于温度。 This effect describes a phenomenon that the polarization voltage on the material and the material is highly dependent on the lattice temperature. 这种材料可以被包括到基板或弹性体中并且被用于检测温度。 Such materials may be included in the substrate or elastomer and is used to detect the temperature. 根据本发明的各实施例,其它电学现象(比如电容和电感)都可以被用于检测温度。 According to various embodiments of the invention, other electrical phenomena (such as capacitance and inductance) can be used to detect temperature. 本领域的技术人员将会认识到许多变体、修改和替换。 Those skilled in the art will recognize many variations, modifications, and alternatives.

[0132] 在一个实施例中,成像系统200按下面的方式来工作。 [0132] In one embodiment, the imaging system 200 to work in the following manner. 首先,微流体器件205被牢固地置于托架207上。 First, microfluidic device 205 is securely placed on the tray 207. 基于微流体器件205的固定的特征(例如,微流体器件的底座的边缘),计算机270使x、y驱动(未示出)移动托架207,以便对准第一x、y位置中的微流体器件。 Based on a fixed feature microfluidic device 205 (e.g., the edge of the base of the microfluidic device), the computer 270 so that x, y drive (not shown) to move the carriage 207, in order to align the first x, y position of the micro- fluidic devices. 在一些实施例中,在对准和定位过程中,使用一个或多个基准标记。 In some embodiments, the alignment and positioning process, using one or more reference marks. 在特定的实施例中,系统用户用成像系统来记录一个或多个基准标记的精确坐标。 In a particular embodiment, the system user with the imaging system to record the exact coordinates of one or more reference marks. 在其它实施例中,该过程是自动执行的,因为通过定位对称的XY基准物体并除去任何非对称成分便可以精确地计算基准的中心。 In other embodiments, the process is performed automatically, as by positioning symmetry XY of the reference object and removing any non-symmetric components can be accurately calculated on the basis of the center. 在一些实施例中,在对准过程中使用了基准的特征(比如边和角)。 In some embodiments, the alignment process, using the criterion of feature (eg edges and corners). 本领域的技术人员将会认识到许多变体、修改和替换。 Those skilled in the art will recognize many variations, modifications, and alternatives.

[0133] 在计算机270的控制下,调节托架207使其定位于光学元件210和212的焦平面中,或者调节光学元件210和212使光学元件210和212的焦平面定位到托架207。 [0133] Under control of the computer 270, the adjusting bracket 207 to position them in the focal plane of the optical element 210 and 212, or adjusting optical element 210 and 212 so that the focal plane of the optical element 210 and 212 positioned to the carriage 207. 在较佳实施例中,视场可以包住整个微流体器件,其中包括微流体器件上存在的许多反应室。 In the preferred embodiment, the field of view can surround the entire microfluidic device, including many of the reaction chamber present on the microfluidic device. [0134] 微流体器件的反应室中正发生的化学过程所发出的荧光、化学发光或光信号均被第一透镜系统210收集。 [0134] fluorescent, chemiluminescent, or chemical process reaction chamber optical signal microfluidic device of CKS occurring emitted the first lens system 210 are collected. 在本发明的一些实施例中,第一透镜系统是多元件光学系统,其中包括一个或多个透镜以及一个或多个孔径。 In some embodiments of the present invention, the first lens system is a multi-element optical system, which includes one or more lenses and one or more apertures. 如图2A所示,第一透镜系统210包括多个单独的透镜元件以及成对的透镜元件等。 Shown, the first lens system in FIG. 2A 210 includes a plurality of individual lens elements and the lens element pairs and the like. 选择第一透镜系统210的光学特性(其中包括焦距f/ft 等)以提供期望的光学性能。 Selecting a first lens system 210 of the optical characteristics (including the focal length f / ft, etc.) to provide the desired optical performance. 本领域的技术人员将会认识到许多变体、修改和替换。 Those skilled in the art will recognize many variations, modifications, and alternatives. 图1A 中示出了发射快门215,用于阻挡离光轴预定距离以外的光线,当然本发明对此不作要求。 Figure 1A shows the emission shutter 215 for blocking a predetermined distance from the outside of the optical axis of the light, of course, the present invention this is not required. [0135] 再次参照图1A,提供了滤光器件213,作为光学组件的一部分。 [0135] Referring again to Figure 1A, the optical device 213 is provided, as part of the optical assembly. 在一些实施例中, 滤光器件是一种包括许多光学元件的滤光轮213,用于使荧光标记试剂或化学发光标记试剂所产生的荧光发射或化学发光发射通过并且对其进行光学处理。 In some embodiments, the filter device is a filter wheel comprising a number of optical element 213, for the fluorescent labeling reagent or chemiluminescent labeled reagent produced by fluorescence emission or chemiluminescence emission and its optically processed through. 作为一个示例,在一个实施例中,该发射滤光轮的第一部分适用于使第一荧光染料CyTM3所产生的荧光发射通过, 该第一荧光染料可以从Amersham Biosciences公司(该公司是新泽西州Piscataway市GE Healthcare的一部分)购买。 As one example, in one embodiment, the first transmitting portion of the filter wheel is suitable for the first fluorescent dye CyTM3 fluorescence emission generated by the first fluorescent dyes available from Amersham Biosciences, Inc. (Piscataway, NJ which is GE Healthcare's part of the city) to buy. 上述发射滤光轮的第二部分适用于使第二荧光染料Cy™5所产生的荧光发射通过,该第二荧光染料也可以从Amersham Biosciences公司购买。 Said second portion of the transmission filter wheel adapted to cause the fluorescent dye Cy ™ 5 second fluorescent emission generated by the second fluorescent dye can also be purchased from Amersham Biosciences Corporation. 当然,这 Of course, this

24些荧光染料的使用不是本发明所要求的。 24 These fluorescent dyes is not required by the present invention. 在可选实施例中,使用一种可以从加州Carlsbad 市的Invitrogen公司买到的Alexa荧光染料。 In an alternative embodiment, one may use from Invitrogen, Carlsbad, California available Alexa fluorescent dye. 作为一个示例,在另一个实施例中,上述发射滤光轮的第一部分适用于使第三荧光染料Alexa Fluor 350所产生的荧光发射通过,该第三荧光染料可以从Invitrogen公司购买。 As one example, in another embodiment, the above-described emission filter wheel is suitable for a first portion of the third fluorescent dye Alexa Fluor 350 fluorescence emission generated by the third fluorescent dye can be purchased from Invitrogen Corporation. 上述发射滤光轮的第二部分适用于使第四荧光染料Alexa Fluor 488所产生的荧光发射通过,该第四荧光染料也可以从Invitrogen公司购买。 Transmitting said second portion adapted to cause the filter wheel fourth fluorescent dye Alexa Fluor 488 fluorescence emission generated by the fourth fluorescent dyes can be purchased from Invitrogen Corporation. 在下文中将提供与该发射滤光轮有关的其它细节。 Provide additional details relating to the emission filter wheel is hereinafter.

[0136] 在一些实施例中,滤光器件213和发射快门215都位于第一透镜系统和第二透镜系统之间。 [0136] In some embodiments, the filter element 213 and the emission shutter 215 is positioned between the first lens system and the second lens system. 在这些实施例中,穿过滤光器件的光线以相对于光轴很小的角度进行传播。 In these embodiments, the light rays pass through the filter device relative to the optical axis of the very small angle spread. 对于本领域的技术人员而言,很明显,在光线入射角很小的各区域中的光谱滤光片(例如,干涉滤光片)更易于设计,并且有可能提供更窄的总光谱带宽,通过这种窄光谱带宽,各种特征和/或滤光片定位都是本发明所要求的。 For the skilled artisan, it is clear that a small angle of incidence of light in the regions of spectral filters (e.g., interference filter) is easier to design, and it is possible to provide a narrower overall spectral bandwidth, By such a narrow spectral bandwidth, the various features and / or filter location is required by the present invention. 如图IA所示,滤光器件和发射快门都耦合到计算机270,从而为这些元件提供了计算机控制。 FIG IA, the filter device and the emission shutter 270 is coupled to a computer, which provides computer control for these elements. 此外,对于本领域的技术人员而言,很明显, 可以在光路中设置多个滤光片且很可能是多个完全一样的滤光片,以增大对激发波长的阻隔。 In addition, for the skilled artisan, it is clear that a plurality of filters may be provided in the optical path, and is likely to be a plurality of identical filters, to increase the excitation wavelength of the barrier. 在一些实施例中,这些滤光片相对于光轴有一定角度,所以被滤光片反射掉的光线会离开光路。 In some embodiments, these filters have a certain angle with respect to the optical axis, so light is reflected off the filter will leave the optical path.

[0137] 在其它实施方式中,某些嵌入染料在结合于双链DNA后会产生剧烈的荧光增强和/或显示出对双链DNA具有强烈的化学亲和力,可用于检测双链扩增DNA。 [0137] In other embodiments, some of the intercalating dye bound to double-stranded DNA in the post will have a dramatic increase in fluorescence and / or double-stranded DNA shown to have a strong chemical affinity, can be used to detect double-stranded amplified DNA. 合适的染料示例包括但不限于SYBRTm和Pico Green (俄勒冈州Eugene市的Molecular Probes公司)、溴化乙锭、碘化丙锭、色霉素、吖啶橙、Hoechst33258、 Toto-l、 Yoyo-1和DAPI(4', 6-二脒基-2-苯基吲哚盐酸盐)。 Suitable dyes Examples include, but are not limited to SYBRTm and Pico Green (Eugene, Oregon City, Molecular Probes Inc.), ethidium bromide, propidium iodide, Chromomycin, acridine orange, Hoechst33258, Toto-l, Yoyo-1 and DAPI (4 ', 6- diamidino-2-phenylindole hydrochloride). 关于嵌入染料的使用,在Zhu等人Anal.Chem.66 : 1941-1948(1994)中进行了另外的讨论,该文章整体引用在此作为参考。 Use about embedding dyes, Zhu et al Anal.Chem.66 in: (1994) 1941-1948 was further discussion, the article reference in its entirety herein by reference. [0138] 图1A也示出了第二透镜系统212。 [0138] Figure 1A also shows a second lens system 212. 穿过滤光器件213和发射快门215的荧光或化学发光发射被第二透镜系统聚焦到检测器260。 Pass through the filter element 213 and the emission shutter 215 fluorescence or chemiluminescence emitted by a second focusing lens system to the detector 260. 在一个实施例中,该检测器是CCD照相机阵列,但本发明对此不作要求。 In one embodiment, the detector array is a CCD camera, but the present invention this is not required. 在特定的实施例中,使用了一种阵列检测器,大小和微流体器件差不多。 In a particular embodiment, the use of an array detector, the size of the microfluidic device, and similar. 较佳地,选择检测器阵列260的像素大小,以提供比微流体器件的反应室面积要小的面积,由此使每个反应室对应多个检测器像素。 Preferably, the detector array 260 to select the pixel size, to provide a microfluidic device than the area of the reaction chamber to a small area, whereby each reaction chamber corresponding to the plurality of detector pixel. 在特定的实施例中,检测器260是一种具有大约15 ii mxl5 ii m像素的CCD阵列。 In a particular embodiment, the detector 260 is a kind of approximately 15 ii mxl5 ii m pixel CCD array.

[0139] 对于本文所提供的微流体器件,可以使用许多不同的检测方案。 [0139] For microfluidic devices provided herein can be used in many different detection schemes. 合适系统的选择部分程度上基于所检测的事件和/或试剂的类型。 And / or the type of reagents suitable choice of the degree of partial systems based on a detected event. 这些检测器可以被设计成检测许多不同的信号类型,包括但不限于来自下列物质的信号:放射性同位素、荧光团、生色团、电子密集颗粒、磁性粒子、自旋标记物、化学发光分子、电化学活性分子、酶、辅因子、连接于核酸探针的酶,以及酶底物。 The detector may be designed to detect a number of different signal types, including but not limited to a signal from the following substances: radioisotopes, fluorophores, chromophores, electron dense particles, magnetic particles, spin labels, chemiluminescent molecules, electrochemically active molecules, enzymes, cofactors, enzymes linked to nucleic acid probes and enzyme substrates.

[0140] 适于本发明微流体器件使用的说明性的检测方法包括但不限于:光散射、多通道荧光检测、UV和可见光波长吸收、发光、示差反射、和激光共聚焦扫描。 [0140] The illustrative detection method suitable for a microfluidic device of the present invention include, but are not limited to: light scattering, multichannel fluorescence detection, UV and visible wavelength absorption, luminescence, differential reflection, and confocal laser scanning. 可用于某些应用中的其它检测方法包括:闪烁亲近测定技术、放射化学检测、荧光偏振各向异性、荧光寿命、荧光相关频谱学(FCS)、时间分辨能量转移(TRET)、荧光共振能量转移(FRET)、以及像生物发光共振能量转移(BRET)这样的变化形式。 Can be used in certain applications of other detection methods include: Scintillation Proximity Assay technology, radiochemical detection, fluorescence polarization anisotropy, fluorescence lifetime, fluorescence correlation spectrum Science (FCS), time-resolved energy transfer (TRET), fluorescence resonance energy transfer (FRET), as well as bioluminescence resonance energy transfer (BRET) in the form of such a change. 其它检测选择包括电阻、电阻率、阻抗和电压的检测。 Other testing options include resistivity, resistivity, impedance, and voltage detection.

[0141] 在一些实施例中,在"检测部分"或"检测区域"进行检测。 [0141] In some embodiments, the "detection section" or the "detection region" for testing. 这些术语和其它相关术语是指微流体器件上进行检测的那部分。 These terms and other related terms refer to that part of the detecting microfluidic device. 在一些微流体器件中,检测部分常常是微流体器件中存在的反应室。 In some microfluidic devices, the detecting portion is often present in the microfluidic device reaction chamber. 其它器件的检测部分可以在与交叉点相邻的流体通道区域内、交点本身、或包括交点及其周围区域的区域。 Detecting part of another device may be in a point of intersection of adjacent fluid channel region, the intersection itself, or its surrounding area including the intersection region.

[0142] 检测部分可以连接到一个或多个显微镜、二极管、光激励器件(例如,激光器)、光电倍增管、处理器、以及上述的组合,它们配合在一起来检测和特定事件和/或试剂相关联的信号。 [0142] detecting portion may be connected to one or more microscopes, diodes, light excitation devices (e.g., lasers), photomultiplier tubes, processors and combinations of the above, and they fit together to detect specific events and / or reagents associated with the signal. 通常,待测信号是光信号,由一个或多个光检测器在检测部分中对其进行检测。 Typically, the measured signal is a light signal, by one or more light detectors detect it in the detecting section. 光检测器可以包括一个或多个光电二极管(例如,雪崩光电二极管)、引向如光电倍增管的光纤光导、显微镜、和/或视频照相机(例如,CCD照相机)。 Light detector may comprise one or a plurality of photodiodes (e.g., avalanche photodiodes), toward the light guide fiber as the photomultiplier tube, a microscope, and / or a video camera (e.g., CCD camera).

[0143] 检测器可以在微流体器件内被微制造,或者可以是分离的元件。 [0143] The detector may be microfabricated within the microfluidic device, or may be a separate element. 如果检测器作为分离的元件存在并且微流体器件包括多个检测部分,则在任何给定的时刻可以在单个检测部分内进行检测。 If the detector exists as a separate element and the microfluidic device comprises a plurality of detecting portions, at any given time can be detected within a single detection section. 作为一个具体的示例,微流体器件可以被附在可平移的工作台上并且在显微镜物镜下进行扫描。 As a specific example, the microfluidic device can be attached to the workbench and translatable scanned under a microscope objective. 如此获得的信号接下来传递到处理器,以便于信号解释和处理。 Next, the signal thus obtained is transmitted to the processor, in order to interpret and process the signal. 也可以使用光电倍增管的阵列。 An array of photomultiplier tubes can also be used. 另外,可以使用这样的光学系统,它们能够从所有不同的检测部分中同时收集信号,而且能够判定来自每一个部分的信号。 In addition, using such an optical system, which can collect signals from all the different detection sections simultaneously, and can be determined from the signal for each section.

[0144] 外部检测器是可以使用的,因为所提供的器件完全或大部分都由对受监控的波长透明的材料制成。 [0144] external detector can be used, as provided by the device is fully or most of a material transparent to the wavelength monitored made. 该特征能够使本发明的器件使用许多光学检测系统,这对于常规的硅基微流体器件而言是不可能的。 This feature enables the device of the present invention is the use of many optical detection system, which for a conventional silicon-based microfluidic device is not possible with.

[0145] 本发明的特定实施例使用了形如CCD照相机的检测器以及可提供大视场和高数值孔径的光路,以使从每一个反应室中收集到的光量最大化,由此增大了检测灵敏度。 [0145] Specific embodiments of the present invention uses a form such as a CCD camera detector, and can provide a large field of view and a high numerical aperture of the optical path, so that the amount of light collected from each reaction chamber to maximize, thereby increasing the detection sensitivity. 在本实施例中,该CCD被用作光电检测器的阵列,其中每一个像素或像素组对应于一个反应室, 而非被用来产生该阵列的图像。 In the present embodiment, the CCD is used as the photodetector array, wherein each pixel or group of pixels corresponds to a reaction chamber, instead of being used to generate an image of the array. 因此,可以设计或改变光学元件,使得在检测器处图像质量下降或图像变得模糊,以增大该光学系统的可用场深,从而从每一个反应室中收集更多的光线。 Thus, the optical element may be designed or changed, such that the detector of the image quality or the image becomes blurred, to increase the depth of field of the optical system can be used to collect more light from each reaction chamber. 特别是,因为本发明的一些实施例中所预期的试验包括使用荧光染料的生物试验,这些染料因暴露于激发光线而使光反差变小(photobleach),因此限制了从给定样品中可以获得的信号光子的总数,有限信号光子的有效收集在仪器中是很重要的。 Specifically, since some contemplated embodiments of the present invention comprises the use of experimental biological tests fluorescent dyes, these dyes upon exposure to excitation light leaving the optical contrast becomes smaller (photobleach), thus limiting from a given sample can be obtained The total number of signal photons, effectively collecting the limited signal photons in the instrument is very important. 对于任何光学系统,Etendue的种种考虑涉及物像NA(数值孔径)和总的系统放大率;因为像方NA通常是有限的(例如,对高入射角光线而言在CCD表面上有反射损耗),对于任意高的放大率而言无法同时实现任意高的物方(样品那边)NA。 For any optical system, Etendue various considerations involved in things like the NA (numerical aperture) and total system magnification; because, like party NA is usually limited (for example, in terms of the high angle of incidence of light on the CCD surface reflection losses) For the purposes of any high magnification can not simultaneously achieve any high object side (sample side) NA. 事实上,更大的系统放大率可以允许更高的物方NA,同时不要求像方NA同时上升(该上升有可能有害,原因如上)。 In fact, larger systems can allow for higher magnification the object side NA, while at the same time does not require the image side NA increased (the rise is likely harmful, as above). 结果,在所描述的系统中,大CCD(例如,30. 7mmx30. 7mm)焦平面阵列被用于允许1 : 1的光学系统(即系统放大率为1)。 As a result, in the described system, a large CCD (.. For example, 30 7mmx30 7mm) focal plane array is used to allow 1: 1 of the optical system (i.e., the system magnification of 1). 这允许选取NA为0. 36,同时CCD上的像方NA为0. 36,这便提供了相对于表面反射损耗的合理的性能。 This allows for selecting NA 0.36, while on the image side NA CCD is 0.36, which would provide a reasonable performance with respect to the surface reflection losses.

[0146] 在一些实施例中,更大的物方NA产生减小的物方焦深,因此对于样品中给定的反应室深度,检测器处的模糊更严重了(假定因焦深引起的模糊大于或等于因透镜像差和其它问题所引起的模糊),从而如果在反应室之间实现了低信号串扰则限制了样品处各反应室之间可允许的最小间隔。 [0146] In some embodiments, the larger the object side to the object side NA produce a reduced depth of focus, so for a given sample in the reaction chamber depth, blur at the detector is more serious (assuming the depth of focus caused by greater than or equal to blur due to lens aberrations and other problems caused by blur), so that, if the reaction chamber between the low signal crosstalk is achieved limits the minimum allowable interval between samples at each reaction chamber. 在与l : l光学系统相结合的情况下,如果想要避免其表面处的反射损耗,则这种物方NA考虑与CCD (或硅检测器)上一般期望的〜0. 5NA最大值很一致。 With l: l case of combining an optical system, if you want to avoid reflection losses at the surface thereof, the object-side NA consider this CCD (or a silicon detector) with the general expectation of the maximum value is ~0 5NA. unanimously. 该l : l成像透镜系统还固有地免受大多数奇数阶像差的影响,从而增大了该特定放大率(M= 1)的优势。 The l: l imaging lens system is also inherently immune to most of the odd-order aberrations, thereby increasing the specific magnification (M = 1) advantage. 作为详细的系统的设计,在本发明的一些实施例中,使用了具有检测器的l : l光学系统,它和待成像的微流体系统一样大或更大。 As the detailed design of the system, in some embodiments of the present invention, using a detector having a l: l of the optical system, and it is as large or larger microfluidic system to be imaged.

[0147] 在其它实施例中,可能存在和检测器(例如,CCD焦平面阵列)尺寸有关的成本限制。 [0147] In other embodiments, there may be and detector (e.g., CCD focal plane array) size limitations associated costs. 例如,目前一些高量子效率、全画面CCD阵列具有27.6mmx27.6mm的尺寸。 For example, the current number of high quantum efficiency CCD array with 27.6mmx27.6mm full screen size. 和尺寸为30. 7mmx30. 7mm的微流体器件相比,该检测器稍微小一点,从而产生0. 88的系统放大率,作为用于上述系统的一种设计。 And size 30. 7mmx30. 7mm compared microfluidic device, the detector is slightly smaller, resulting 0.88 magnification system, as a design for the system described above. 因为接近系统放大率M二l,所以针对这种放大率,与上述检测器(像方)入射NA有关的种种约束条件均得到满足。 Because close to the system magnification M two l, so for this magnification, and said detector (like square) NA incident related to various constraints are met.

[0148] 在其它实施例中,可以指定各反应室的给定XY平面(和光轴相垂直)间隔和大小(例如,在XY平面中实现期望的样品-反应室密度),同时反应室的最小总体积仍然受到约束(例如,实现最小所需化学体积,以避免因试剂或目标分子数目太小而导致的过大的统计波动,或简单地实现所需的最少数目的荧光或其它发光分子或物体)。 [0148] In other embodiments, each reaction chamber can specify a given XY plane (perpendicular to the optical axis) and the size of the interval (e.g., to achieve a desired sample in the XY plane - the density of the reaction chamber), while minimizing the reaction chamber The total volume would remain bound (e.g., to achieve the minimum desired chemical volume, in order to avoid excessive statistical fluctuations due to the number of reagents or target molecule is too small and or simply achieve the required minimum number of fluorescent or luminescent molecules or other objects). 在这种情况下,可能有必要平行于Z轴(光轴)来延伸反应室,使得每一个反应室的总体积仍然等于或大于一些最小数字。 In this case, it may be necessary parallel to the Z axis (optical axis) to extend the reaction chamber, such that the total volume of each reaction chamber remains equal to or greater than some minimum number. 假定焦深所引起的模糊大于或等于由透镜像差和其它问题所导致的模糊, 考虑到焦深,对于给定的物方NA而言,沿Z轴作更大的延伸(从而产生高纵横比或柱状反应室,这使待询问的样品沿Z轴集中)通常将使检测器处的反应腔图像更模糊。 Fuzzy assumed that the depth of focus caused by the lens is greater than or equal to the aberration and the blur caused by other problems, taking into account the focal depth, for a given object-side NA is concerned, along the Z axis for the greater extension (to produce high aspect than or cylindrical reaction chamber, which allows the sample to be interrogated along the Z-axis focus) will cause the reaction chamber is generally at the image detector more ambiguous. 在一些情况下,这将导致使用更低的物方NA。 In some cases, this will result in the use of a lower object-side NA. 使用更低的NA透镜系统可以允许焦深更大,因此,从平行于光轴延伸的反应室中更好地进行光线收集,同时通常不会引起相邻或相近的反应室之间的光信号串扰。 Using a lower NA lens system may allow a greater depth of focus, therefore, parallel to the optical axis extending from the reaction chamber for better light collection, but usually do not cause adjacent or close to the optical signal between the reaction chamber crosstalk. 这样,可以使XY平面(存在于光轴的地方)中反应室的密度更大,却没有过度的串扰,同时通过在Z方向(平行于光轴)延伸,使总的反应室体积保持很大。 This can make the XY plane (where present in the optical axis) of greater density in the reaction chamber, but not excessive crosstalk, while by the Z direction (parallel to the optical axis) extends, so that the total volume of the reaction chamber to maintain a large . 在这种情况下,或者在更低的物方NA尚可接受的情况下(例如,情况一,各反应室之间更大的XY 间隔使检测器处的反应腔_图像更模糊,同时却没有过多的串扰;情况二,在非光发射应用中,更高的NA不是必需的;情况三,有足够多的样品以至于光白化不是问题;情况四,没有光白化的样品,像降低可接受的系统灵敏度这样的情形),较低的系统放大率(M< 1)可能是合适的,尤其是当M > NA。 In this case, or in the case of a lower object-side NA is acceptable (e.g., a case, a larger reaction chamber between the reaction chamber spaced _ XY image at the detector is more blurred, while they no excessive crosstalk; Case 2, the non-light emitting applications, higher NA is not required; three cases, there is enough light albino specimen that is not a problem; four cases, there is no light albino specimen, such as lower Acceptable case sensitivity of such a system), the lower the system magnification (M <1) may be appropriate, especially when M> NA. /0. 5时,M > NA。 / 0. 5, M> NA. /0. 36更佳,其中NA。 / 0.36 is better, which NA. 二物方NA,或者M > NA。 Two things side NA, or M> NA. / NAdet,其中NAdet等于可允许到检测器表面上的最大NA,同时对于检测器而言没有过大的反射/插入损耗(对于典型的CCD而言,NAdet = 0. 36〜0. 5)。 / NAdet, wherein NAdet NA is equal to the maximum allowable on the detector surface, and for the purposes of the detector without excessive reflection / insertion loss (for a typical CCD is concerned, NAdet = 0. 36~0. 5).

[0149] 当物方焦深和/或模糊要求并不需要物方NA《0. 36或0. 5或者更一般地讲NA。 [0149] When the object-side depth of focus and / or vague requirement does not require the object side NA "0. 36 or 0.5, or more generally speaking NA. 《NAdet时,基于Etendue的种种考虑(如上所述)期望用更大的检测器,因为对于给定的NA。 "NAdet when, based on various considerations Etendue (as described above) with a larger detector desired, because for a given NA. ,更大的M(对于给定的样品尺寸,通常需要更大的检测器)将允许更小的NAJ像方NA)。 , A larger M (for a given sample size, typically requires more detectors) would allow smaller NAJ image-side NA). 因此,当光收集要求(例如,实现某些试验灵敏度)需要很大的NA。 Thus, when the light collection requirements (e.g., to achieve certain test sensitivity) requires a large NA. (由NA。 > NAdet来定义)并且其它设计考虑(例如,成本)允许很大的时NA。 (.> NAdet defined by NA) and other design considerations (e.g., cost) when allowing a large NA. ,期望M更大,使得检测器处的损耗最小化。 Expected M greater, so that the loss at the detector is minimized. 在这种实施例中,有用的是,使用一个或多个CCD器件等检测器件,其尺寸要大于待成像的微流体器件的面积。 In such embodiments, it is useful to use one or more CCD devices, and other detection devices, which is greater than the size of the microfluidic device to be imaged area. 使用这种大检测器可以增大光学系统的放大率,因此(通过Etendue的种种考虑),对于到检测器的固定入射NA,从样品中收集光线的NA可以更高(例如,通过CCD表面处更高入射角的入射光的反射损耗来实现后者) Use of such a large detector may increase the magnification of the optical system, therefore, is incident to the detector is fixed NA, to collect light from the sample (by considerations Etendue of) the NA may be higher (e.g., through the CCD surface reflection loss of incident light to achieve the latter a higher angle of incidence)

[0150] —种较佳的检测器使用CCD照相机和可以提供大视场和高数值孔径的光路,从而使从各反应室中收集的光的量最大化,由此增大了检测灵敏度。 [0150] - Maximize species preferred detector uses a CCD camera and can provide a large field of view and a high numerical aperture of the optical path, so that the collected light from each reaction chamber volume, thereby increasing the detection sensitivity. 关于这一点,CCD是作为光电检测器的阵列而被使用的,其中每一个像素或像素组对应于一个反应室,而非被用来产生该阵列的图像。 In this regard, CCD is used as the photodetector array is used, wherein each pixel or group of pixels corresponds to a reaction chamber, instead of being used to generate an image of the array. 因此,可以改变光学系统,使得图像质量下降或散焦,从而增大光学系统的场深,以便从每一个反应室中收集更多的光线。 Thus, the optical system can be changed, so that the image quality, or defocus, thereby increasing the depth of field of the optical system to collect more light from each reaction chamber. 在一些实施例中,有用的是,使用高纵横 In some embodiments, it is useful to use a high aspect

27比或圆柱形反应室,使要由检测器询问的样品沿光学系统的光轴集中,并且较佳地通过使图像散焦来增大场深。 Than or cylindrical reaction chamber 27, so that the optical axis to focus the sample interrogated by the detector along the optical system, and preferably by defocusing the image to increase depth of field. 使用低NA透镜系统,较佳地使用两侧对称的透镜系统。 Use low NA lens system, preferably using two-sided symmetrical lens system. 有用的是, 使用一个或多个CCD器件等检测器件,其尺寸大于待成像的微流体器件的面积。 It is useful to use one or more CCD devices, and other detection devices, the size of the area to be imaged is larger than the microfluidic device. 与低NA光学系统结合使用,可以实现改进的检测灵敏度。 In combination with a low NA optics, improved detection sensitivity can be achieved.

[0151] —种检测系统可以包括光源,用于激发报道物来产生可检测的信号。 [0151] - seed detection system may include a light source for exciting the reporter to produce a detectable signal. 所用光源的类型取决于待激发的报道物的性质。 On the type of light source depends on the nature of the reporter to be excited. 合适的光源包括但不限于激光器、激光二极管、白光源、和高强度灯。 Suitable light sources include, but are not limited to, lasers, laser diodes, white light source, and high-intensity lamp. 如果使用激光器,则激光器可以用来在一组检测部分上或单个检测部分上进行扫描。 If a laser is used, the laser can be used to scan on a group or individual detecting portion detecting portion. 激光二极管可以被微制造成微流体器件本身。 Laser diodes can be microfabricated into the microfluidic device itself. 或者,激光二极管可以被微制造成位于微流体器件附近的另一个器件,它被用来进行热循环反应,使得该二极管的激光被引至检测部分。 Alternatively, laser diodes can be microfabricated into the other device is located in the microfluidic device, which is used to carry out a thermal cycling reaction such that the diode laser is directed to the detection portion.

[0152] 检测可以包括许多非光学方法。 [0152] Detection may include many non-optical methods. 例如,检测器可以包括温度传感器、电导传感器、 电势传感器(例如,pH电极)和/或测量电流的传感器(例如,监测氧化和还原反应)。 For example, the detector may include a temperature sensor, a conductivity sensor, a potential sensor (e.g., pH electrode) and / or measurement of the current sensor (e.g., to monitor oxidation and reduction reactions). [0153] 某些嵌入染料在结合于双链DNA时会产生剧烈的荧光增强和/或显示出对双链DNA具有强烈的化学亲和力,它们可用于检测双链扩增DNA。 [0153] In certain intercalating dye bound to double-stranded DNA will produce intense fluorescence enhancement and / or double-stranded DNA shown to have a strong chemical affinity, they may be used to detect double-stranded amplified DNA. 合适的染料示例包括但不限于SYBir和Pico Green(俄勒冈州Eugene市的Molecular Probes公司)、溴化乙锭、碘化丙锭、色霉素、吖啶橙、Hoechst 33258、Toto-l、Yoyo-l以及DAPI (4' , 6- 二脒基-2-苯基吲哚盐酸盐)。 Suitable dyes Examples include, but are not limited to SYBir and Pico Green (Eugene, Oregon City, Molecular Probes Inc.), ethidium bromide, propidium iodide, Chromomycin, acridine orange, Hoechst 33258, Toto-l, Yoyo- l and DAPI (4 ', 6- diamidino-2-phenylindole hydrochloride). 关于嵌入染料的使用,在Zhu等人Anal. Chem. 66 :1941-1948(1994)中进行了另外的讨论,该文章整体引用在此作为参考。 Use about embedding dye 66 in Zhu et al. Anal Chem:. 1941-1948 (1994) carried out a further discussion, the article reference in its entirety herein by reference.

[0154] 如图1A所示,本发明的一些实施例提供了1 : l成像系统,用于产生并检测荧光、 化学发光、生物发光以及来自微流体器件的其它信号。 [0154] As shown in Figure 1A, some embodiments of the present invention provides a 1: l imaging system for generating and detecting fluorescence, chemiluminescence, bioluminescence, and other signals from the microfluidic device. 在所使用的图像检测器件和待成像的样品一样大的一些实施例中,提供了l : l成像系统。 As in the image detection device used and the sample to be imaged larger some embodiments, there is provided a l: l imaging system. 通过提供大视场(量级在若干个平方厘米)的l : l成像,本发明的各实施例提供了增大的数值孔径(NA)光学系统。 By providing a large field of view (in the order of several square centimeters) l: l imaging, various embodiments of the present invention provides increased numerical aperture (NA) of the optical system. 因为光收集效率大约正比于NA、所以本发明的一些实施例所提供的NA增大能够从反应室中收集到合适的荧光信号,这些反应室包括量级在一纳升到数十纳升的反应体积以及量级为1. 0 纳摩尔的活性荧光团浓度。 Since the light collection efficiency of about proportional to NA, NA so some embodiments of the present invention there is provided an increase can be collected from the reaction chamber to the appropriate fluorescent signal, in which the order of the reaction chamber comprises a nanoliters to several tens of nanoliters reaction volume and the order of the active fluorophore concentration of 1.0 nM. 在其它的实施例中,浓度为皮摩尔范围中的活性荧光团提供合适的荧光信号。 In other embodiments, the concentration of the fluorescent signal for providing suitable activity in the picomolar range fluorophore.

[0155] 另外,本发明的各实施例提供了这样的成像系统,它们稍微减小并构成的图像范围从大约和物体大小一样到大约是物体大小的一半。 [0155] Further, various embodiments of the present invention provides such imaging systems, and they are slightly reduced range image and formed from about the same size as the object to about half the size of the object. 例如,在一个实施例中,微流体器件的空间区域的图像被传输且被拍摄,该空间区域和不止96个反应室相关联。 For example, in one embodiment, an image of the spatial region of the microfluidic device is transmitted and captured, the spatial region and more than 96 of the reaction chamber is associated. 通过使用比微流体器件的空间区域稍小或大致相等的图像检测空间区域,图像检测器件被用于拍摄上述空间区域的图像。 By using slightly less than the spatial region of the microfluidic device is substantially equal to or spatial image detection area, the image sensing device is used to image the spatial region of the image. 仅作为示例,微流体器件的空间区域的面积比上该空间区域的像的面 Only as an example of image surface area ratio, the spatial region of the microfluidic device in the space area

积得到的比例可以是i : i、i : o.99、i : o.95、i : o.9、i : o.88、i : o.85、i : o.8、 i : o. 7、i : o.6和i : o. 5。 Volume ratio can be obtained i: i, i: o.99, i: o.95, i: o.9, i: o.88, i: o.85, i: o.8, i: o. 7, i: o.6 and i:. o 5. 在一些实施例中,该比例小于i : 0.5。 In some embodiments, the ratio is less than i: 0.5. 这些特定的比例只是示例性的,因为针对成像系统来选择的比例将取决于特定的应用。 These specific ratio are exemplary only, since the ratio for the imaging system to choose will depend on the particular application.

[0156] 在一些实施例中,光学成像系统包括大约3cmx3cm的视场。 [0156] In some embodiments, the optical imaging system comprises a field of view of approximately 3cmx3cm. 在其它实施例中,光学成像系统包括范围从大约lcmxlcm到大约5cmx5cm的视场。 In other embodiments, the optical imaging system comprises a range from about lcmxlcm to the field of view of approximately 5cmx5cm. 在特定的实施例中,提供大小为2cmx2cm、2. 5cmx2. 5cm、2. 76cmx2. 76cm、3. 07cmx3. 07cm、3. 5cmx3. 5cm禾口4cmx4cm的物体视场。 In a particular embodiment, there is provided a size of 2cmx2cm, 2. 5cmx2. 5cm, 2. 76cmx2. 76cm, 3. 07cmx3. 07cm, 3. 5cmx3. 5cm Wo port field 4cmx4cm objects. 通常,光学成像系统的视场被选择成对应于微流体器件的空间区域(例如,包括许多感兴趣的反应室的区域)。 Typically, the field of view of the optical imaging system is selected to correspond to the spatial region of the microfluidic device (e.g., a reaction chamber comprising a plurality of regions of interest). [0157] 此外,本发明的各实施例提供了具有许多数值孔径的光学成像系统。 [0157] In addition, embodiments of the present invention provides an optical imaging system having a plurality of numerical aperture. 作为一个示例,根据各种实施例,提供了范围从O. 1到0.5的NA。 As one example, in accordance with various embodiments, there is provided a range of from O. 1 to 0.5 NA. 在特定的实施例中,提供了大小为0. 15、0. 18、0. 2、0. 23、0. 25、0. 3、0. 36和0. 4的多种NA。 In a particular embodiment, there is provided a size of 0. 15,0. 18,0. 2,0. 23,0. 25,0. 3,0. 36, and 0.4 of a variety of NA.

[0158] 光学成像系统的空间分辨率通常是图像检测器件中像素大小的函数。 Spatial resolution [0158] optical imaging system is usually a function of image detection device pixel size. 在本发明的一些实施例中,放大率(在一些实施例中等于1)和检测器中的像素大小将决定和每一个反应室相关联的像素的数目。 In some embodiments of the present invention, the magnification (equal to 1 in some embodiments) and the detector pixel size will determine the number of each of the reaction chamber and associated pixels. 通常,每一个反应室最好和多个检测器像素相关联。 Typically, each reaction chamber and a plurality of detectors is preferably associated with pixels. 例如,在1 : l成像系统中,如果反应室一边是45微米,则它将与多达9个方形像素(其边等于15 微米)重叠。 For example, in 1: l imaging system, if the reaction chamber side is 45 microns, it will with up to 9 square pixels (whose side is equal to 15 micrometers) overlap. 因此,根据本发明的各实施例,与每一个反应室相关联的像素数目介于从l到100的范围中。 Thus, according to various embodiments of the invention, the number of pixels associated with each reaction chamber is between the range from l to 100. 例如,根据本发明的一些实施例,与每一个反应室相关联的有4个像素区域、 9个像素区域、16个像素区域、25个像素区域、36个像素区域、49个像素区域、64个像素区域和81个像素区域。 For example, according to some embodiments of the present invention, and each reaction chamber has four associated pixel area, the pixel area 9, 16 pixel regions, 25 pixel regions, 36 pixel regions, 49 pixel regions, 64 a pixel region and a 81-pixel region.

[0159] 在本发明的各实施例中,使用范围介于lyi^〜900iii^之间的像素大小。 [0159] In various embodiments of the present invention, the use range is pixel size lyi ^ ~900iii ^ between. 例如, 在本发明的各种实施例中,使用了边长为1 ym、2 ii m、3 ii m、4 ii m、5 ii m、 10 ii m、 13. 5 ii m、 15 ii m、20 ii m、25 ii m、30 ii m的正方形像素。 For example, in various embodiments of the present invention, the use of a side length of 1 ym, 2 ii m, 3 ii m, 4 ii m, 5 ii m, 10 ii m, 13. 5 ii m, 15 ii m, 20 ii m, 25 ii m, 30 ii m square pixels. 对于本领域的技术人员而言,很明显,像素大小、 检测器阵列尺寸、和每个阵列的像素数目是彼此相关的。 For the skilled person that, obviously, the number of pixels in the pixel size, the size of the detector array, and each array is related to each other. 在可选的实施例中,使用矩形像素,其像素面积介于1 y m2〜900 ii m2之间。 In an alternative embodiment, the use of rectangular pixels, its pixel area is between 1 y m2~900 ii m2.

[0160] 此外,根据本发明各实施例,使用了检测器阵列(也被称为图像检测器件,其中包括不同范围的像素数)。 [0160] Further, according to various embodiments of the present invention, using a detector array (also referred to as an image detection device, which includes the number of pixels different ranges). 阵列大小介于512x512像素区域到3, 000x3, 000像素区域。 The array size is between 512x512 pixel area to 3, 000x3, 000 pixel area. 根据检测器阵列的可获得性,在一些实施例中可以使每个阵列具有更多的像素数目。 According to the availability of the detector array, in some embodiments, may each array having a greater number of pixels. 在特定的实施例中,使用大小为1, 024x1, 024像素区域和2, 048x2, 048像素区域的阵列。 In a particular embodiment, the use of size 1, 024x1, 024 pixel area and array 2, 048x2, 048 pixel area. [0161] 本发明的各实施例提供了一种光学成像系统,它具有若干系统参数。 [0161] Embodiments of the present invention provides an optical imaging system having a plurality of system parameters. 例如,在本发明的各实施例中,可以使用大于35mm的工作距离,例如45. 92mm。 For example, in various embodiments of the present invention may be used more than 35mm working distance, e.g., 45. 92mm. 在另一个实施例中,提供了平均为13.44iim的均方根(RMS)光斑直径,其最大值为17.85ym。 In another embodiment, there is provided a root mean square average 13.44iim (RMS) spot diameter, a maximum value of 17.85ym. 此外,在本发明的各实施例中,实现约5%的照明变化。 In addition, in various embodiments of the present invention, to achieve approximately 5% of the illumination variation. 在一些实施例中,光学成像系统总长是542. lmm,其最大滤光片AOI为12. 56度,在滤光片处最大光束直径为76mm,失真< 0. 10% ,并且最大透镜直径为5. 512英寸。 In some embodiments, the total length of the optical imaging system is 542. lmm, a maximum filter AOI of 12.56 degrees, the maximum beam diameter at the filter of 76mm, distortion <0.10%, and the maximum diameter of the lens 5.512 inches.

[0162] 图1B是本发明一实施例的成像系统的简化图。 [0162] Figure 1B is a simplified diagram of an image forming system according to an embodiment of the present invention. 在一些实施例中,图1B所示的成像系统被用于给微流体器件成像,该微流体器件包括用于执行蛋白质结晶处理的器件。 In some embodiments, the imaging system shown in Figure 1B is used for forming a microfluidic device, the microfluidic device comprises a device for performing the processing of protein crystallization. 关于图1B所示成像系统及相关联的微流体器件的其它细节在下列文献中可以找到:共同待批且共同拥有的美国专利申请10/902, 494 (2004年7月28日提交),以及美国专利申请10/851, 777(2004年5月20日提交),这两个专利申请引用在此作为参考。 On the imaging system shown in FIG. 1B, and other details associated with the microfluidic devices can be found in the following documents: co-pending and commonly owned U.S. Patent Application 10/902, 494 (July 28, 2004 submitted), and U.S. Patent Application 10/851, 777 (May 20, 2004 submitted), these two patent applications are incorporated herein by reference. 特别是,在这两个专利申请中,可以找到关于本发明各实施例所提供的微流体器件及其和图1B所示成像系统结合使用的其它细节。 In particular, in these two patent applications may find additional details regarding microfluidic device provided by its embodiment and various embodiments of the present invention shown in Figure 1B in combination with the imaging system. 这些图只是示例,不应该过多限制权利要求书的范围。 These figures are only examples and should not be overly restrictive rights scope of the claims. 本领域的技术人员将会认识到许多变体、替换和修改。 Those skilled in the art will recognize many variations, alternatives, and modifications.

[0163] 在一个实施例中,成像系统(10)以下面的方式操作。 [0163] In one embodiment, the imaging system (10) in the following manner. 首先,微流体器件(30)牢固地设置在工作台(20)上。 First, microfluidic device (30) fixedly disposed on the table (20). 基于微流体器件(30)的固定特征,计算机(110)使x, y驱动(25)去移动工作台(20),以便使第一x, y位置中的微流体器件(30)与多个基准标记中的第一基准标记对准,其中基准标记被嵌入微流体器件内(离反应室中心点的z维度距离已知),基于该固定特征的推算定位通过成像设备(60)达到聚焦。 Based microfluidic device (30) is fixed characteristic, the computer (110) so that x, y drive (25) to the movable table (20), so that the first x, y position in the microfluidic device (30) and a plurality of reference mark of the first reference mark is aligned, wherein the reference mark is embedded within the microfluidic device (from the center of the reaction chamber from a known z-dimension), based on the projected location of the fixed feature reaches focused through the imaging device (60). 然后,系统用户使精确的 Then, the system user to make precise

29基准坐标与像系统重合。 29 reference coordinate system coincides with the image. 在用户的辅助下,两个或更多个其它的基准标记也同样地被绘制。 In aid of the user, two or more other reference marks are similarly drawn.

在其它实施例中,该过程是自动的,因为通过定位对称的XY基准物并除去任何非对称的成分,便可以精确地计算这些基准的形心。 In other embodiments, the process is automatic, and remove any non-symmetric because a component, it is possible to accurately calculate the centroid of these reference by positioning symmetric XY matrix. 成像设备(60)在计算机(110)的控制下接下来调节焦平面(100)的z维度定位以便聚焦到基准标记。 The image forming apparatus (60) in a computer (110) followed by adjusting the focal plane of the control (100) is positioned so that the z-dimension is focused onto the reference mark. 例如,一旦聚焦到第一基准标记,成像系统就获得了微流体器件(30)的第一x, y坐标图像,从而寻找成像设备(60)的视场之内其它的基准标记。 For example, when focused to a first reference mark, the imaging system to obtain a microfluidic device (30) in a first x, y coordinate of the image, in order to find the field of view of the image forming apparatus (60) of the other reference mark. 在较佳实施例中,该视场可以包括整个测定单元。 In the preferred embodiment, the field of view may include the entire measurement unit. 然后,计算机分析该第一x, y坐标图像以确定微流体器件是否具有偏斜和延伸,并且如果确定有偏斜或延伸,则变换该第一x,y图像以使微流体器件的图像和坐标图成为理想的坐标图。 The computer then analyzes the first x, y coordinate of the image to determine whether the microfluidic device has deflection and extension, and if it is determined there is deflection or extension, then converting the first x, y image so that the image of the microfluidic device and graph of the ideal graph. 随后在图像减影和掩模步骤中使用该理想的坐标图。 Then use the ideal graph in image subtraction and masking steps.

[0164] 在较佳实施例中,当微流体器件x, y坐标图像与理想的坐标图对准时,系统接下来通过比较x, y坐标图中的基准标记位置和理想的存储图像图的x, y坐标图中的基准标记位置,来确定微流体器件中是否存在延伸、扭曲、或各微流体层没有共同重合。 [0164] In the preferred embodiment, when the microfluidic device x, y coordinates of the image and the ideal graph of time, followed by comparing the system x, y coordinates of the reference marks in FIG ideal storage location and the image of x in Fig. , y coordinate position of the figure of the reference mark, to determine the presence or absence of the microfluidic device extends, distorted, or each of the microfluidic layer is not common overlap. 如果在实际基准位置和成像基准位置之间有差异,则执行矩阵变换(最好是仿射变换),以便将测定单元的成像形状变换成理想测定单元形状的虚拟形状。 If there are differences between the actual reference position and the imaging reference position, the matrix transformation is performed (preferably affine transformation), so that the shape of the imaging unit is converted into the desired shape of the virtual shape measurement unit will be determined. 通过使用矩阵变换(该矩阵变换是从测得的实际基准位置和所存储的理想基准位置之间的差异中计算出来的)将实际图像转换成已知且固定的理想图像,便可以使图像减影和其它图像分析成为可能。 By using the matrix transformation (transformation matrix is calculated from the difference between the actual reference position of the measured and the ideal reference position between the stored out of) the actual image into an ideal image is known and fixed, so that the image can be reduced video and other image analysis possible. [0165] 通过矩阵分析计算坐标图之间的差异,矩阵变换可以演化成将实际图像重新构造成理想的图像以便作进一步的图像处理。 [0165] Analysis calculated by the difference matrix between the graph, the actual matrix transformation may be developed as an ideal image reconstructed image for further image processing. 通过使成像的微流体器件与标准形状一致,图像减影和掩模便可能使测定单元反应室的可视区域最大化。 By microfluidic device consistent with the standard shape of the imaging, image subtraction and can mask the measurement unit maximizes the viewable area of the reaction chamber. 此外,如果在一系列基于时间的图像中时间零处的反应室内存在缺陷或碎片,则在接下来的图像中可以遮掉这种缺陷或碎片以避免施加自动分析时的伪信号。 In addition, if there is defect or debris in time series based on the image of the reaction chamber at time zero, then the next image can cover off the defect or debris in order to avoid a dummy signal is applied when the automatic analyzer. 除了遮掉反应室中含缺陷或碎片的区域,还可以从接下来的图像中减去反应室的壁,同样是为了在接下来的分析中不引起伪读数。 In addition to the reaction chamber cover off region containing defects or debris, can also subtracted from the walls of the reaction chamber in a next image, the same is read in order not to cause pseudo-following analysis. 基于控制层中发现的物体(比如控制线自身)的位置,也可以计算各层之间(比如控制层和通道层之间)的差异。 Based on an object found in the control layer (such as the control line itself) position, the difference may be calculated between the layers (such as control layer and the channel layer) of. 在另一个示例中,这种校正是基于控制层基准自身而确定的。 In another example, this correction is the control layer determined based on the reference itself. 对于某些实施例,这种额外的变换是重要的,因为控制层将蛋白质反应室与其它控制线隔离开。 For certain embodiments, such additional transformation is important because the reaction chamber control layer protein isolate with other control lines. [0166] 图2A-2C是示出了本发明一实施例的透镜组件的简化示意图。 [0166] Figures 2A-2C is a simplified schematic diagram illustrating a lens assembly according to an embodiment of the present invention. 如图2A-2C所示, 每张图都提供了微流体器件205。 Shown in FIG. 2A-2C, each figure provides microfluidic device 205. 尽管在图2A-2C中没有示出热控制器和其它系统元件,但是本领域的技术人员将会理解这两种图之间的关系。 Although not shown, the thermal controller and other system components in Figure 2A-2C, but those skilled in the art will understand the relationship between these two FIG. 因此,在合适的地方,标号都是取自图1A,以进一步澄清各图之间的关系。 Therefore, where appropriate, the label is taken from Figure 1A, in order to further clarify the relationship between the figures.

[0167] 图2A示出了第一透镜系统210,其中包括第一孔径211。 [0167] Figure 2A illustrates a first lens system 210, which includes a first aperture 211. 如图所示,第一孔径定位于单独的透镜元件之间,然而,这不是本发明所要求的。 As illustrated, the first aperture is positioned between the individual lens elements, however, this is not required by the present invention. 在其它光学设计中,选择第一孔径的位置、大小和其它特征,以实现预定的光学设计目标。 In other optical design, selecting a first aperture position, size, and other characteristics, to achieve a predetermined optical design goals. 图2A还示出了第二透镜系统212, 其中包括第二孔径213。 Figure 2A also shows a second lens system 212, which includes a second aperture 213. 如结合第一透镜系统210所讨论的那样,光学元件都根据预定的光学设计目标来排列。 As with the first lens system 210, as discussed, the optical elements are based on a predetermined optical design goals arranged.

[0168] 参照图1A和2A-2C,示出了和滤光器件有关的其它细节。 [0168] Referring to FIG. 1A and 2A-2C, illustrate further details relating to the device and a filter. 通常,透镜系统210、212 以及滤光设备213都设置在成像通路的一部分中。 Typically, the lens system 210, 212 and a filter device 213 are provided in a portion of the imaging path. 在一些实施例中,成像通路是一种发射通路,用于将来自微流体器件的一个或多个荧光发射信号传输到检测器。 In some embodiments, the imaging path is a transmission path for a microfluidic device from the one or more fluorescent emission signals to the detector. 如下文更全面的描述,滤光器件适用于使一个或多个荧光发射信号中的选定光谱带宽通过,并且还适用于将与一个或多个荧光发射信号相关联的一种或多种色差处理到确定的水平。 As more fully described, the filter device is adapted to cause one or more fluorescent emission signals through the selected spectral bandwidth, and also for the one or more fluorescent emission signals associated with one or more color to determine the level of treatment. 在一些实施例 In some embodiments

30中,处理一种或多种色差包括减小这种色差 30, the processing comprises reducing one or more of such color difference chrominance

[0169] 在特定的实施例中,如图2A所示,提供了第一光谱滤光片214和零屈光度光学元件261。 [0169] In a particular embodiment, shown in Figure 2A, a first spectral filter 214 and zero refractive optical element 261. 参照图1,当滤光器件213(在一些实施例中是发射滤光轮)处于第一工作位置时, 在微流体器件和检测器之间的光路中存在滤光片/零屈光度元件的组合。 When referring to FIG. 1, when the optical device 213 (in some embodiments is emission filter wheel) is in the first operative position, the combination filter / zero diopter element exists in the microfluidic device and the optical path between the detector . 针对大约位于光谱中心的波长的传输和聚焦,使图2A的光学元件最佳化。 For located about the center wavelength of the transmission spectrum and focusing, the optical element of Figure 2A optimized. 因此,在一个实施例中,光谱滤光片214以570纳米波长为中心,该波长和荧光团fluorophore Vic (从加州Foster市Applied Biosystems公司那儿可以买到)有关。 Thus, in one embodiment, the spectral filter 214 to 570 nm as the central wavelength and the fluorophore fluorophore Vic (Foster City, California, from Applied Biosystems. There can buy) related. 另外,该光谱滤光片的特征在于其30纳米的光谱带宽。 In addition, the spectral filter is characterized by its spectral bandwidth of 30 nm. 在一些实施例中,如下文更全面地描述那样,还提供了适用于校正色差的滤光片/零屈光度元件,但以其它波长(通常比570纳米要短或长)为中心。 In some embodiments, as more fully described above, also provides a suitable correction of chromatic aberration filter / zero diopter elements, but other wavelengths (usually shorter than 570 nanometer or long) as the center. [0170] 如结合图1中的光谱滤光片244所讨论的那样,滤光器件并不限于轮子这种形状。 [0170] The combination of Figure 1 as discussed spectral filter 244, the filter device is not limited to this shape the wheel. 例如,在本发明的一些实施例中,包括光谱滤光片、像差校正元件及其组合(它们落在滤光片支架中)。 For example, in some embodiments of the invention, including the spectral filter, the aberration correcting element, and combinations thereof (they come within filter holder). 此外,根据本发明的实施例,还包括与像差校正元件组合在一起的电光滤波器和被驱动器置入光路中的滤光片。 Further, according to embodiments of the present invention, further comprising a combination together with the aberration correcting element and the drive electro-optical filter placed in the optical path of the filter.

[0171] 图2B示出了第一和第二透镜系统,其中发射滤光轮位于第二工作位置。 [0171] Figure 2B illustrates the first and second lens systems, wherein the emission filter wheel is in the second operating position. 在图2B 所示的实施例中,光谱滤光片224以518纳米波长为中心(该波长与荧光团FamTM有关,可以从Applied Biosystems公司买到)并且其特征在于25纳米的光谱带宽。 In the embodiment shown in Figure 2B, the spectral filter 224 centered on 518 nm (the wavelength of the fluorophore FamTM related, commercially available from Applied Biosystems.) And is characterized by a spectral bandwidth of 25 nanometers. 通常,光谱滤光片224适用于透射和在"蓝色"波长处发光的荧光团有关的荧光信号,通常该荧光信号和光谱短波长部分附近的波长相关。 Typically, the spectral filter 224 and is suitable for transmission in the "blue" wavelength light-emitting fluorophore-related fluorescence signal, usually near the wavelength dependence of the fluorescence signal and the short-wavelength part of the spectrum.

[0172] 图2B示出了充当零屈光度双透镜的光学元件226。 [0172] Figure 2B shows a dual function as a zero diopter lens optical element 226. 在一些实施例中,滤光片/零屈光度双透镜是作为复合光学元件而提供的,而在其它实施例中,滤光片和零屈光度双透镜是彼此分开的。 In some embodiments, the filter / zero refractive power as a composite of two-lens optical element is provided, while in other embodiments, the double filter and zero diopter lens are separated from each other. 此外,在一些实施例中,使发射滤光轮旋转以调整滤光片/零屈光度光学元件的位置,从而从第一工作位置变到第二工作位置。 Further, in some embodiments, so that the filter wheel is rotated to adjust the transmit filter / zero diopter position of the optical element, thereby changed from the first operating position to the second work position. 图2B所示的零屈光度双透镜226被设计成用于校正由光学系统在"蓝色"波长处引入的色差。 Figure 2B double zero diopter lens 226 shown is designed for correction by the optical system in the "blue" wavelengths introduce chromatic aberration. 在特定的实施例中,选择零屈光度双透镜,以便校正与特定荧光团(比如FanT)的发光相关的波长处的色差。 In a particular embodiment, the select zero diopter double lens, in order to correct chromatic aberration with a particular fluorophore (such FanT) emitting at a wavelength associated. [0173] 在一些实施例中,零屈光度双透镜是由多种折射率数值不同的单独的光学材料制成的。 [0173] In some embodiments, the double-zero diopter value of the refractive index of the lens is a combination of different individual optical material. 仅作为示例,如图2B所示,平凹透镜耦合到凸平透镜。 Only as an example, shown in Figure 2B, is coupled to the plano-concave lens convex plano lens. 在特定的实施例中,零屈光度双透镜是一种由多个光学元件胶合在一起的夫琅和费消色差透镜。 In a particular embodiment, the zero-diopter lens is a double of a plurality of optical elements are cemented together Fraunhofer achromatic lens. 在其它实施例中,使用对本领域技术人员而言很明显的可替换的设计。 In other embodiments, the use of the skilled artisan obvious alternative design. 在一些实施例中,使用本说明书中所示的嵌入式双透镜,来减小蓝色和红色滤光波带中的轴向色差。 In some embodiments, the use of an embedded lenticular lens as shown in the present specification, the blue and red filters to reduce the axial chromatic band.

[0174] 图2C示出了第一和第二透镜系统,其中发射滤光轮处于第三工作位置。 [0174] Figure 2C shows the first and second lens systems, wherein the emission filter wheel is in the third operating position. 在图2C 所示的实施例中,光谱滤光片228以645纳米波长为中心(该波长和荧光团RoxTM有关,这种荧光团可以从Applied Biosciences公司买到)并且特征在于75纳米的光谱带宽。 In the embodiment shown in Figure 2C, the spectral filter 228 is centered on 645 nm (the wavelength and fluorophore RoxTM related, such fluorophores available from Applied Biosciences Company), and wherein the spectral bandwidth of 75 nm . 通常,光谱滤光片228适用于透射与在"红色"波长处发光的荧光团有关的荧光信号,该荧光信号通常与光谱的长波长部分附近的波长有关。 Typically, the spectral filter 228 and applied to the fluorescent signal transmission in the "red" wavelength light emitting fluorophore-related, the fluorescence signal is typically near the long-wavelength part of the wavelength spectrum related.

[0175] 如图2B所示,图2C示出了充当零屈光度双透镜的光学元件230。 [0175] shown in Figure 2B, Figure 2C shows a dual function as a zero diopter lens optical element 230. 在一些实施例中,滤光片/零屈光度双透镜是作为复合光学元件来提供的,而在其它实施例中,滤光片和零屈光度双透镜彼此分开。 In some embodiments, the filter / zero diopter lenticular lens is provided as a compound optical element, whereas in other embodiments, the double filter and zero diopter lens separated from each other. 此外,在一些实施例中,使发射滤光轮旋转,以便调整滤光片/ 零屈光度光学元件的位置,从而从第一或第二工作位置变为第三工作位置。 Further, in some embodiments, the emitter filter wheel is rotated in order to adjust the filter / zero diopter position of the optical element, whereby the work from the first or second position to the third operating position. 图2C所示的零屈光度双透镜230被设计成校正由光学系统在"红色"波长处所引入的色差。 Figure 2C double zero diopter lens 230 is designed as shown in the correction by the optical system in the "red" wavelength premises introduce chromatic aberration. 在特定的实 In a particular real

31施例中,选择零屈光度双透镜,以便校正与特定的荧光团(例如RoxTM)发光有关的波长处的色差。 31 embodiment, select double zero diopter lens to correct chromatic aberration at the wavelength of a specific fluorophore (eg RoxTM) luminescence related. 比较图2B和2C,图2C所示的零屈光度双透镜包括耦合到凹平透镜的平凸透镜。 Comparing Figures 2B and 2C, FIG. 2C zero diopter lens shown comprises a double concave plano lens coupled to a plano-convex lens. [0176] 如结合图2A、2B和2C所讨论的那样,与各个滤光片有关的校正光学元件216、226 和230被设计成,它们帮助校正在上述各个滤光片所透射的波长处的色差。 [0176] The combination of Figures 2A, 2B and 2C as discussed, correction optical element associated with the respective filters 216, 226 and 230 are designed so that they help in the above-mentioned respective correction filters at the wavelength of the transmitted color. 这种设计允许在不同的滤光片(透射不同的波长区域)上使透镜系统的光斑大小、模糊和其它光学特性更均匀和更一致。 This design allows different filters (transmitted wavelengths different regions) of the lens system of the spot size, fuzzy, and other optical properties of more uniform and more consistent. 这些益处可用于许多应用,例如,微流体器件中的反应室紧密填充。 These benefits can be used in many applications, e.g., microfluidic device tightly packed in the reaction chamber. 另外,针对来自不同反应室的光信号之间的可允许串扰而定的设计目标通常对图像平面上可允许的最大光斑或模糊的尺寸给出限制。 Further, for the allowable crosstalk given design objective optical signals from different reaction chambers between usually spot on the image plane may be the maximum allowable limit or blurred given size. 像差校正光学元件减小一个波长极限(例如,蓝色波长区域中的滤光透射)处的模糊或光斑大小,并且还减小不同波长区域处的模糊或光斑大小(例如,对于其通带位于红色波长区域中的滤光片)。 An aberration correction optical element to reduce the limit wavelength (e.g., blue wavelength region optical transmission) or blur spot size at, and also to reduce the blur or spot size at a different wavelength region (e.g., for its passband located in the red wavelength region filter). 该益处可用于下列情形:整个测量是一种比例度量,这取决于在红色和蓝色波长区域中检测到的单独信号,由此提高了整个试验的灵敏度。 This benefit can be used in the following situations: the whole measurement is a measure of the ratio, depending on the individual signals detected in the red and blue wavelength regions, thereby improving the sensitivity of the entire trial. 上文提供的讨论涉及特定的实施例,但是本领域的技术人员应该理解, 有许多校正零屈光度光学元件的变体,它们以相似的方式和具有不同波长通带的滤光片结合起来被置于光学系统中,这些都被包括在本发明的范围中。 Discussion provided above relates to specific embodiments, those skilled in the art will appreciate, there are many variations of the zero-diopter correction optical element, which in a similar manner and pass-band filters having different wavelengths are set to combine in the optical system, which are included in the scope of the present invention.

[0177] 图3是本发明一实施例的微流体器件中存在的许多反应室中发生的反应所产生的以第一波长为中心的荧光发射的照片。 [0177] FIG. 3 is a reaction of many reaction chambers of the microfluidic device of one embodiment of the invention in the presence of the generated first fluorescence emission wavelength of the center photograph. 如图3所示,许多反应室产生第一波长〃 波长1〃 的发光。 As shown in Figure 3, many of the reaction chamber to produce a first wavelength 1〃 〃 emitting wavelength. 在图3所示的本发明的实施例中,10x16的反应室阵列被成像。 In the embodiment shown in Figure 3 of the present invention, the reaction chamber 10x16 array is imaged. 在一些实施例中, 第一波长与上述荧光团相关联。 In some embodiments, the first wavelength is associated with said fluorophore. 对于本领域的技术人员而言,很明显,第一波长的发光强度是反应室中发生的化学过程的函数。 For the skilled person that, obviously, the emission intensity of the first wavelength is a function of the chemical processes occurring in the reaction chamber. 如图所示,用根据本发明一实施例提供的光学成像系统给10x16反应室的二维阵列成像。 As shown, to a two-dimensional array of reaction chambers according to the imaging 10x16 optical imaging system provided in an embodiment of the present invention. 如上所述,在本发明的一些实施例中,反应室处于流体隔离。 As described above, in some embodiments of the invention, the reaction chamber is fluidly isolated. 此外,根据可替代的实施例,反应室的特征在于,体积在纳升范围,和/或反应室密度的量级为每平方厘米有几百个反应室。 Further, according to an alternative embodiment, the reaction chamber is characterized in that the volume in the nanoliter range, and / or density of the order of the reaction chamber for a few hundred per square centimeter of the reaction chamber.

[0178] 图4是本发明一实施例的微流体器件中存在的许多反应室中发生的反应所产生的以第二波长为中心的荧光发射的照片。 [0178] FIG. 4 is a reaction of many reaction chambers of the microfluidic device according to the present in the generated second fluorescence emission wavelength of the center photograph an embodiment of the present invention. 如图4所示,许多反应室产生第二波长〃 波长2〃 处的发光比较图3和4, 一些反应室产生很少的光或不产生光,而其它反应室产生第一波长的光、第二波长的光、或第一和第二波长的光。 4, as shown in many of the reaction chamber to produce a second wavelength emitting comparing Figures 2 〃 〃 wavelength at 3 and 4, some of the reaction chamber to produce a first wavelength of light or no light, while the other reaction chamber to produce very small, a second wavelength, or the first and second wavelengths. 当然,荧光活性的收集和分析可以对反应室中发生的化学反应的本质作出理解。 Of course, the collection and analysis of the nature of the fluorescent activity may be a chemical reaction occurring in the reaction chamber to be understood.

[0179] 图5-7是用本发明一实施例产生的选定波长的光点图。 [0179] Figure 5-7 is a diagram of a selected wavelength to produce a light spot diagram one embodiment of the present invention. 参照这些图的说明,图中所示的波长范围通常分成三个波带:绿色波长,蓝色波长和红色波长。 Described with reference to these figures, the wavelength range is generally shown divided into three wavelength bands: green wavelength, blue wavelength and a red wavelength. 图5示出了525、550 和575nm的波长,它们通常与光谱的绿色区域相关联。 Figure 5 shows a 525,550 and 575nm wavelengths, they are usually associated with the green region of the spectrum is associated. 图6示出了486、501和516nm的波长,它们通常与光谱的蓝色区域相关联。 Figure 6 shows a 486,501 and 516nm wavelength, and they are usually the blue region of the spectrum is associated. 图7示出了616、636和656nm的波长,它们通常与光谱的红色区域相关联。 Figure 7 shows 616,636 and 656nm wavelengths, they are usually associated with the red region of the spectrum. 图5是针对图2A所示的透镜系统而计算的,图6是针对图2B所示的透镜系统而计算的,而图7是针对图2C所示的透镜系统而计算的。 Figure 5 is for the lens system shown in Figure 2A and calculated, Fig. 6 for the lens system shown in Figure 2B calculated, and Fig. 7 is for the lens system shown in Figure 2C calculated. [0180] 图8是用本发明一实施例产生的照度图,该图示出了作为位置的函数的相对均匀性。 [0180] Figure 8 is an embodiment of the present invention is produced by illumination diagram, which shows the relative position as a function of the uniformity. 在图8中,针对图2A所示的光学系统,绘出了作为Y段(毫米)的函数的相对照度。 In Figure 8, for the optical system shown in FIG. 2A, plotted as a Y section (mm) as a function of relative illuminance. 在0. 550 ii m的波长处,超过距离21. 4mm的相对照度均匀性大于90% 。 At a wavelength of 0. 550 ii m, exceeds the distance 21. 4mm illuminance uniformity of greater than 90%.

[0181 ] 图9-11是本发明若干实施例的正方块块内能量图。 [0181] Figures 9-11 are within the present invention, a number of square Kuaikuai energy diagram of an embodiment. 在一些光学系统中,光学系统性能的测量是正方块块内能量百分比,这是指定中心区域中的总能量的百分比。 In some optical systems, the optical measuring system performance is within the square Kuaikuai energy percentage, which is the central area of a specified percentage of the total energy. 参照图9, 通过使用图2A所示的透镜系统,所包括的能量的百分率被绘制成各位置的形心的半宽(以微米为单位)的函数。 Referring to Figure 9, the lens system shown in FIG. 2A by using, included the percentage of energy is plotted as a centroid position of the respective half-width (in microns) of the function. 作为一个示例,对于离中心14. 8mm的位置,约50%的能量被包括在离形心约7. 5 m处,而对于离中心21. 4mm的位置,约90 %的能量被包括在离形心相同距离处。 As one example, for the position from the center 14. 8mm, approximately 50% of the energy from being included in the centroid at about 7. 5 m, and for the position from the center of 21. 4mm, about 90% of the energy is included in the off centroid same distance. 在图9-11中,包括衍射,通常用快速傅立叶变换(FFT)算法来执行上述计算。 In Figures 9-11, including diffraction, usually using Fast Fourier Transform (FFT) algorithm to perform the above calculations. 图IO 和11分别是图2B和2C所示透镜系统的正方块块内能量图。 Figure IO and 11, respectively, Fig. 2B and 2C, the lens system within the square tranches Energy. 在这些图中,如图9所示,包括衍射。 In these figures, as shown in Figure 9, including diffraction.

[0182] 图12是示出了本发明一实施例所提供的光学系统的场曲和畸变的图。 [0182] FIG. 12 is a diagram illustrating an embodiment of the present invention, curvature of field and distortion in Example provided optical system. 针对波长0. 525iim、0. 550iim和0. 575 ym示出了场曲,它们都标记在图中。 For wavelength 0. 525iim, 0. 550iim and 0. 575 ym shows field curvature, they are marked in the figure. 对于不同的所示波长,畸变不相同,其中各波长之间可忽略的差异在0. 05%的水平。 For different wavelengths, the distortion is not the same, wherein the respective wavelengths between negligible differences in the level of 0.05%.

[0183] 图13是本发明一实施例的系统所产生的"双波长-焦点"的图。 [0183] FIG. 13 is generated by the system according to an embodiment of the present invention "dual wavelength - focus" FIG. 在该图中,色焦点移动是波长(以微米为单位)的函数。 In the figure, the focus movement is color wavelength (in microns) of the function. 图13所示的波长范围覆盖了从480纳米到650纳米的波长范围。 Wavelength range shown in FIG. 13 covers from 480 nm to 650 nm wavelength range. 如图所示,最大焦点移动范围是214. 3702 ii m,而衍射限制范围是4. 243 ym。 As shown, the biggest focus of the range of movement is 214. 3702 ii m, while the diffraction limits is 4. 243 ym. 参照图13,超过214iim长的颜色焦点跨越所示范围。 Referring to FIG. 13, the color of the focus over 214iim long spans the range indicated. 因此,系统性能的分析通常包括要考虑士100iim的散焦处的光斑大小。 Therefore, the analysis of system performance usually includes consideration disabilities 100iim spot size at the defocus. 通常,散焦光斑的大小将超过焦点中的光斑的大小。 Typically, the defocused spot size exceeds the size of the focus spot. 根据本发明各实施例,通过使用上述嵌入式双透镜,来校正轴向色差。 According to various embodiments of the present invention, by using the embedded double lens, the axial chromatic aberration is corrected. 在可选的实施例中,使用特殊的玻璃类型来实现复消色差性能(通常在比其它玻璃类型要贵的情况下获得)。 In an alternative embodiment, the use of a special type of glass to achieve apochromatic performance (normally under expensive than other types of glass obtained in a case). [0184] 图14-16是用本发明一实施例产生的针对选定波长的光点图。 [0184] Figures 14-16 is a dot plot for the wavelength of light generated by a selected embodiment of the present invention in one embodiment. 如图14的说明所示,当上述系统在最佳焦点位置时,提供了光谱绿色区域中若干个波长(525、550和575nm) 的光点图。 Description As shown in FIG. 14, when said system is in the best focus position, there is provided a plurality of spectral wavelength in the green region (525,550 and 575nm) light spots in FIG. 为了产生图14-16所示的光点图,使用了图2A所示的透镜系统。 To generate the spot diagram shown in Fig. 14-16, the use of the lens system shown in Figure 2A. 图15和16分别是在+100iim的散焦和-100iim的散焦处计算出的光点图。 Figures 15 and 16 are in the + defocus 100iim and -100iim defocus calculated at the spot FIG. 在图15和16中,考虑光谱绿色区域中相同的波长。 In Figures 15 and 16, in consideration of the green spectral region of the same wavelength.

[0185] 图17-19是用本发明一实施例产生的针对选定波长的光点图。 [0185] Figures 17-19 is a dot plot for the wavelength of light generated by a selected embodiment of the present invention in one embodiment. 如图17的说明所示,当系统位于最佳焦点位置时,提供了光谱蓝色区域中若干个波长(486、501和516nm)的光点图。 Description As shown in Figure 17, when the system is located at the best focus position, there is provided a plurality of spectral wavelength in the blue region (486,501 and 516nm) light spots in FIG. 为了产生图17-19所示的光点图,使用了图2B所示的透镜系统。 To generate the spot diagram shown in Fig. 17-19, the use of the lens system shown in Figure 2B. 图18和19分别是在+100iim的散焦和-100iim的散焦处计算的光点图。 Figures 18 and 19 respectively in FIG. + Spot defocus 100iim and -100iim defocus calculated at. 在图18和19中,考虑光谱的蓝色区域中相同的波长。 In Figure 18 and 19, consider the blue region of the spectrum the same wavelength. 如图19所示,场4包括501nm处的光线,延伸到100iim盒子以外。 As shown in Figure 19, the light field 4 comprises at 501nm, extend beyond 100iim box. [0186] 图20-22是用本发明一实施例产生的针对选定波长的光点图。 [0186] Figure 20-22 is a wavelength of a light spot diagram for a selected embodiment of the present invention to produce an embodiment. 如图20的说明所示,在系统位于最佳焦点位置时,提供了光谱的红色区域中若干个波长(616、636和656nm) 的光点图。 Description of FIG. 20, in the best focus position when the system is located, is provided in the red region of the spectrum a plurality of wavelengths (616,636 and 656nm) light spots in FIG. 为了产生图20-22所示的光点图,使用了图2C所示的透镜系统。 To produce the light spot shown in Fig. 20-22, the use of the lens system shown in Figure 2C. 图21和22分别是在+100 ii m散焦和-100 ii m散焦处计算出的。 Figures 21 and 22, respectively, in the +100 ii m defocus and defocus at -100 ii m calculated. 在图21和22中,考虑光谱的红色区域中相同的波长。 In Figures 21 and 22, in consideration of the red region of the spectrum in the same wavelength.

[0187] 在本发明的可选实施例中,提供了另一个l : l光学中继成像系统,其中包括对图2A-2C所示光学元件的修改。 [0187] In an alternative embodiment of the present invention, there is provided another l: l relay optical imaging system, including modification of the optical element shown in Figures 2A-2C. 尽管保留了一般的光学系统,但是特定元件(包括滤光片/零屈光度双透镜的组合)的特性被修改了。 While retaining the general optical system, a particular element (including a combination filter / double zero diopter lens) characteristic is modified. 在这种可选实施例中,提供了大于35mm(例如,46.12mm)的工作距离。 In this alternative embodiment, there is provided greater than 35mm (e.g., 46.12mm) working distance. 此外,提供了平均为11. 28 ym的RMS光斑直径,其中最大值是14. 73iim,光学成像系统的总长是542. 2mm,其中最大滤光片AOI为12. 59度,且滤光片处的最大光束直径为76mm。 In addition, there is provided an average of 11. 28 ym of RMS spot diameter, wherein the maximum value is 14. 73iim, the total length of the optical imaging system is 542. 2mm, wherein the maximum filter AOI of 12.59 degrees, and filters at The maximum beam diameter of 76mm.

[0188] 图23-25是用本发明一可选实施例产生的针对选定波长的光点图。 [0188] Figures 23-25 is optional with the present invention is a wavelength of light generated dot pattern for the selected example embodiment. 参照这些图的说明,图中所示波长范围通常分成三个波带:绿色波长,蓝色波长,和红色波长。 Referring to these figures, in this figure the wavelength range is usually divided into three wavelength bands shown: green wavelength, blue wavelength and a red wavelength. 图23示出了波长525、550和575nm,它们通常和光谱的绿色区域相关。 Figure 23 shows a wavelength 525,550 and 575nm, and they are generally related to the green regions of the spectrum. 图24示出了波长486、501和516nm,它们通常和光谱的蓝色区域相关。 Figure 24 shows a wavelength 486,501 and 516nm, and they are usually related to the blue region of the spectrum. 图25示出了波长621、636和651nm,它们通常和光谱的红色区域相关。 Figure 25 shows a wavelength 621,636 and 651nm, and red regions of the spectrum are typically related. 图23是基于图2A针对透镜系统而计算的,图24是基于图2B针对透镜系统而计算的,图25是基于图2C针对透镜系统而计算的。 Figure 23 is based on FIG. 2A calculated for a lens system, Fig. 24 is based on FIG. 2B calculated for the lens system, FIG. 25 is a lens system for Figure 2C calculated based.

[0189] 图26-28是根据本发明的若干实施例的正方块块内能量图。 [0189] FIG. 26-28 square lumps within the energy diagram in accordance with several embodiments of the present invention. 参照图26,通过使用图2A所示的透镜系统,针对各个位置,绘出了所包括的能量的百分率,作为形心半宽(以微米为单位)的函数。 Referring to FIG. 26, by using the lens system shown in Figure 2A, for each position, the percentage of energy plotted included, as a function of the centroid of the half width (in microns) of. 作为一个示例,对于离中心14. 8mm的位置,约80%的能量被包括在离形心约7. 5 ii m处,而对于离中心21. 4mm的位置,约90%的能量被包括在离形心相同距离处。 As one example, for the position from the center 14. 8mm, approximately 80% of the energy is included in the centroid from about 7. 5 ii m place, and for the position from the center of 21. 4mm, about 90% of the energy is included in the at the same distance from the centroid. 在图26-28中,包括衍射,通常用快速傅里叶变换(FFT)算法来执行计算过程。 In Fig. 26-28, including diffraction, usually Fast Fourier Transform (FFT) algorithm to perform the calculation process. 图27 和28分别是图2B和2C所示透镜系统的正方块块内能量图。 27 and 28 are within the square as shown in Figures 2B and 2C Kuaikuai lens system energy diagram. 在这些图中,像在图26中那样,包括衍射。 In these figures, like that, in Fig. 26 comprises a diffraction.

[0190] 还应该理解,上文所描述的示例和实施例只用于解释说明,并且各种修改和变化对于本领域的技术人员而言都是很明显的并且应该被包括在本申请的精神和权限以及所附权利要求书的范围之内。 [0190] It should also be understood that the above described exemplary embodiments only serve to explain and illustrate, and that various modifications and variations to persons skilled in the art are very significant and should be included in the spirit of the present application and competence as well as within the scope of the appended claims.

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Classifications
International ClassificationG01N27/447, G01N21/64, B01L3/00, B01L7/00, G02B21/02, G02B21/16
Cooperative ClassificationG01N21/64, C12Q1/686, G02B21/36, G01N21/75, G01N2201/061, G01N21/8483, G02B21/16, B01J2219/00704, B01L3/5027, B01J2219/00576, G01N27/44721, B01L7/52, G01N21/6452, G01N2021/6421, G01N21/6456, G01N21/6486
European ClassificationG01N21/64P2, G01N27/447B3A, G01N21/64P4, G02B21/16
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