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Publication numberCN101711257 A
Publication typeApplication
Application numberCN 200880009136
PCT numberPCT/US2008/000860
Publication date19 May 2010
Filing date22 Jan 2008
Priority date22 Jan 2007
Also published asCA2677833A1, CA2677833C, EP2109627A1, EP2109627A4, US8252581, US9132427, US20080176290, US20130053272, US20160186235, WO2008091626A1
Publication number200880009136.1, CN 101711257 A, CN 101711257A, CN 200880009136, CN-A-101711257, CN101711257 A, CN101711257A, CN200880009136, CN200880009136.1, PCT/2008/860, PCT/US/2008/000860, PCT/US/2008/00860, PCT/US/8/000860, PCT/US/8/00860, PCT/US2008/000860, PCT/US2008/00860, PCT/US2008000860, PCT/US200800860, PCT/US8/000860, PCT/US8/00860, PCT/US8000860, PCT/US800860
InventorsA希夫吉, A赫达, V约瑟夫
Applicant瓦弗根公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Apparatus for high throughput chemical reactions
CN 101711257 A
Abstract
Apparatus, systems, chips, and methods of performing a large number of simultaneous chemical reactions are provided herein. The chips of the invention comprise addressable units that can be addressed according to the temperature of the reaction to be run. The subject apparatus, systems, and chips are particularly suited for performing polymerase chain reactions on thousands of nucleic acid sequences, up to and including sequences of an entire genome of an organism of interest.
Claims(57)  translated from Chinese
  1. 一种包含至少一个设置为与芯片形成热接触的加热元件的装置,所述芯片包括片基和纳米孔阵列,其中,该至少一个加热元件设置为相对于芯片移动。 A set of at least one heating element in thermal contact with the chip forming means comprises a base sheet and a chip comprises a nanopore array, wherein the at least one heating element is arranged to move with respect to the chip.
  2. 2. 如权利要求1所述的装置,其中,所述至少一个加热元件从芯片的上面和下面与芯片形成热接触,而且其中,从芯片下面热接触的加热元件的温度设定得比从芯片上面热接触的加热元件的温度低。 2. The apparatus according to claim 1, wherein said at least one heating element in thermal contact with the chip is formed above and below the chip, and wherein the temperature of the heating element in thermal contact with the chip below the set lower than from the chip low temperature heating element of the thermal contact of the above.
  3. 3. 如权利要求1所述的装置,其中,所述芯片包括上表面和下表面,并且其中,第一系列的纳米孔在沿上表面上的一个方向排列,第二系列的纳米孔垂直于第一系列的纳米孔定向。 3. The apparatus according to claim 1, wherein the chip includes an upper surface and a lower surface, and wherein the nano-holes of the first series arrangement of a direction along the surface of the nano-second series of holes perpendicular to the The first series of nanopores orientation.
  4. 4. 如权利要求1所述的装置,其中,所述加热元件位于包括纳米孔阵列的静止芯片的上面或下面。 4. The apparatus according to claim 1, wherein said heating element is located above or below the stationary chip comprises nanopore arrays.
  5. 5. 如权利要求1所述的装置,其中,所述加热元件能够加热和冷却。 5. The apparatus according to claim 1, wherein said heating element capable of heating and cooling.
  6. 6. 如权利要求1所述的装置,包括对应于多个温度带的多个加热元件。 6. The apparatus as claimed in claim 1, comprising a plurality of heating elements corresponding to a plurality of temperature zones.
  7. 7. 如权利要求6所述的装置,其中,所述多个温度带在大约52t:-大约951:的范围。 7. The apparatus according to claim 6, wherein said plurality of temperature zone of about 52t: - About 951: range.
  8. 8. 如权利要求7所述的装置,其中,所述多个温度带提供温度梯度。 8. The apparatus of claim 7, wherein said plurality of temperature zones provides a temperature gradient.
  9. 9. 如权利要求6所述的装置,其中,至少一个温度带设定为处于大约52C -大约65C 范围的温度,而且至少一个另外的温度带设定为处于大约9(TC _大约951:范围的温度。 9. The apparatus according to claim 6, wherein the at least one temperature zone is set at about 52 C - about 65 C temperature range, and at least one further temperature zone set at approximately 9 (TC _ About 951: range of temperatures.
  10. 10. 如权利要求9所述的装置,进一步包括至少一个设定为处于大约68C -大约72C 范围的温度的另外的温度带。 10. The apparatus of claim 9, further comprising at least one set at about 68 C - about 72 C temperature range an additional temperature zone.
  11. 11. 如权利要求1所述的装置,其中,所述至少一个加热元件设置为提供包括温度随时间变化的尖峰波形的输出。 11. The apparatus of claim 1, wherein said at least one heating element arranged to provide a time varying temperature comprising spike output waveforms.
  12. 12. 如权利要求l所述的装置,其中,所述阵列中的单个纳米孔具有长度为大约250 ii m、宽度为大约250 ym和深度为大约525 ym的尺寸或者更小。 12. The apparatus as claimed in claim l, wherein said single nanopore array having a length of about 250 ii m, a width and a depth of about 250 ym to about 525 ym in size or smaller.
  13. 13. 如权利要求1所述的装置,其中,所述芯片可操作地与检测光学信号的光学系统偶联。 13. The apparatus of claim 1, wherein said chip detection system operatively associated with the optical coupling of the optical signal.
  14. 14. 如权利要求13所述的装置,其中,所述光学系统包括多个光学检测器。 14. The apparatus of claim 13, wherein said optical system comprises a plurality of optical detectors.
  15. 15. 如权利要求1所述的装置,其中,所述纳米孔的数目大于大约30,000。 15. The apparatus of claim 1, wherein the number of the nano-pores greater than about 30,000.
  16. 16. 如权利要求1所述的装置,其中,所述纳米孔设置为容纳大约100nl的体积。 16. The apparatus of claim 1, wherein said nanopore is configured to receive a volume of approximately 100nl.
  17. 17. —种进行化学反应的方法,包括: 提供设置用于容纳反应样品的芯片;提供至少一个可相对于芯片定位的加热元件,以在该至少一个加热元件和芯片之间提供热接触;禾口通过改变芯片温度在反应样品中进行化学反应,其中,通过相对于芯片移动该至少一个加热元件以使得加热元件与芯片形成热接触,从而实现所述温度改变。 17. - chemically reactive species, comprising: providing a sample of the reaction provided for receiving the chip; providing at least one heating element chips positioned relative to the at least one heating element providing thermal contact between the chip and; Wo port by varying the chip temperature in the reaction sample in a chemical reaction, wherein, by moving the chip with respect to the at least one heating element such that the heating element is in thermal contact with the chip is formed, in order to achieve the temperature changes.
  18. 18. 如权利要求17所述的方法,其中,所述化学反应是核酸扩增反应。 18. The method of claim 17, wherein said chemical reaction is a nucleic acid amplification reaction.
  19. 19. 如权利要求17所述的方法,其中,所述至少一个加热元件的移动受到可操作地与芯片连接的温度传感器产生的信号控制。 19. The method of claim 17, wherein said at least one heating element by moving the control signal and a temperature sensor operatively connected to the chip produced.
  20. 20. 如权利要求18所述的方法,其中,所述反应样品能够产生光学信号,并且其中,芯片可操作地与设置为检测反应样品发射的光学信号的光学系统偶联。 20. The method of claim 18, wherein said reaction sample to produce an optical signal, and wherein, the chip is operably coupled to the optical system of the optical signal and configured to detect a reaction samples transmitted.
  21. 21. 如权利要求20所述的方法,其中,所述光学信号与化学反应产物的量成比例。 21. The method of claim 20, wherein the amount of chemical reaction with the optical signal is proportional to the product.
  22. 22. 如权利要求21所述的方法,其中,所述改变温度通过移动多个加热元件实现,各加热元件设定在不同的温度。 22. The method of claim 21, wherein said temperature change is achieved by moving a plurality of heating elements, each heating element is set at different temperatures.
  23. 23. 如权利要求21所述的方法,其中,至少一个加热元件设定为处于大约52C -大约65t:范围的温度,至少一个另外的加热元件设定为处于大约9(rc -大约951:范围的温度。 23. The method of claim 21, wherein the at least one heating element is set to be in about 52 C - about 65t: temperature range, at least one additional heating element is set to be in about 9 (rc - about 951 : range of temperatures.
  24. 24. —种进行反应的芯片,包括可编址的单元的阵列,各单元设置为进行化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中的单个单元的尺寸设计为容纳小于大约1 P 1的化学反应混合物。 24. - kind of a reaction of the chip, including an array of addressable units, each unit is set to undergo a chemical reaction, which can be programmed array settings addressing unit corresponding to a predetermined temperature zone, and wherein the array dimensioned to receive a single cell is less than about 1 P 1 chemical reaction mixture.
  25. 25. 如权利要求24所述的芯片,包括多个阵列。 25. The chip according to claim 24, comprising a plurality of arrays.
  26. 26. 如权利要求24所述的芯片,包括各对应于不同的温度带的多个阵列。 26. The chip according to claim 24, comprising an array of a plurality of different temperatures for each zone corresponds to.
  27. 27. 如权利要求24所述的芯片,包括多个阵列,其中,至少一个阵列设定为处于用于支持核酸扩增反应的退火温度,和至少一个另外的阵列设定为处于用于支持核酸扩增反应的变性温度。 27. The chip according to claim 24, comprising a plurality of arrays, wherein the at least one array is set to be in the nucleic acid amplification reaction for supporting the annealing temperature, and at least one other set in the array is used to support nucleic acid denatured amplification reaction temperature.
  28. 28. 如权利要求24所述的芯片,其中,对所述带编址以表明预定的温度带。 28. The chip of claim 24, wherein said belt addressed to indicate the predetermined temperature zone.
  29. 29. 如权利要求24所述的芯片,其中,所述可编址单元的阵列设置为对应于6个或更多个预定的温度带。 29. The chip of claim 24, wherein said programmable array settings addressing unit corresponding to six or more predetermined temperature band.
  30. 30. 如权利要求24所述的芯片,其中,所述芯片与加热元件形成热接触。 30. The chip according to claim 24, wherein said chip is formed in thermal contact with the heating element.
  31. 31. —种用于进行需要在至少两个温度水平循环的化学反应的装置,包括:(a) 用于进行反应的包含可编址单元的阵列的芯片,各单元设置为用于化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中的单个单元的尺寸设计为容纳小于大约1 P 1的化学反应混合物;禾口(b) 与芯片形成热接触的加热元件。 31. - the need for the kind of chemical reactions in the device at least two cycles of temperature levels, comprising: (a) for the reaction chip comprises an array of addressable units, each unit arranged for the chemical reaction, wherein an array of addressable units set to correspond to a predetermined temperature zone, and wherein the array is sized to accommodate a single cell is less than about 1 P Chemistry reaction mixture; Hekou (b) and chip form heating element thermal contact.
  32. 32. 如权利要求24或权利要求31所述的装置,其中,所述可编址单元的阵列大于大约30, 000。 31 or apparatus according to claim 24, wherein said programmable array addressing unit is greater than about 30, 000 as claimed in claim 32..
  33. 33. 如权利要求31所述的装置,进一步包括:(c)可操作地与芯片偶联的光学系统,其中,所述光学系统检测来自编址的可热控制的单元的光学信号。 33. The apparatus according to claim 31, further comprising: (c) is operatively coupled to the chip with an optical system, wherein the optical signal from the optical detection system can be addressed by thermal control units.
  34. 34. 如权利要求33所述的装置,其中,所述光学系统包括多个光学检测器。 34. The apparatus of claim 33, wherein said optical system comprises a plurality of optical detectors.
  35. 35. 如权利要求31所述的装置,进一步包括多个加热元件。 35. The apparatus according to claim 31, further comprising a plurality of heating elements.
  36. 36. 如权利要求35所述的装置,其中,所述多个加热元件包括6个或更多个加热元件。 36. The apparatus according to claim 35, wherein said heating element comprises a plurality of six or more heating elements.
  37. 37. 如权利要求24或权利要求31所述的装置,其中,所述阵列中的单个单元包括用于容纳和限制样品的纳米孔,所述孔在填充样品后被密封。 31 or apparatus according to claim 24, wherein said array comprises a single unit for receiving and limitations nanopores samples, after filling the sample in the aperture 37. The seal of claim.
  38. 38. 如权利要求24或权利要求31所述的装置,其中,所述化学反应是核酸扩增反应。 31 or apparatus according to claim 24, wherein said chemical reaction is a nucleic acid amplification reaction as claimed in claim 38.
  39. 39. 如权利要求38所述的装置,其中,单元的预定温度设置为从化学反应产生至少90%的同质产物。 39. The apparatus according to claim 38, wherein the predetermined temperature setting unit is generated from the chemical reaction product of at least 90% homogeneity.
  40. 40. —种进行涉及多个反应样品并需要在至少两个温度水平循环的反应的方法,包括:(a) 提供进行反应的芯片,包括可编址单元的阵列,各个单元设置为用于化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中的单个单元的尺寸设计为容纳小于大约1P 1的化学反应混合物;(b) 根据预定温度的设定,将多个反应样品放置于芯片的单元中;禾口(c)控制加热元件以实现至少两个温度水平的循环。 40. - involving more than one kind of reaction sample in the process of the reaction and requires at least two cycles of temperature levels, comprising: (a) providing a reaction chip comprising an array of addressable units, each unit provided for Chemicals The reaction, wherein the array of addressable units corresponding to a predetermined set temperature zone, and wherein the size of the array is designed to accommodate a single cell of less than about 1P 1 Chemical reaction mixture; (b) according to a predetermined temperature, setting a plurality of reaction sample is placed on the chip in the unit; Hekou (c) controls the heating element in order to achieve at least two temperature levels of circulating.
  41. 41. 如权利要求40所述的方法,其中,所述芯片的阵列中的单个单元包括用于容纳并限制样品的纳米孔,所述孔在填充样品后被密封。 41. The method of claim 40, wherein said array of a single chip unit comprises means for receiving a sample and to limit the nanopore, the sample aperture is sealed after filling.
  42. 42. 如权利要求40所述的方法,其中,所述化学反应是核酸扩增反应。 42. The method of claim 40, wherein said chemical reaction is a nucleic acid amplification reaction.
  43. 43. 如权利要求42所述的方法,其中,单元的预定温度设置为从化学反应产生至少90%的同质产物。 43. The method of claim 42, wherein the predetermined temperature setting unit is generated from the chemical reaction product of at least 90% homogeneity.
  44. 44. 一种用于进行涉及至少两个温度水平的循环的反应的装置,包括:(a) 设置为容纳芯片的主体,所述芯片包括多个用于容纳化学反应物的纳米孔;禾口(b) 提供第一温度的第一加热器和提供第二温度的第二加热器;其中,第一加热器和第二加热器设置为可在第一和第二方位之间移动,且其中,第一方位使加热器与样品座形成热接触,和第二方位不使加热器与样品座形成热接触。 44. An apparatus for performing at least two temperature levels involves circulating reaction, comprising: (a) to accommodate the body of the chip, the chip includes a plurality of nano-holes for receiving chemical reactant; Wo port (b) providing a first temperature of a first heater and a second temperature of a second heater; wherein the first and second heaters to be disposed between the first and the second position of the mobile, and wherein The first orientation of the heater in thermal contact with the sample holder is formed, and a second orientation does not form the heater in thermal contact with the sample holder.
  45. 45. 如权利要求44所述的装置,其中,所述多个纳米孔是可编址的,其中,纳米孔按照预定的温度设置排列,使得至少一个纳米孔编址为表明用于在所述纳米孔内进行化学反应的预定温度。 45. The apparatus of claim 44, wherein said plurality of nanopores is addressable, wherein nanopores are arranged in accordance with a predetermined temperature setting, such that the at least one nanopore is addressed for the show nano-holes predetermined temperature chemical reaction.
  46. 46. 如权利要求44所述的装置,其中,所述多个纳米孔包括超过大约30, 000纳米孔。 46. The apparatus according to claim 44, wherein said plurality of nanopores comprises more than about 30, 000 nanopores.
  47. 47. 如权利要求44所述的装置,其中,所述第一加热器包括多个温度带。 47. The apparatus of claim 44, wherein said first heater comprises a plurality of temperature zones.
  48. 48. 如权利要求47所述的装置,其中,所述温度带包括6个或更多个温度带。 48. The apparatus of claim 47, wherein the temperature zones comprise six or more temperature zones.
  49. 49. 如权利要求48所述的装置,其中,所述多个温度带对应于预定的温度设置,可热控制的单元根据该温度设置形成阵列。 49. The apparatus of claim 48, wherein said plurality of temperature zones corresponding to a predetermined set temperature, the thermal control unit can form an array based on the temperature setting.
  50. 50. 如权利要求44所述的装置,其中,所述第一加热器和第二加热器按照方案在第一和第二方位之间移动。 50. The apparatus of claim 44, wherein said first and second heaters according to the program to move between the first and second orientation.
  51. 51. 如权利要求44所述的装置,进一步包括用于在第一和第二方位之间移动第一加热器和第二加热器的电动机。 51. The apparatus of claim 44, further comprising means for moving between the first and second orientation of the first and second heaters motor.
  52. 52. 如权利要求44所述的装置,其中,所述第一加热器提供温度梯度。 52. The apparatus of claim 44, wherein the first heater provides a temperature gradient.
  53. 53. 如权利要求44所述的装置,进一步包括与第一加热器形成热接触的散热器。 53. The apparatus of claim 44, further comprising a heat sink in thermal contact with the first heater is formed.
  54. 54. 如权利要求44所述的装置,进一步包括与第二加热器形成热接触的散热器。 54. The apparatus of claim 44, further comprising forming a radiator and a second heater in thermal contact.
  55. 55. 如权利要求53或权利要求54所述的装置,进一步包括用于从散热器散热的风扇。 55. The apparatus of claim 54 or claim 53, further comprising a fan for cooling the radiator claims.
  56. 56. 如权利要求44所述的装置,进一步包括多个可操作地与芯片连接的温度传感器。 56. The apparatus of claim 44, further comprising a plurality of temperature sensors operatively connected with the chip.
  57. 57. 如权利要求56所述的装置,其中,所述多个温度传感器具有至少一个指定为测量各温度带的温度的温度传感器。 57. The apparatus of claim 56, wherein said plurality of temperature sensors having at least one temperature zone is designated as measuring a temperature of each temperature sensor.
Description  translated from Chinese

用于高通量化学反应的装置 Means for high-throughput chemical reactions

[0001] 交叉引用 [0001] The cross-reference

[0002] 本申请要求2007年1月22日提交的美国临时申请第60/886, 067号和2007年12 月21日提交的美国临时申请第61/016, 377号的优先权,本文完整引入这些申请作为参考。 [0002] This application claims priority to U.S. Provisional Application January 22, 2007, filed on 60/886, US Provisional Application No. 067 and December 21, 2007 filed on 61/016, 377, filed the complete article introduced These herein by reference. [0003] 发明背景 [0003] Background of the Invention

[0004] 聚合酶链反应(PCR)自1983年出现和后来的发展通过极大地扩展其鉴定、操作和复制DNA的能力已彻底改变了分子生物学。 Ability [0004] Polymerase chain reaction (PCR) since 1983 and subsequent developments appear to expand its appraisal, operation and replication of DNA by significantly has revolutionized molecular biology. 已开发了利用PCR的许多不同的应用,如科学研究、临床诊断、法医鉴定和环境研究。 PCR has been developed using a number of different applications, such as scientific research, clinical diagnosis, forensic and environmental research.

[0005] 继对人类基因组测序之后,对估计30,000个人类基因的基因组分析已成为基础和应用生物化学和药物学研究的主要焦点。 [0005] Following on after the human genome sequencing, genome analysis of the estimated 30,000 human genes has become a major focus of basic and applied research in biochemistry and pharmacology. 可以从基因的分析和操作开发用于多种疾病的诊断、药物和治疗。 It can be used for diagnostics, drugs and treat a variety of diseases from analytical and operational development genes. 诊断设备经常利用来自患者的小样品。 Diagnostic equipment often use a small sample from the patient. 为诊断目的收集的患者样品典型地具有有限的数量和体积,因而在单一样品上只可以进行少量的测试。 For the collection of patient samples for diagnostic purposes typically have limited the number and volume, and thus can only be carried out on a single sample a small amount of testing. 因此,需要能够对来自单一小样品的大量基因或核酸序列进行分析的小型化的设备。 Therefore, a large number of possible nucleic acid sequences from a single gene or a small sample of the miniaturization of equipment analysis.

[0006] 开发基于基因的疗法也已经成为研究人员和制药公司的主要焦点。 [0006] Based on the development of gene therapy has become researchers and the pharmaceutical company's main focus. 为了开发新的治疗方法和识别新的治疗靶标,理想的是利用生物体全基因组的大部分(如果不是全部) 进行高通量筛选。 In order to develop new treatments and identify new therapeutic targets, the ideal is to use most of the genome of an organism (if not all) of high-throughput screening. 另外,测序和扩增来自个体样品的全基因组的能力可以为个性化医学的发展铺平道路。 In addition, the ability to amplify the whole genome sequencing of a sample from an individual and can pave the way for the development of personalized medicine.

[0007] 目前可得到的许多PCR微板和热循环仪不能以合理的成本进行大量的PCR。 [0007] Many currently available PCR microplates and thermal cycler is not at a reasonable cost a lot of PCR. 在许多反应中,用来分析各个单序列所需要的样品体积是微升的级别。 In many reactions, the volume of the sample used to analyze each single sequence is required microliters level. 当对数以千计的基因进行测序或扩增时,所需要的来自个体或个体组的样品的量通常是不可能得到的。 When thousands of gene sequencing or amplification, the required amount of the sample from the individual or groups of individuals is often impossible to get. 另外,当处理大量的序列时,反应的灵敏度和特异性成为进行PCR时的主要关注点。 Furthermore, when dealing with a large number of sequences, reaction sensitivity and specificity are the primary concerns when performing PCR. 序列PCR扩增必需的退火温度在不同的序列之间的变化可能达15t:之多。 PCR amplification of sequences required annealing temperature changes between different sequences may reach 15t: as much. 为了对来自相对小的样品的数以千计的基因进行测序,热循环装置需要适应不同温度的范围。 To thousands from a relatively small sample of gene sequencing, thermal cycling device need to adapt to different temperature ranges.

[0008] 近年来,纳米制造技术的进步使得能够生产整合了电学元件、光学元件、化学元件或机械元件的小型化的设备。 [0008] In recent years, advances in nano-manufacturing technology enables the production of integrated electrical components, optical components, mechanical components, or chemical components of the device miniaturization. 技术体现为众多的制造技术,包括低压气相沉积、光刻术和蚀刻。 Technology embodied in many manufacturing techniques, including a low pressure vapor deposition, photolithography and etching. 基于这些技术,已经提出了包含与纳米加热器偶联的硅通道的小型化设备(参见,例如,美国专利第6, 962, 821、6, 054, 263、5, 779, 981和5, 525, 300号)。 Based on these technologies it has been proposed comprising a heater coupled to the silicon nano channel miniaturized devices (see, e.g., U.S. Patent No. 6, 962, 821,6, 054, 263,5, 779, 981 and 5, 525 No. 300). 尽管基于通道或室的设计原则上减少了热质量和反应体积,它仍然具有其它的实际缺陷。 Although based on the design principles of the channel or chamber decreases the thermal mass and the reaction volume, it still has other practical drawbacks. 特别地,设计的通道或室在控制温度和蒸发方面受到限制。 In particular, the design of channels or chambers are limited in terms of controlling the temperature and evaporation.

[0009] 这样的设备或系统将大大利于诊断测试、药物开发和个人医疗。 [0009] Such a device or system will be greatly beneficial diagnostic testing, drug development and personal health. 本发明满足这种 The present invention satisfies this

需要,而且还提供了相关的优势。 It needs, but also provides related advantages.

[0010] 发明概述 [0010] Summary of the Invention

[0011] 总的说来,一方面,本发明提供包含至少一个设置为与芯片形成热接触的加热元件的装置,所述芯片包括片基和纳米孔阵列,其中,该至少一个加热元件设置为可相对于芯片移动。 [0011] In general, one aspect, the present invention provides a set comprising at least one heating element with the chip forming means in thermal contact, said chip comprises a base sheet and a nanopore array, wherein the at least one heating element is set to can move relative to the chip.

[0012] 在一种实施方式中,加热元件从芯片的上面和下面与芯片形成热接触,而且其中, 从芯片下面形成热接触的加热元件的温度设定得比从芯片上面形成热接触的加热元件的温度低。 [0012] In one embodiment, the heating element is formed from thermal contact with the chip above and below the chip, and wherein the temperature of the thermal contact is formed below the heating element from the chip set higher than the thermal heating of the contact is formed from above the chip low temperature of the element. 在另一种实施方式中,芯片包括上表面和下表面,并且其中,第一系列的纳米孔沿上表面上的一个方向排列,第二系列的纳米孔垂直于第一系列的纳米孔定向。 In another embodiment, the chip includes an upper surface and a lower surface, and wherein the nano-holes of the first series along one direction on the surface of the arrangement, a second series of nanopores perpendicular to the first series of nanopore orientation. 加热元件可以位于包括纳米孔阵列的静止芯片的上面或下面,且加热元件可以是能够加热和冷却的。 The heating element may be located above or below the stationary nanopore array chips, and the heating element may be capable of heating and cooling. [0013] 在一种实施方式中,该装置包括对应于多个温度带的多个加热元件。 [0013] In one embodiment, the apparatus comprises a plurality of heating elements corresponding to a plurality of temperature zones in. 该多个温度 The plurality of temperature

带可以在大约52t:-大约95t:的范围之内。 Band may be about 52t: -: within range of about 95t. 在另一种实施方式中,该多个温度带提供了温度梯度。 In another embodiment, the plurality of temperature zones provides a temperature gradient. 温度带中的至少一个可以设定为处于大约52t:-大约65t:范围的温度,而且至少 At least one temperature zone can be set at about 52t: - about 65t: range of temperatures, and at least

一个另外的温度带可以设定为处于大约90C -大约95t:范围的温度。 A further temperature zone can be set at about 90 C - about 95t: range of temperatures. 在进一步的实施方 In further embodiments

式中,至少一个另外的温度带设定为处于大约68t:-大约721:范围的延伸温度。 Wherein at least one further temperature zone is set to be in approximately 68t: - About 721: stretching temperature range.

[0014] 在特定的实施方式中,该至少一个加热元件设置为提供包括温度随时间变化的尖 [0014] In a particular embodiment, the at least one heating element arranged to provide a sharp temperature changes over time comprising

峰波形的输出。 Output peak waveform.

[0015] 在一种实施方式中,所述阵列中的单个纳米孔具有长度为大约250ym、宽度为大约250iim和深度为大约525iim的尺寸或者更小。 [0015] In one embodiment, the single nanopore array having a length of about 250ym, a width and a depth of about 250iim 525iim size of about or less.

[0016] 在另一种实施方式中,芯片可操作地与检测光学信号的光学系统偶联。 [0016] In another embodiment, the chip detector operatively associated with the optical system of the optical signal coupling. 光学系统可以包括多个光学检测器。 The optical system may include a plurality of optical detectors.

[0017] 在一种实施方式中,纳米孔的数目大于大约30, 000。 [0017] In one embodiment, the number of nanometer pores greater than about 30, 000. 在另一种实施方式中,纳米孔设置为容纳大约100nl的体积。 In another embodiment, the nano-holes configured to receive a volume of approximately 100nl.

[0018] 总的说来,另一方面,本发明提供了进行化学反应的方法,包括提供设置为容纳反应样品的芯片;提供至少一个可相对于芯片定位的加热元件以在该至少一个加热元件和芯片之间提供热接触;和通过改变芯片的温度在反应样品中进行化学反应,其中,所述改变温度通过相对于芯片移动该至少一个加热元件以使得加热元件与芯片形成热接触而实现。 [0018] In general, another aspect, the present invention provides a chemical reaction comprising providing to the reaction a sample receiving chip; providing at least one heating element chip positioned relative to the at least one heating element and providing thermal contact between the chip; and by varying the chip temperature chemical reaction in the reaction sample, wherein the change with respect to temperature by moving the chip to the at least one heating element such that the heating element is in thermal contact with the chip formation is achieved. [0019] 在一种实施方式中,化学反应是核酸扩增反应。 [0019] In one embodiment, the chemical reaction is a nucleic acid amplification reaction.

[0020] 在另一种实施方式中,该至少一个加热元件的移动受到可操作地连接芯片的温度传感器产生的信号的控制。 [0020] In another embodiment, the at least one heating element is controlled by moving operably linked to the chip temperature sensor generates a signal.

[0021] 在进一步的实施方式中,反应样品能够产生光学信号,并且其中,芯片可操作地与设置为检测从反应样品发射的光学信号的光学系统偶联。 [0021] In a further embodiment, the reaction sample to produce an optical signal, and wherein, the chip is operably configured to detect optical signals emitted from the reaction samples coupling optical system. 光学信号可以与化学反应的产物量成比例。 The optical signal can be proportional to the amount of a product of chemical reactions.

[0022] 在一种实施方式中,通过移动多个加热元件实现改变温度,各个加热元件设定为 [0022] In one embodiment, a plurality of heating elements is achieved by moving the temperature change, the respective heating element is set to

处于不同的温度。 At different temperatures. 在另一种实施方式中,至少一个加热元件设定为处于大约52t:-大约65t:范围的温度,且至少一个另外的加热元件设定为处于大约9(rc -大约951:范围的温度。 In another embodiment, the at least one heating element is set to be in approximately 52t: - About 65t: range of temperatures, and at least one additional heating element is set to be in about 9 (rc - about 951: range of temperatures.

[0023] 总的来说,另一方面,本发明提供了进行反应的芯片,包括可编址的(addressable)单元的阵列,各个单元设置用于化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中单个单元的尺寸设计为容纳小于大约lPl的化学反应混合物。 [0023] In general, on the other hand, the present invention provides the reaction of the chip, including an array of (addressable) addressable units, each unit is provided for the chemical reaction, which can be made available in an array of settings addressing unit corresponding to a predetermined temperature zone, and wherein the array is sized to accommodate a single cell of less than about lPl chemical reaction mixture. 在一种实施方式中,所述装置包括多个阵列。 In one embodiment, the apparatus comprises a plurality of arrays. 在另一种实施方式中,所述装置包括各对应于不同的温度带的多个阵列。 In another embodiment, the apparatus comprises a plurality of arrays of different temperature zones, each corresponding to. 在一种实施方式中,至少一个阵列设定为处于用于支持核酸扩增反应的退火温度,和至少一个另外的阵列设定为处于用于支持核酸扩增反应的变性温度。 In one embodiment, the array is set to at least one nucleic acid amplification reaction for supporting the annealing temperature, and at least one other set in the array is used to support the denaturation temperature nucleic acid amplification reaction.

[0024] 在特定的实施方式中,对温度带编址以表明预定的温度带。 [0024] In a particular embodiment, the temperature band addressed to indicate the predetermined temperature zone. 在另一种实施方式中, 可编址单元的阵列设置为对应于6个或更多个预定的温度带。 In another embodiment, an array of addressable units set to correspond to six or more predetermined temperature band.

6[0025] 在一种实施方式中,芯片与加热元件形成热接触。 6 [0025] In one embodiment, the chip is formed in thermal contact with the heating element.

[0026] 总的来说,另一方面,本发明提供了用于进行需要在至少两个温度水平循环的化学反应的装置,包括:(a)用于进行反应的包含可编址单元的阵列的芯片,各个单元设置用于化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中的单个单元的尺寸设计为容纳小于大约1 P 1的化学反应混合物;和(b)与芯片热接触的加热元件。 Array (a) for the reaction of the addressable unit comprises: [0026] In general, another aspect, the present invention provides apparatus for performing chemical reactions need at least two cycles of temperature levels, comprising chips, each unit is provided for chemical reactions, which can be made available for array addressing unit disposed corresponding to a predetermined temperature zone, and wherein the array is sized to accommodate a single cell is less than about 1 P 1 of Chemicals The reaction mixture; heating element and (b) in contact with the hot chips.

[0027] 在一种实施方式中,可编址单元的阵列大于大约30, 000。 [0027] In one embodiment, an array of addressable units is greater than about 30, 000.

[0028] 在特定的实施方式中,所述装置进一步包括:(C)可操作地与芯片偶联的光学系统,其中,光学系统检测来自编址的可热控制的单元的光学信号。 [0028] In a particular embodiment, the apparatus further comprises: (C) is operatively coupled to the chip with an optical system, wherein the optical system of the optical signal detecting means may be addressed from the thermal control. 在一种实施方式中,光学系统包括多个光学检测器。 In one embodiment, the optical system comprises a plurality of optical detectors.

[0029] 在一种实施方式中,所述装置进一步包括多个加热元件。 [0029] In one embodiment, the apparatus further comprises a plurality of heating elements. 在特定的实施方式中,所述多个加热元件包括6个或更多个加热元件。 In certain embodiments, the plurality of heating elements comprises six or more heating elements. 在一种实施方式中,阵列中的单个单元包括用于容纳并限制样品的纳米孔,所述孔在填充样品后被密封。 In one embodiment, the array comprises a single unit for receiving the sample and limits the nanopore, the sample aperture is sealed after filling. 在另一种实施方式中,化学反应是核酸扩增反应。 In another embodiment, the chemical reaction is a nucleic acid amplification reaction. 在一种实施方式中,单元的预定温度设置为从化学反应产生至少90% 的同质产物。 In one embodiment, the predetermined temperature unit is arranged to generate at least 90% of the product from the chemical reaction homogeneous.

[0030] 总的来说,在另一方面,本发明提供了进行涉及多个反应样品并需要在至少两个温度水平上循环的反应的方法,包括:(a)提供进行反应的包括可编址单元的阵列的芯片, 各个单元设置用于化学反应,其中,可编址单元的阵列设置为对应于预定的温度带,并且其中,所述阵列中单个单元的尺寸设计为容纳小于大约lPl的化学反应混合物;(b)根据预定温度的设置将多个反应样品放置于芯片的单元中;和(c)控制加热元件以实现至少两个温度水平的循环。 [0030] In general, in another aspect, the present invention provides a method involving reacting a plurality of reaction samples and requires at least two cycles of temperature levels, comprising: (a) providing a reaction comprising programmable address array chip units, each unit is provided for the chemical reaction, wherein the array of addressable units provided corresponding to a predetermined temperature zone, and wherein the array is sized to accommodate a single cell of less than about lPl Chemical reaction mixture; (b) in accordance with a predetermined temperature setting the plurality of reaction samples placed in chip units; and (c) controlling the heating element to effect at least two temperature levels of the cycles.

[0031] 在一种实施方式中,芯片的阵列中的单个单元包括用于容纳并限制样品的纳米 [0031] In one embodiment, a single array chip unit comprises means for receiving the sample and limits nano

孔,所述孔在填充样品后被密封。 A bore seal in the sample after filling. 在另一种实施方式中,化学反应是核酸扩增反应。 In another embodiment, the chemical reaction is a nucleic acid amplification reaction. 在进一 In into a

步的实施方式中,单元的预定温度设置为从化学反应产生至少90%的同质产物。 Step embodiment, the predetermined temperature setting unit to generate at least 90% of homogeneous product from the chemical reaction.

[0032] 总的来说,在再另一方面,本发明提供了用于进行涉及在至少两个温度水平上的 [0032] In general, in another aspect, the present invention provides a method for involving at least two temperature levels

循环的反应的装置,包括:(a)设置为容纳芯片的主体,芯片包括多个用于容纳化学反应物 Cycling reaction apparatus, comprising: (a) to accommodate the body of the chip, the chip includes a plurality of chemical reactants for receiving

的纳米孔;和(b)提供第一温度的第一加热器和提供第二温度的第二加热器;其中,第一加 Wherein the first addition; nanopore; and (b) providing a first temperature of a first heater and a second temperature of second heater

热器和第二加热器设置为可在第一和第二方位(orientation)之间移动,且其中,第一方 Heat and second heater is set to be between the first and second azimuth (orientation) movement, and wherein the first party

位使加热器与样品座形成热接触,和第二方位不使加热器与样品座形成热接触。 Place the heater in thermal contact with the sample holder is formed, and a second orientation does not form the heater in thermal contact with the sample holder.

[0033] 在一种实施方式中,多个纳米孔是可编址的,其中,根据预定的温度设置排列纳米 [0033] In one embodiment, a plurality of nanopores is addressable, which are arranged according to a predetermined temperature setting nano

孔以使得至少一个纳米孔编址为表明用于在所述纳米孔内进行化学反应的预定温度。 Such that the at least one aperture nanopore addressed to indicate a predetermined temperature for a chemical reaction in the nano-pores. 在特 Laid

定的实施方式中,多个纳米孔包括超过大约30, 000个纳米孔。 Given embodiment, it comprises a plurality of nanopores in excess of about 30, 000 nanopores.

[0034] 在一种实施方式中,第一加热器包括多个温度带。 [0034] In one embodiment, the first heater comprises a plurality of temperature zones. 在另一种实施方式中,所述温度带包括6个或更多个温度带。 In another embodiment, the temperature zone comprises six or more temperature zones. 在进一步的实施方式中,多个温度带对应于预定的温度设置, 可热控制的单元按照该温度设置形成阵列。 In a further embodiment, a plurality of temperature zones corresponding to a predetermined temperature setting, can form an array of thermal control unit in accordance with the temperature setting. 在一种实施方式中,第一加热器和第二加热器根据方案在第一和第二方位之间移动。 In one embodiment, the first and second heaters under the program moving between the first and second orientation.

[0035] 在进一步的实施方式中,所述装置包括用于在第一和第二方位之间移动第一加热器和第二加热器的电动机。 [0035] In a further embodiment, the apparatus comprises means for moving between the first and second orientation of the first and second heaters motor.

[0036] 在一种实施方式中,第一加热器可以提供温度梯度。 [0036] In one embodiment, a first heater temperature gradient can be provided. 在另一种实施方式中,所述装置进一步包括与第一加热器热接触的散热器(heatsink)。 In another embodiment, the apparatus further comprises a heater in contact with the first heat sink (heatsink). 在不同的实施方式中,所述装置进一步包括与第二加热器热接触的散热器。 In various embodiments, the apparatus further comprises a heater in contact with the second heat sink.

[0037] 在某些实施方式中,所述装置进一步包括用于从散热器除去热量的风扇。 [0037] In certain embodiments, the apparatus further comprising means for removing heat from the radiator fan. 在其它实施方式中,所述装置进一步包括多个可操作地连接芯片的温度传感器。 In other embodiments, the apparatus further comprises a plurality of operatively connecting the chip temperature sensor. 在一种实施方式中,多个温度传感器具有至少一个指定用于测量各个温度带的温度的温度传感器。 In one embodiment, a plurality of temperature sensors has at least one temperature sensor for measuring the respective temperature zones specified temperature. [0038] 参考文献的引入 The introduction of [0038] references

[0039] 在本说明书中提到的所有出版物和专利申请引入本文作为参考,如同各单独的出 [0039] All publications and patent applications mentioned in this specification are incorporated herein by reference as if each individual out

版物或专利申请具体地和单独地表明引入作为参考一样。 Version or patent application was specifically and individually indicated to be incorporated by reference.

[0040] 附图简述 [0040] Brief Description

[0041] 在所附的权利要求书中特别说明了本发明的新型特征。 [0041] In the appended claims particularly illustrate the novel features of the invention. 通过参考下面阐明示例性的实施方式的详细说明书和附图可以获得对于本发明的特征和优势的更好的理解,所述实施方式利用了本发明的原理: By reference to the following exemplary embodiments illustrate the detailed specification and drawings can be obtained for a better understanding of the features and advantages of the present invention, the embodiment utilizes the principles of the invention:

[0042] 图1说明本发明的芯片的示例性的实施方式,其包括6个较小的芯片或6个代表不同的编址预定温度的可编址单元的阵列。 [0042] FIG. 1 illustrates the chip of the present invention, an exemplary embodiment, which includes six smaller chips or a predetermined temperature array 6 represent different addressing addressable unit.

[0043] 图2是圆形片基上的一个示例性的芯片布局的顶视图。 [0043] FIG. 2 is a top view of an exemplary chip layouts based on a circular sheet.

[0044] 图3是显示芯片上的纳米孔的一个示例性的芯片布局的顶视图。 [0044] FIG. 3 is a top view of the nanopore chip on display exemplary chip layout.

[0045] 图4是示例性的纳米孔芯片的侧视纵剖面图。 [0045] FIG. 4 is an exemplary longitudinal sectional side view of the nanopore chip FIG.

[0046] 图5说明包括一系列较小芯片的本发明的示例性的芯片,各个较小芯片代表不同的温度带。 [0046] Figure 5 illustrates an exemplary chip includes a series of small chips of the present invention, each of the smaller chips represent different temperature zones.

[0047] 图6描述具有至少一个带有纳米孔芯片的加热元件的说明性的装置设计。 [0047] Figure 6 depicts an illustrative device is designed with at least one heating element with the nanopore chip.

[0048] 图7是具有超过一个温度带的本发明的一个实施方式的装置的示意图。 [0048] FIG. 7 is a schematic view of an apparatus having a temperature zone more than one embodiment of the present invention.

[0049] 图8是本发明的具有顶部安装的加热元件和光学扫描器的实施方式的一个装置 [0049] FIG. 8 is an embodiment of the top-mounted device having a heating element and an optical scanner of the present invention.

实施方式的侧视图。 A side view of the embodiment.

[0050] 图9是描绘装置的温度(y-轴)在不同时间(x-轴)的变化的曲线图。 [0050] FIG. 9 is a graph depicting temperature (y- axis) plot at different times (x- axis) of the device changes.

[0051] 图10描述一系列能够由弧光灯产生的热温度分布图。 [0051] Figure 10 depicts a series of hot temperature profile can be produced by arc lamps. 各个温度分布图显示芯片 Each temperature profile shows chip

对加热元件的响应。 Response to the heating element. 上面一行(a)的曲线图显示温度(y-轴)随时间(x-轴)的变化。 Top row (a) shows the temperature graph (y- axis) versus time (x- axis). Next

面一行(b)的曲线图显示芯片的温度(y-轴)随芯片的深度(x-轴)的变化。 Surface line (b) of the graph shows changes in the depth (x- axis) of the chip with the chip temperature (y- axis).

[0052] 图lla-c代表热循环装置的不同加热元件配置的例子。 [0052] FIG. Lla-c represents the thermal cycle apparatus different heating element configuration examples.

[0053] 图12显示热循环装置的示例性的实施方式,其中,两个加热器可在第一方位和第二方位之间移动,其中在第一方位中加热器与样品座形成热接触。 [0053] FIG. 12 shows an exemplary embodiment of the thermal cycling apparatus, wherein, between two heaters may move first orientation and a second orientation wherein the first orientation is formed in the heater in thermal contact with the sample holder.

[0054] 图13显示当加热器处于与具有可编址单元的芯片热接触的第一方位时,图12的实施例的第一加热器的侧视图。 When the [0054] Figure 13 shows when the heater is in a first orientation and chip heat having addressable unit contact, a side view of a first embodiment example of a heater 12.

[0055] 图14显示由本发明具有分为不同温度带的加热器的热循环装置提供的温度分布。 [0055] FIG. 14 shows the temperature of the heater is divided into different temperature zones of the thermal cycle apparatus provided by the present invention has a distribution.

[0056] 图15显示本发明的示例性的装置或系统,其中,所述装置包括顶端盖片和能够提供将芯片或化学反应物与顶端盖片形成光接触和/或热接触的力的加热器。 [0056] Figure 15 shows an example of a device or system of the present invention, wherein the device comprises a top cover sheet and to provide the chip or chemical reactants and the top cover sheet to form a light touch and / or thermal contact with the heating power device. [0057] 图16是本发明的热循环系统的一个实施方式的示意图。 [0057] FIG. 16 is a schematic diagram of one embodiment of the thermal cycle system of the present invention.

[0058] 图17说明本发明的示例性的系统,包括芯片、加热装置和用于分析反应结果的光学系统。 [0058] FIG. 17 illustrates an exemplary system of the present invention comprises a chip, a heating device and for analyzing the reaction results in an optical system.

8[0059] 图18说明本发明的示例性的系统,包括光学系统、加热装置和用于进行化学反应的芯片。 8 [0059] FIG. 18 illustrates an exemplary system of the present invention, including chip optical systems, heating devices and for carrying out chemical reactions.

[0060] 图19是方框示意图,显示与本发明的特定实施方式的系统进行通讯的逻辑设备 [0060] FIG. 19 is a block diagram showing a particular embodiment of communication system of the present invention will be logical device

的代表性的例子。 Representative examples.

[0061] 发明详述 [0061] DETAILED DESCRIPTION

[0062] 本发明提供进行多个化学反应和用于单个分子的多重分析的芯片、热循环装置、 系统及方法。 [0062] The present invention provides a plurality of chips, heat cycle apparatus, system and method of multiple chemical reactions and analysis for a single molecule. 本发明也提供允许精确控制反应物质、条件和温度的操作的小型化、高度自动化的设备和方法。 The present invention also provides precise control of the reaction mass allowed miniaturization conditions and temperatures of operation, equipment and methods are highly automated.

[0063] 本发明可能涉及用于同时分析生物体的全基因组的方法、芯片和装置。 [0063] The present invention relates to a method for the simultaneous analysis of possible organisms genome, chips and devices. 许多方法涉及使用在非常小的反应体积中进行的聚合酶链反应或类似的扩增方法对核苷酸的基因组混合物进行的定性和/或定量分析。 Many qualitative methods involve the use of very small reaction volume carried by polymerase chain reaction amplification method or similar mixture of genomic nucleotide conducted and / or quantitative analysis.

[0064] 预计的30, 000人类基因的分析可以提供用于应用药物研究和开发对于许许多多 [0064] estimated 30, 000 analysis of human genes can be provided for use in drug research and development for many

疾病的诊断、药物和治疗方法。 Diagnosis, medications and treatments. 例如,通过认识正常和患病个体之间的遗传差异,可以确定 For example, by understanding normal and diseased genetic differences between individuals can be determined

细胞和组织的生物化学组成和功能之间的差异,并确认适当的治疗干预。 Differences in biochemical composition and function of cells and tissues between and confirm the appropriate therapeutic intervention.

[0065] 在一种实施方式中,基因组可以来自人类、哺乳动物、小鼠、拟南芥(Arabidopsis) [0065] In one embodiment, the genome can be derived from human, mammalian, mouse, Arabidopsis (Arabidopsis)

或任何其它植物、细菌、真菌或动物种类。 Or any other plant, bacterial, fungal or animal species. 本发明可以用于药物发现和用于特定个体、动物 The present invention may be used in drug discovery and for a specific individual, animal

或植物的诊断。 Or diagnosis of a plant.

[0066] 在许多情况下,可能需要从生物体的所有基因的测试确定基因表达谱。 [0066] In many cases, you may need to determine the gene expression profile of the test organisms from all genes. 这样的测 Such a measure

试也可以用于从来自单一个体的DNA或RNA筛选与相同的或不同的基因的不同突变(例 Test can also be used for screening DNA or RNA from a single individual or different mutations in the same gene from a different (e.g.

如,单核苷酸多态性或〃 SNPs〃 )有关的序列变异,或筛选作为来自亲本的不同染色体片 For example, a single nucleotide polymorphism or 〃 SNPs〃) the sequence variation, or screening as different chromosomes from the parent sheet

段的遗传性标记的序列变异。 Sequence Variation genetic marker segment. 这样的测试也可以用于例如,预测疾病的易感性,确定个体是 Such a test may also be used, for example, predict susceptibility to the disease, the individual is determined

否为遗传突变的载体,确定个体是否对某些药物的副作用敏感或对于某些药物耐受,或用 No genetic mutation carrier, to determine whether an individual susceptible to the side effects of certain drugs or for certain drugs, or

于其它诊断、治疗或研究目的。 In other diagnostic, therapeutic or research purposes.

[0067] [0067]

[0068] 本发明的芯片的总体大小可以变化,且它的厚度范围可以是从几微米到几厘米, 宽度或长度范围可以是从几厘米到50厘米。 [0068] The overall size of the chip of the present invention may be varied, and its thickness may range from a few microns to a few centimeters, width or length may range from a few centimeters to 50 centimeters. 典型地,整个芯片的大小,宽度和/或长度范围是大约10mm-大约200mm,厚度范围是大约lmm-大约10mm。 Typically, the size of the entire chip, the width and / or length range is about from about 10mm- 200mm, a thickness ranging from about lmm- about 10mm. 在某些实施方式中,芯片是大约40mm宽,大约40mm长,大约3mm厚。 In certain embodiments, the chip is about 40mm wide and approximately 40mm long, about 3mm thick.

[0069] 芯片也可以是一系列较小的芯片。 [0069] chip may be a series of smaller chips. 例如,芯片可以包括6个较小的芯片(例如,6 个可编址单元的阵列),在各个较小的芯片之间有热缓冲。 For example, the chip may include six smaller chips (for example, an array of 6 addressable unit) between each smaller chip thermal buffer. 作为较小芯片系列的芯片在本文也被称为板(plate)。 As a small chip series chips also referred to herein plate (plate). 在该实施例的一种实施方式中,6个较小芯片各对应于不同的预定温度,整个芯片中的单元阵列相对于不同的预定温度编址。 In one embodiment of this embodiment, six smaller chips each corresponding to a different predetermined temperature, the entire chip cell array with respect to addressing different predetermined temperature.

[0070] 芯片上的单元的总数随采用所述芯片的特定应用的不同而变化。 The total number of units of different chip [0070] With the use of the chip application-specific change. 芯片表面上的单 On a single chip surface

元的密度随特定的应用变化。 Dollar change in density with the particular application. 单元的密度(例如,片基每单位表面积的室数)及单元的大 Density unit (for example, film base number of cells per unit surface area) and a large unit

小和体积随需要的应用和如本发明的方法采用的生物体种类这样的因素而变化。 And with the application of small volumes required and such factors as the type of organisms used in the method of the present invention varies.

[0071] 大量的单元可以引入本发明的芯片中。 [0071] a large number of units can be introduced into the chip of the present invention. 在各种实施方式中,芯片上的单元的总数 Total In various embodiments, the chip unit

是大约1000-大约200, 000,更优选大约5000-大约100, 000。 About 1000- about 200, 000, and more preferably about 5,000 to about 100, 000. 在其它实施方式中,芯片包 In other embodiments, the chip package

括较小的芯片,其中各较小的芯片包括大约5, 000-大约20, 000个单元。 Including smaller chips, wherein each smaller chip includes about 5, 000- about 20, 000 units. 因此,如果较大的 Accordingly, if larger

芯片包括大约3-大约20个较小的芯片,那么它包括大约15, 000-大约400, 000单元。 Chip comprising about 3 to about 20 small chips, it comprises about 15, about 000- 400, 000 units. 在一定的实施方式中,芯片包括大约100, 000单元。 In certain embodiments, the chip comprising about 100, 000 units.

[0072] 例如,方形芯片可以包括125X 125个纳米孑L纳米孔具有O. lmm的直径。 [0072] For example, a square chip may include 125X 125 nanometers larvae L nanopores having O. lmm diameter. 表I显 Table I was

示本发明的特定的示例性芯片的孔排列格式的某些例子。 Holes present invention shown certain exemplary arrangement of some examples chip format. 板相当于包括本文所述的多个较 A plurality of panels including those described herein compared with the equivalent

小芯片的芯片。 Small chip chips. 在表I的例子中,板包括6个较小的芯片。 In the example of Table I, the plate comprising six smaller chips.

[0073] 表I. [0073] Table I.

[0074] [0074]

(大约) n (大约) 深度(mm) 〜孑L /芯片(大约) 孔/板(大约) (Approximately) n (about) Depth (mm) ~ larvae L / chip (approximately) hole / plate (approximately)

125 125 0. 1 15743 94459 125 125 0. 11574394459

122 122 0. 11 14790 88742 122 122 0. 111479088742

118 118 0. 12 13921 83527 118 118 0. 121392183527

115 115 0. 13 13126 78756 115 115 0. 131312678756

111 111 0. 14 12397 74381 111 111 0. 141239774381

108 108 0. 15 11726 70358 108 108 0. 151172670358

105 105 0. 16 11108 66651 105 105 0. 161110866651

103 103 0. 17 10538 63228 103 103 0. 171053863228

100 100 0. 18 10010 60060 100 100 0. 181001060060

98 98 0. 19 9521 57124 9898 0. 19952157124

95 95 0. 2 9066 54396 9595 0. 2906654396

93 93 0. 21 8643 51858 9393 0. 21864351858

91 91 0. 22 8249 49492 9191 0. 22824949492

89 89 0. 23 7881 47283 8989 0. 23788147283

87 87 0. 24 7536 45218 8787 0. 24753645218

85 85 0. 25 7214 43285 8585 0. 25721443285

83 83 0. 26 6912 41472 8383 0. 26691241472

[0075] [0075]

[0076] 在表I中,m是沿水平轴方向的孔的近似数,n是沿垂直轴方向的孔的近似数,深度以mm计量,且孔数/芯片和孔数/板是近似的。 [0076] In Table I, m is the approximate number of pores in the horizontal axis, n is an approximation of the number of the vertical axis of the bore, the depth measured in mm, and the number of holes / chip, and the number of holes / board is approximated .

[0077] 芯片可以是任意大小,或具有任何数量的单元。 [0077] chip can be any size, or have any number of elements. 在一种实施方式中,接收本发明的芯片的使用者或消费者对大小、单元和单元是否包括纳米孔进行选择。 In one embodiment, the present invention receives user or consumer of the size of the chip, cells and cell selection is included nanopores. 在优选的实施方式 In a preferred embodiment,

10中,当用本发明的芯片上的单元进行核酸扩增化学反应时,使用者可以根据需要对物种的整个基因组测序的基因数目选择单元的数目。 10, when the chip by means of the present invention on the nucleic acid amplification chemistry, the user can be the number of species throughout the genome sequencing of genetic selection based on the number of units required.

[0078] 图1中说明包括6个较小芯片110的实施方式的示例性的芯片100。 [0078] Figure 1 illustrates an embodiment comprising six smaller chip 110 of an exemplary chip 100. 较小芯片110的面积是38mmX39. 5mm,且包括6个较小芯片110的芯片100的总体尺寸是大约85mmX129mm的面积。 Smaller chip area 110 is 38mmX39. 5mm, and includes overall chip size 6 small chips is about 110 100 85mmX129mm area. 在本实施例中,较小芯片110包括大约400 ym的较大的孔尺寸(在图中不是按比例显示)。 In the present embodiment, the smaller chip 110 comprises approximately 400 ym larger pore size (not shown to scale in the figure). 较小芯片110可以包括如表I所示的一系列mXn个纳米孔120。 Smaller chip 110 may include a series as shown in Table I mXn holes 120 nanometers. 在本实施方式中,t-mm的壁厚122可以确定芯片上的纳米孔的数目。 In the present embodiment, the number of nanopores t-mm wall thickness 122 may be determined on the chip. 各较小芯片IIO可以代表整体芯片100的预定温度,而且因此,各较小芯片110可以根据在各个纳米孔120中进行的反应的温度编址。 Each smaller chip IIO can represent the overall chip predetermined temperature of 100, and therefore, each small chip 110 may be in accordance with the temperature of each nanopore 120 response addressing. 另外,总的较大的芯片100中采用多个较小芯片110时,热绝缘框架130可以相互热隔离各个可编址的预定温度区域。 In addition, the total of the larger chip 100 when multiple smaller chips 110, heat insulating frame 130 may be thermally isolated from each addressable predetermined temperature region. 在本实施例中,热绝缘框架130厚度为3mm,且可以由云母、聚乙烯或本领域的技术人员显而易见的任何其它绝缘材料制造。 In the present embodiment, the thickness of the thermal insulation frame 130 of 3mm, and may be formed of mica, polyethylene or any other insulating material of the present apparent to those skilled in the art. [0079] 在一种实施方式中,对于全基因组高通量基因表达实时PCR,芯片可以进行33, 750项分析。 [0079] In one embodiment, for high-throughput genome-wide gene expression in real time PCR, the chip can be 33, 750 analysis.

[0080] 纳米孔可以制成任何方便的大小、形状或体积。 [0080] The nanopore may be made of any convenient size, shape or volume. 孔的长度可以是大约lOOym-大约lmm,宽度可以是大约100iim-大约lmm,和深度可以是大约100 y m-大约lmm。 The length of the holes may be about lOOym- about lmm, a width may be about 100iim- about lmm, and a depth of about 100 y m- may be about lmm. 在各种实施方式中,各个纳米孔具有大约l-大约4的深宽比(深度和宽度的比)。 In various embodiments, each of approximately l- nanopores having an aspect ratio (ratio of the depth and width) of about 4. 在一种实施方式中,各个纳米孔具有大约2的深宽比。 In one embodiment, each of nanopores having aspect ratio of about 2. 横截面可以是圆形、椭圆形、卵形、圆锥形、矩形、三角形、多边形或任何其它形状。 Cross-section may be circular, elliptical, oval, conical, rectangular, triangular, polygonal or any other shape. 孔在任何给定深度的横截面积也可以随大小和形状变化。 Hole sectional area at any given depth may also vary with the size and shape. [0081] 在一种实施方式中,纳米孔可以具有大约lnl-大约1 ii 1的体积。 [0081] In one embodiment, the nanopores may have about lnl- volume of about 1 ii 1. 纳米孔典型地具有小于1 y 1的体积,优选小于500nl的体积。 Nanopores typically have a volume of less than 1 y 1, preferably less than the volume 500nl of. 体积可以是小于200nl,或甚至小于100nl。 Volume may be less than 200nl, or even less than 100nl. 在一种实施方式中,纳米孔的体积是大约100nl。 In one embodiment, the volume of nanopores is about 100nl. 需要时,可以制造纳米孔以增加表面积与体积的比例,从而促进经过单元的热转换,其可以减少热循环的斜面变化时间(ramptime)。 If desired, to increase the manufacturing nanoporous surface area to volume ratio, thereby promoting heat transfer unit through which heat can reduce cycle time ramp changes (ramptime). [0082] 各个纳米孔的空腔可以采取各种构型。 [0082] cavity of each nanopore can take a variety of configurations. 例如,纳米孔中的空腔可以通过线性或弯曲的壁分割以形成分离但相邻的隔室,或通过圆形壁分割以形成内和外环形隔室。 For example, nanopores cavity can be linear or curved wall is divided to form separate but adjacent compartments, or by dividing the circular wall to form inner and outer annular compartment. [0083] 可以用材料涂覆具有高内表面_体积比的纳米孔,以减少其中包含的反应物与孔的内表面相互作用的可能性。 [0083] may be coated with a material having a high inner surface _ nanopore volume ratio, to reduce the likelihood of reactants contained therein with the inner surface of the hole interactions. 如果试剂倾向于不利地与内表面相互作用或附着于内表面,那么涂层尤其有用。 If the reagent tends to adversely interacting with the inner surface or attached to the inner surface, the coating is particularly useful. 根据反应物的性质,可以选择疏水或亲水涂层。 Depending on the nature of the reactants, hydrophobic or hydrophilic coating can be selected. 各种合适的涂层材料是本领域中可得的。 Various suitable coating materials are available in the art. 某些材料可以共价附着于表面,其它可以经非共价相互作用附着于表面。 Some material may be covalently attached to the surface, the other via non-covalent interactions can be attached to the surface. 涂层材料的非限制性的例子包括硅烷化试剂,如二甲基氯硅烷、二甲基二氯硅烷、六甲基二硅氮烷或三甲基氯硅烷、聚马来酰亚胺,和硅化试剂,如氧化硅、AquasilTM和Surfasil"1。另外的合适的涂层材料是封闭剂,如氨基酸或包括但不限于聚乙烯吡咯烷酮、多聚腺苷酸和聚马来酰亚胺的聚合物。 Non-limiting examples of coating materials include silylating agent, such as dimethyl chlorosilane, dimethyl dichlorosilane, hexamethyl disilazane or trimethyl chlorosilane, polymaleimide, and silicification agent, such as silica, AquasilTM and Surfasil "1. Another suitable coating material is a sealant, such as amino acids or include, but are not limited to polyvinylpyrrolidone, and poly polymers polymaleimide .

[0084] —定的涂层材料可以经广泛的加热、辐射和通过化学反应与表面交联。 [0084] - given a wide range of coating materials can be heated by radiation and by chemical reaction with the surface cross-linking. 本领域的技术人员了解用于涂覆芯片的纳米孔的其它合适的方法,或者能够不需要过多实验确定这样的方法。 Those skilled in the art will appreciate other suitable method for coating the chip nanopores, or can not require undue experimentation to determine such methods.

[0085] 在一种实施方式中,芯片的单个单元包括用于容纳和限制样品的纳米孔,所述孔在填充样品后被密封。 [0085] In one embodiment, the chip comprises a single unit for receiving and limitations nanopores samples, the sample aperture is sealed after filling.

[0086] 阵列中的单个单元可以通过抗液体通过的物理屏障彼此分隔。 [0086] array can be separated from each other by a single unit a physical barrier against liquids pass. 一方面,这些单元可以包括称为纳米孔的锯齿状的区域。 On the one hand, these units may include a nanopore referred serrated region. 纳米孔可以是顶部开放的,但与其它孔物理地分离以限制液体通过。 Nanopores can be open at the top, but physically separated from the other hole to limit the liquid through. 因此,纳米孔具有至少一个适于容纳并限制反应样品的空腔。 Thus, at least one nanopore having a cavity adapted to receive and limit the reaction of the sample. 为了将一个纳米孔与环境分离以限制液体通过,可以密封纳米孔。 In order to separate a nanopore and the environment to restrict fluid pass through the nanopore can be sealed. 在优选的实施方式中,密封纳米孔的方法是将矿物油沉积于孔中的样品的顶部,以封闭样品。 In a preferred embodiment, the method is to seal the top of the nanopore mineral oil deposited in the hole in the sample, to close the sample. 矿物油可以是纳米分配的。 Mineral oil may be assigned nanometer. 可以通过本领域的技术人员显而易见的任何方法密封纳米孔。 Nanopore may be sealed by any method apparent to those skilled in the art.

[0087] 在许多应用中,需要密封纳米孔以防止液体蒸发,因而在整个热循环中保持优选的反应浓度。 [0087] In many applications, we need to be sealed to prevent liquid evaporation nanopores, thus maintaining the preferred reaction concentration throughout the thermal cycle. 因此,可以采用密封纳米孔阵列的技术。 Accordingly, the sealing technology can be used nanopore arrays. 有用的密封技术考虑了几种因素。 Useful sealing technology takes into account several factors. 首先,该方法应该顺应于大量纳米孔的高通量处理。 First, the method should conform to a large number of high-throughput processing nanopores. 其次,该方法应该允许单个纳米孔的选择性密封。 Second, the method should allow for selective sealing of individual nanopore. 如此,该方法可以以任何需要的类型或模式产生包含散布于密封的纳米孔间的开放纳米孔的芯片。 Thus, the method may comprise generating scattered seal between the open nanopores nanopore chips or any type of desired pattern. 开放的和/或未填充的孔不仅可以允许被动热消散,而且可以减少邻近的纳米孔之间的热传递。 Open and / or unfilled holes not only allows passive heat dissipation, and can reduce heat transfer between adjacent nanopores.

[0088] —种可替代的密封方法导致含有至少一个开放孔的纳米孔阵列。 [0088] - kind of an alternative sealing method leads nanopore array having at least one open hole. 该方法可以包括以下步骤:(a)沿限定该至少一个开放的纳米孔的开放表面的外周维度应用可辐射固化的粘合剂;(b)放置盖子以涵盖限定待密封的该至少一个开放的纳米孔的开放表面的外周维度;和(c)将阵列暴露于辐射线以实现密封。 The method may include the steps of: (a) defining the at least one opening along nanopores open surface of the outer peripheral dimension of a radiation-curable adhesive application; (b) placing a lid to cover qualified to be sealed at least one opening the outer peripheral surface of the nano dimension open bore; and (c) the array is exposed to radiation in order to achieve a seal.

[0089] 如本文所用,"可辐射固化的粘合剂"指在暴露于辐射线时而不需要深度加热就固化和结合到粘附表面的任何组合物。 [0089] As used herein, "radiation-curable adhesive" refers to any composition sometimes does not require exposure to radiation to cure the depth of heating and bonded to the adhesion surface. "辐射线"指电磁波能量,按频率的递增顺序包括红外辐射、可见光、紫外线(uv)、x-射线和Y射线。 "Radiation" refers to electromagnetic energy, in ascending order of frequencies, including infrared radiation, visible light, ultraviolet (uv), x- rays and Y-rays. 大量的辐射固化粘合剂可商购得到(参见, A large number of radiation-curable adhesives are commercially available (see,

例如,来自ThomasNet⑧的网站的销售辐射固化粘合剂和辐射系统的公司的列表)。 For example, sales from ThomasNet⑧ site radiation and radiation curable binder system lists companies). 这样 This

的材料包括各种丙烯酸树脂、丙烯酸酯、聚氨酯(pur)、聚酯、乙烯基、乙烯酯类,和大量通过各种频率的辐射线固化的环氧树脂类。 The materials include acrylic resins, acrylate, polyurethane (pur), polyester, vinyl, vinyl esters, and epoxies through a large number of various frequencies of radiation curing. 这些和其它辐射固化材料以液体或如糊剂、粉末、树脂和带的固体的形式商业供应。 These and other radiation curable material in the form of a liquid or as a paste, powder, resins and with a solid commercial supply.

[0090] 辐射固化粘合剂的选择取决于构成待附着的表面的材料。 [0090] The choice of binder depends on the radiation-curable material having a surface to be adhered. 前述种类的粘合剂适于 Adapted to the aforementioned types of adhesive

将芯片片基附着于由各种材料制成的盖子。 The chip is attached to the cover sheet substrate from a variety of materials. 例如,丙烯酸树脂和环氧树脂类可用于辐射密 For example, acrylic resins and epoxy resins can be used in radiation density

封任两个由选自玻璃、陶瓷、类金属(metalloids)、半导体(例如,硅、硅酸盐、氮化硅、二氧化硅、石英和砷化镓)、塑料和其它有机聚合材料的任一种材料制成的表面。 Selected from any of the two sealing glass, ceramics, metals (metalloids), semiconductors (e.g., silicon, silicates, silicon nitride, silicon dioxide, quartz, and gallium arsenide) either, plastics and other organic polymeric materials made of a material surface. 显示低使用温度和快速固化时间的性质的辐射固化材料对于密封所述芯片可能是需要的。 Display radiation curable material properties of low temperature and fast curing time for sealing the chip may be needed. 这些材料允许快速密封以避免对于芯片中包含的化学或生物试剂的辐射损伤。 These materials allow rapid sealed to prevent radiation damage of chemical or biological agents contained in the chip.

[0091] 可以通过任何机械方法沿限定纳米孔的开放表面的外周维度应用辐射固化粘合剂。 [0091] defined nanopores along the open surface of the outer periphery by any mechanical method Dimension application of radiation-curable adhesive. "外周维度"可以是芯片片基或盖子上的边界。 "Outer peripheral dimension" to the boundary chip film base or cover. 在任一情况下,粘合剂一旦固化,外周维度结合片基或盖子的相应的附着表面。 In either case, the adhesive once cured, the outer circumferential dimension of the corresponding attachment surface binding group or the cover sheet. 可以使用任何合适的工具在外周维度上涂覆、印刷、分配或喷雾辐射固化粘合剂。 You can use any suitable tool outer peripheral dimension of coating, printing, distribution or radiation curable adhesive spray. 机械方法可以在外周维度上产生均匀的粘合剂层。 Mechanical methods can produce a uniform outer peripheral dimension of the adhesive layer. 提供均一分布的一种方法是使用在有孔的印网掩模(screen mask)上的涂刷器直接将粘合剂涂布到开放孔的外周维度上。 A method for providing a uniform distribution is used in the perforated screen mask (screen mask) on the squeegee adhesive is applied directly to the open hole on the outer periphery dimension. 或者,可以直接在已经使用有孔的印网掩模以外周维度标记的盖子上应用辐射固化粘合剂。 Or, on the outside you have been used directly in the screen mask with holes dimension week marks cover the application of radiation-curable adhesive. 在除去掩模时,获得均一的粘合剂层。 When the mask is removed, to obtain a uniform adhesive layer.

[0092] —经应用辐射固化粘合剂,在纳米孔上放置盖子以包围限定孔的开放表面的外周维度。 [0092] - by the application of radiation-curable adhesive, place the lid on the nanopores to surround the outer circumferential dimension defining the open surface of the hole. 通常由允许辐射线通过的材料制造合适的盖子。 Usually made by a suitable cap to allow radiation through the material. 用如玻璃、石英、塑料、本领域的技术人员已知的任何合适的有机聚合材料或其任意组合的透明材料制造优选的盖子。 Used such as glass, quartz, plastics, known to those skilled in the art of any suitable organic polymeric material, or any combination of the transparent material lid preferred. [0093] 可以通过将孔暴露于辐射线完成密封覆盖的纳米孔。 [0093] can be produced by exposure to radiation holes completed nanopores sealed cover. 取决于选择的粘合剂的类 Class depends on the choice of adhesive

型,辐射线可以来自常规的白炽光源、激光器、激光二极管、uv灯泡、x-射线仪或Y-射线仪等。 Type, radiation can come from conventional incandescent light, lasers, laser diodes, uv lamp, x- ray apparatus or Y- ray detector. 需要时,允许来自辐射源的辐射线仅到达纳米孔阵列上的所选择的位置,使得仅特定的所选择孔被密封。 If desired, allowing only radiation from the radiation source to the position of the selected hole array Dana Mi, such that only certain selected hole is sealed. 经常通过使用以纳米孔位置形成图案的光掩模实现选择性的密封。 Often nanopores position patterned photomask to achieve selectivity by using a seal. 为光掩模提供分别对应于待密封的和保持开放的纳米孔的透明的位置和不透明的位置。 Provided corresponding respectively to be sealed opaque and transparent position and remain open nanopores location photomask. 辐射线自由通过透明区域,但被不透明区域反射或吸收。 Free radiation through the transparent regions, but is reflected or absorbed by the opaque region. 因此,仅所选择的纳米孔暴露于光线,因此通过固化粘合剂进行密封。 Thus, only a selected nanopore exposed to light, and therefore sealed by curing the adhesive. 光掩模可以形成图案以使得没有两个邻接的开放纳米孔被密封。 Photomask pattern can be formed so that no two adjacent open nanopores is sealed. 光掩模可以形成图案以使得产生的纳米孔阵列包含交替密封和未密封的孔。 Photomask pattern can be formed so that the generated nanopore array comprises alternating sealed and unsealed holes. 本领域的技术人员可以形成具有任何模式的不限数量的光掩模,以产生包含任何形式的开放和密封的纳米孔的芯片。 Those skilled in the art can form an unlimited number of photomask has any pattern to create an open and seal the nanopore chip contains any form. 本领域中已良好地建立了制造这样的光掩模的方法,因而本文不再详述。 The art has been well established method for producing such photomasks, thus we will not elaborate. [0094] 图2是由硅晶片200制成的可选择的芯片300的代表性示意图。 [0094] FIG. 2 is a schematic representation of an alternative chip 300 by 200 made of a silicon wafer. 优选的硅芯片的整体大小为厚度大约500微米且取决于需要的纳米孔的数目,可以具有任何宽度或长度。 Preferably the overall size of the silicon chip and a thickness of about 500 microns depending on the number of desired nanopore, may have any width or length. 这样的硅晶片200直径为6英寸(150mm),厚度为626 ym。 Such a silicon wafer 200 having a diameter of 6 inches (150mm), having a thickness of 626 ym. 可以从这样的晶片200制造芯片300,这样的芯片沿一侧210为大约85. 48mm,沿另一侧220为127. 76mm,和沿芯片的对角线250的长度为150mm。 200 can be manufactured from such a wafer chip 300, 210 along one side of such a chip is about 85. 48mm, along the other side 220 127. 76mm, and 250 along the length of the diagonal chip is 150mm. 制造时,芯片是SBS顺应性(compliant)的qPCR芯片。 Manufacturing, the chip is SBS compliant (compliant) of qPCR chip. 在芯片上制造的纳米孔的总数随采用所述芯片的特定应用而变化。 The total number of on-chip manufacturing nanopores varies with the use of a specific application of the chip. 为了适应同时进行大量反应的需要,所述芯片一般包括至少100个纳米孔,更通常为超过30,000个纳米孔。 In order to meet simultaneously requires a lot of reaction, the chip 100 typically includes at least nanopore, more typically more than 30,000 nanopores. 芯片表面上的纳米孔的密度可以随特定的应用而变化。 Density nanopore chip on the surface may vary with the particular application. 例如,芯片表面上的纳米孔的密度可以是大约1-大约1000个纳米孔/mm2。 For example, the density nanopore on the surface of the chip may be from about 1 to about 1000 nanometer holes / mm2. 在另一实施例中,芯片上的纳米孔的密度可以是大约10-大约100个纳米孔/mm2。 In another embodiment, the density nanopore chip may be about 10 to about 100 nano-holes / mm2.

[0095] 图3是具有代表性的纳米孔302的芯片300的顶面的示意图。 [0095] FIG. 3 is a schematic diagram of the top representative of the nanopore chip 300 302. 在一种示例性的实施方式中,芯片300的纳米孔302为长度0. 25mm、宽度0. 25mm,且纳米孔302的中心间隔0. 348mm。 In one exemplary embodiment, the nanopore chip 300 of length 302 is 0. 25mm, the width of 0. 25mm, and the center hole 302 nanometer intervals 0. 348mm. 可以想见,纳米孔302的中心按需要间隔,包括例如间隔2mm-0. Olmm。 It is conceivable that the central nanopore 302 interval as needed, including for example the interval 2mm-0. Olmm. 以横越或遍布芯片表面的任何形式使所述芯片的纳米孔形成阵列,如以排和列的形式形成栅格,以特定的图案形式等,如在图3中可见的。 In any form or traverse across the surface of the chip so that the chip is formed nanopore array, such as in the form of rows and columns forming a grid, in order to form a specific pattern, etc., as visible in FIG. 3. 在优选的实施方式中,纳米孔以与已存在的用于分配试剂和/或读数分析的仪器相容的形式形成阵列,以至于不需要对可商购获得的液体操作设备进行工程处理。 In a preferred embodiment, the nanopore with the existing distribution agent and / or reading apparatus compatible form for analysis form an array that does not require liquid handling equipment commercially available in the engineering process. 如同图3中的实施例,芯片可以具有至少246个纳米孔,更优选至少367个纳米孔,更优选至少45, 141个纳米孔,甚至更优选至少90, 282个纳米孔。 As shown in Example 3, the chip may have at least 246 nanopores, more preferably at least 367 nanopores, more preferably at least 45, 141 nano-holes, even more preferably at least 90, 282 nanopores. 尽管芯片的纳米孔的数目可以多达90, 282或更多,但是可以预计,纳米孔的数目通常不超过大约1, 444, 512个纳米孔。 Although the number of chips can be up nanopore 90, 282 or more, but can be expected, the number of nanopores usually not more than about 1, 444, 512 nanopores. 芯片的优选实施方式的纳米孔的数目足够各列容纳一式三份的82个基因,所有的行足够容纳一式三份的30,094个基因。 The number of preferred embodiments of the chip nanopores enough to accommodate triplicate each column of 82 genes, enough to accommodate all of the rows in triplicate 30,094 genes. 如同这样的一种优选实施方式,孔的数目足够一式三份地筛选整个人类基因组。 As such a preferred embodiment, a sufficient number of holes in triplicate to screen the entire human genome. 可以预计,芯片上的纳米孔的数目也可以包括用于扩增其它生物体的基因组中表达基因的完整集合的充足的反应孔。 It is expected that the number of nanopore chip may also include wells for sufficient amplification of the genome of other organisms expressing the complete set of genes. [0096] 图4是如图3所示的示例性的芯片300的第二层的横截面侧视图。 [0096] FIG. 4 is a cross-sectional side view of the second layer of the chip 300 shown in the example of FIG. 3. 图4显示厚度为0. 625mm的芯片300的非限制性的例子。 Figure 4 shows the thickness of non-limiting example of the chip 300 0. 625mm. 图4也显示尺寸为长度和宽度0. 25mm(250 ym)的单孔302。 Figure 4 also shows the size of the length and width of 0. 25mm (250 ym) of the hole 302. 如图所示,纳米孔深度可以为0. 525mm(525iim),在给定的孔之下留下0. lmm的芯片。 As shown, the depth of the nanopores can be 0. 525mm (525iim), under a given hole left 0. lmm chips. 可以预计,纳米孔的开口可以包括如圆形、方形、矩形或任意其它希望的几何形状的形状。 It can be expected, the opening of the nanopore may include, for example circular, square, rectangular or any other shape desired geometry. 例如,纳米孔可以包括大约100 ii m-大约lmm的直径或宽度,大约150 y m-大约lmm的孔距(pitch)或宽度和大约lOym-大约lmm的深度。 For example, nanopores can include about 100 ii m- diameter or width of about lmm, about 150 y m- about lmm the pitch (pitch) or about the width and depth of lmm of about lOym-. 所制得的各纳米孔的空腔采取各种构型。 Each prepared cavity nanopores take various configurations. 例如,纳米孔中的空腔可以通过线性或弯曲的壁分割,以形成分隔但相邻的隔室。 For example, the nanopore cavity by linear or curved dividing wall, to form a separated but adjacent compartments. [0097] 可以使用通常已知的光刻技术形成芯片的纳米孔。 [0097] may be used generally known photolithographic techniques to form the nanopore chip. 可以使用湿KOH蚀刻技术或各向异性干蚀刻技术形成纳米孔。 Nanopores can be formed using a wet KOH etching technique or an anisotropic dry etching techniques.

[0098] 可以用材料涂覆具有高的内表面和体积比例的纳米孔,以减少其中包含的反应物与纳米孔的内表面相互作用的可能性。 [0098] may be coated with a material having a high volume fraction of the inner surface of the nanopore in order to reduce the likelihood of reactants contained therein with the inner surface of the nanopore interaction. 也可以用电阻加热材料制造芯片。 You can also use a resistive heating material chips. 材料的非限制性的例子包括金属板,例如铝和不锈钢基质,如SS-316。 Non-limiting examples of materials include a metal plate, such as aluminum and stainless steel substrates, such as SS-316. 当所用的基质是金属时,通常优选用绝缘层涂覆表面,以防止腐蚀和/或在液体样品的操作期间样品成分的电泳现象。 When the metal substrate is used, it is usually electrophoresis sample components during operation of a liquid sample is preferably coated with an insulating layer surface to prevent corrosion and / or. 在非金属加热材料的情况下,涂层通常不是必需的。 In the case of a non-metallic material to be heated, the coating is generally not necessary. 可得到多种保护涂层,包括那些由Si02、Si3N4和特氟纶(Teflon)制成的涂层。 To obtain a variety of protective coatings, including those produced by coating Si02, Si3N4 and Teflon (Teflon) made. 图4显示芯片300,其中,单个孔302用K0H蚀刻,并用Si02涂层。 Figure 4 shows the chip 300, which, with a single hole 302 K0H etched with Si02 coating.

[0099] 图4也显示包括至少两个相对的纳米孔阵列的说明性的芯片。 [0099] Figure 4 also shows an illustrative chip includes at least two opposing nanopore arrays. 在该图中,芯片300具有顶面360和底面362。 In this figure, the chip 300 having a top surface 360 and bottom 362. 沿顶面364排列一个阵列,而沿底面368排列另一个相对的阵列。 364 are arranged along the top surface of an array of arrays are arranged along the bottom surface opposite the other 368. 底面阵列的纳米孔以倒置的方式排列,使得各个单元的开放表面指向芯片的相对单元的开放表面的相反方向。 The bottom surface of the nanopore array are arranged in an inverted manner, such that the open surface of each unit in the opposite direction of the open surface of the chip pointing to the opposite unit. 可以排列两个相对的阵列,使得各纳米孔的基部与相对阵列的基部直接相对。 Two opposing arrays may be arranged such that the base of each nanopore with the base portion opposite the array directly opposite.

[0100] 尽管图4中没有具体描述,但是顶面364阵列和/或底面368阵列中的任何纳米孔可以是密封或非密封的。 [0100] Although not specifically described in Figure 4, but the top 364 array and / or the bottom surface 368 of any nanopore arrays can be sealed or non-sealed. 另外,顶面阵列的任何纳米孔可以用或不用反应样品填充或不填充。 In addition, the top surface of any nanopore array may be with or without reaction samples filled or not filled. 接着通过将芯片与外部加热元件接触,所述芯片与加热元件形成热接触。 Followed by contacting the chip with an external heating element, the heating element is formed with a chip thermal contact. [0101] 可以进一步改变本发明的芯片的纳米孔的表面以建立反应试剂的吸附位点。 [0101] may further alter the surface of the nanopore chip of the present invention to establish adsorption sites reagents. 这些位点可以包括用于连接生物或化学化合物(如简单的或复杂的有机或无机分子、肽、蛋白质(例如,抗体)或多核苷酸)的连接体部分。 These sites may include a linker moiety biological or chemical compound (such as a simple or complex organic or inorganic molecules, peptides, proteins (e.g., antibody) or polynucleotide) of. 本领域的技术人员可以理解:有许多建立固定化学或生物反应物的吸附位点的方法。 Those skilled in the art will appreciate: establishing a fixed many adsorption sites of biological or chemical reactants approach. 例如,许多技术可用于经共价键或非共价键将核酸和氨基酸直接固定在芯片上,将它们锚定(anchoring)于连接体部分上,或将它们连接(tethering)于固定部分上(参见,例如,MethodsMol. Biol. Vol. 20 (1993) , Beier等人,Nucleic Acids Res. 27 :1970-1-977 (1999) , Joos等人,Anal. Chem. 247 :96-101(1997),Guschin等人,Anal.Biochem. 250 :203-211 (1997))。 For example, many techniques can be used by covalent or non-covalent bond to nucleic acids and amino acids directly on the chip, they anchor (anchoring) on the connector body portion, or connect them (tethering) on a fixed part ( . See, e.g., MethodsMol Biol Vol 20 (1993), Beier et al., Nucleic Acids Res 27:... 1970-1-977 (1999), Joos et al., Anal Chem 247:. 96-101 (1997) ., Guschin et al., Anal.Biochem 250: 203-211 (1997)). 纳米孔的表面可以进行等离子体蚀刻以允许固定探针或引物。 Surface nanopores plasma etching may be fixed to allow the probe or primer.

[0102] 如本文所用,术语"化学反应"指包括物质的化学性质改变的任何过程。 [0102] As used herein, the term "chemical reaction" refers to any chemical process including material properties altered. 这样的过程包括涉及生物分子(如蛋白质、糖蛋白、核酸、脂质)和无机化学物或其任意组合的形形色色的反应。 Such a process including those involving biological molecules (such as proteins, glycoproteins, nucleic acids, lipids) and any combination of inorganic chemicals or the kinds of reactions. 所述芯片在需要不同温度的化学和生物学应用中具有广泛的用途。 The chip has a wide range of uses require different temperature chemical and biological applications. 化学反应也可以包括核酸分子之间、蛋白质之间、核酸和蛋白质之间、蛋白质和小分子之间的相互作用。 Chemical reaction between the nucleic acid molecules may also include protein-protein interactions between nucleic acids and proteins proteins and small molecules,,,. 通过酶催发生的过程也称为"酶促反应"。 By enzymatic process occurs also known as "enzymatic reaction."

[0103] 所述芯片和其它装置在进行酶促反应中尤其有用,因为大部分酶只在一定温度下发挥功能。 [0103] The chips and other devices during the enzymatic reaction is particularly useful because most of the enzymes play only function at a certain temperature. 尤其依赖温度的代表性的酶促反应包括,但不限于核酸扩增(如定量聚合酶链反应(qPCR))、核酸测序、逆向转录和核酸连接。 In particular, the temperature dependence of the enzymatic reaction typically include, but are not limited to nucleic acid amplification (e.g., quantitative polymerase chain reaction (qPCR)), nucleic acid sequencing, reverse transcription and nucleic connection. 在一种实施方式中,在本发明的芯片上进行的核酸扩增反应是实时聚合酶链反应。 In one embodiment, the nucleic acid amplification reaction on a chip of the present invention is a real-time polymerase chain reaction. 在另一种实施方式中,核酸扩增反应是逆转录偶联的聚合酶链反应。 In another embodiment, the nucleic acid amplification reaction is reverse transcription-polymerase chain reaction coupled.

[0104] 本发明的芯片提供经济的用于扩增核酸的方法。 [0104] chip of the present invention provide an economical method for nucleic acid amplification. 不同于常规的微滴定板和热循环仪,所述芯片高度小型化,能够在小体积中和在独立的热程序下进行大量的目标核酸的快速扩增。 Unlike conventional microtiter plates and thermal cycler, a highly miniaturized chip, capable of small volume and rapid amplification of a large number of target nucleic acid in the independent thermal program.

[0105] 如本文所用,术语"核酸扩增"指其中目标核酸副本数目增加的酶促反应。 [0105] As used herein, the term "nucleic acid amplification" refers to a copy of the target nucleic acid which increases the number of enzymatic reactions. 这样的增加可以以线性或几何方式发生。 Such an increase may be linear or geometric manner. 可以通过天然或重组的DNA聚合酶(如Taq聚合酶、Pfu 聚合酶、T7DNA聚合酶、大肠杆菌DNA聚合酶的克林诺片段(Klenow fragment))和/或RNA 聚合酶(如逆转录酶)进行扩增。 By natural or recombinant DNA polymerase (e.g. Taq polymerase, Pfu polymerase, T7DNA polymerase, Ke Linnuo fragment of E. coli DNA polymerase (Klenow fragment)) and / or the RNA polymerase (e.g. reverse transcriptase) amplification.

[0106] —般地,聚合酶链反应(PCR)的目的是制造大量与初始供应的少量目标或种子DNA相同的DNA。 [0106] - In general, the purpose of the polymerase chain reaction (PCR) is to make a large number of the initial supply of a small amount of target DNA or seed same DNA. 该反应包括复制DNA的链,然后使用这些副本在随后的循环中产生另外的副本。 The reaction involves copying DNA strand, and then use these copies to produce additional copies in subsequent cycles. 各循环使存在的DNA量加倍,因而导致反应混合物中存在的目标DNA链的副本量成几何级数增长。 Each cycle to double the amount of DNA present, resulting in a copy of the amount of the reaction mixture in the presence of the target DNA strand geometric growth.

[0107] PCR的一般过程在美国专利第4, 683195号(Mullis)和第4, 683, 202号(Mullis 等人)中有记载。 [0107] The general procedure are described in U.S. Patent PCR 4th, 683,195 numbers (Mullis) and 4, 683, No. 202 (Mullis et al.). 简单地说,通过PCR的核酸扩增包括以下的重复循环:DNA的热变性,使两个引物退火结合到与待扩增的目标核酸片段侧邻的序列,和用聚合酶延伸退火的引物。 Briefly, by PCR amplification of a nucleic acid comprising repeated cycles of: heat denaturation of the DNA, the two primers anneal to the target nucleic acid fragments to be amplified flanking sequences, and extension of the annealed primers by the polymerase. 引物与目标核酸的相反链杂交并定向,以使得聚合酶的合成跨越引物之间的片段进行,从而有效地倍增目标片段的量。 Primers hybridize to opposite strands of the target nucleic acid and oriented such that the polymerase synthetic fragment spanning between the primers were, effectively doubling the amount of target fragments. 而且,因为延伸产物也与引物互补并能够结合引物,各个后续的循环基本上加倍前一循环中合成的目标核酸的量。 Moreover, because the extension products are also complementary to and capable of binding primers primers, each successive cycle essentially doubles the amount synthesized in the previous cycle of the target nucleic acid. 这导致特定的目标核酸以大约2n的速度指数累积,其中,n是循环的次数。 This leads to a specific target nucleic acid at a rate of approximately 2n accumulation index, where, n is the number of cycles.

[0108] 典型的常规PCR热循环方案包括以下的30个循环:(a)9(TC-95t:范围内的变性, (b)在50C -68"的温度范围退火,和(c)在68C _75"延伸。使用所述芯片,热循环时间可以急剧地减少,部分是由于小的反应体积、小的加热质量和有效散热特征的设计。 [0109] 可以在逆转录PCR反应(RT-PCR)中采用所述芯片。RT-PCR是常规PCR的另一种变型,其中逆转录酶首先将RNA分子转化为双链的cDNA分子,其随后在聚合酶链反应中用作后续扩增的模板。在进行RT-PCR时, 一般地在目标核酸加热变性之后,将逆转录酶加到反应样品中。然后在适当的温度下(例如,30-45t:)保持反应足够长的时间(例如,5-60 分钟),以在预定的扩增循环发生之前产生cDNA模板。这样的反应尤其用于检测其遗传信息储存于RNA分子中的生物实体。这类型的生物实体的非限制性的例子包括RNA病毒,如HIV和导致肝炎的病毒。通过本发明实施的RT-PCR的另一种重要的应用是基于在测试样品中检测到的mRNA水平对生物实体进行同步定量。本领域的技术人员可以理解:如果需要定量结果,那么必须注意使用保持或控制扩增的核酸的相对副本的方法。 [0110] 核酸"定量"扩增的方法是本领域的技术人员熟知的。例如,定量PCR(qPCR)可以包括使用同样的引物同时共扩增已知量的对照序列。这提供了可以用来校准PCR反应的内标。其它进行qPCR的方法是本领域已知的。 [0108] A typical conventional PCR thermal cycling program includes the following 30 cycles: (a) 9 (TC-95t: the range of variability, (b) at 50 C -68 "annealing temperature range, and (c) at 68 C _75 "extends the use of the chip, thermal cycle time can be reduced dramatically, in part due to the small reaction volume, small effective heating and cooling characteristics of the quality of the design. [0109] The reaction may reverse transcription PCR ( RT-PCR) used in the chip .RT-PCR is another variant of the conventional PCR, in which a reverse transcriptase is first converted to double-stranded RNA molecule cDNA molecule, which is then used in the subsequent expansion of the polymerase chain reaction growing template during RT-PCR, generally after heating the target nucleic acid denaturation, the reverse transcriptase reaction was added to the sample. Then at the appropriate temperature (e.g., 30-45t :) maintaining the reaction for a time sufficient (e.g., 5-60 minutes), prior to the occurrence of a predetermined amplification cycles generated cDNA template used for this reaction, especially biological entities which genetic information is stored in the detection of RNA molecules. Non-limiting types of biological entities which examples include RNA viruses such as HIV and hepatitis viruses cause. Another important application by the embodiment of the invention is based on RT-PCR detected mRNA levels in test samples of biological entities for Quantitative this field technical staff can understand: If you need quantitative results, so be careful to maintain or control the amplification of nucleic acids is relatively copy [0110] nucleic acid "quantitative" amplification methods are well known to those skilled in the art, such as quantitative. PCR (qPCR) can include using the same primers simultaneously co-amplifying a known quantity of a control sequence. This provides an internal standard that can be used to calibrate the PCR reaction. qPCR Other methods are known in the art.

[0111] —般使用扩增试剂进行核酸扩增。 [0111] - like nucleic acid amplification using amplification reagents. 扩增试剂典型地包括酶、水性缓冲液、盐、引物、 目标核酸和三磷酸核苷。 Typically, amplification reagents include enzymes, aqueous buffers, salts, primers, target nucleic acid, and nucleoside triphosphates. 根据环境设置,扩增试剂可以是完全的或不完全的扩增反应混合物。 According to the environment setting, amplification reagents may be complete or incomplete amplification reaction mixture.

[0112] 包含在本发明的芯片中的试剂取决于待进行的反应。 Reagents [0112] included in the chip of the present invention depends on the reaction to be performed. 在一种实施方式中,可编址单元的阵列的至少一个单元包含用于进行核酸扩增反应的试剂。 In one embodiment, programmable array addressing unit is at least one unit containing reagents for nucleic acid amplification reactions. 试剂可以是用于免疫分析、核酸检测分析(包括但不限于核酸扩增)的试剂。 Reagent may be used for immunoassay, nucleic acid detection (including but not limited to nucleic acid amplification) reagents. 试剂在芯片单元中可以处于干燥状态或液体状态。 Reagent in the chip unit may be in a dry state or a liquid state.

[0113] 在一种实施方式中,能够进行核酸扩增反应的芯片的可编址单元阵列中的至少一个单元包含以下的至少一种:探针、聚合酶和dNTP。 [0113] In one embodiment, the nucleic acid amplification reaction can be performed chips addressable cell array in at least one unit containing at least one of the following: probe, polymerase, and dNTP. 在另一种实施方式中,芯片的纳米孔包含包括探针、引物和聚合酶的溶液。 In another embodiment, the chip contains nanopores include probes, primers and polymerase solution. 在各种实施方式中,各个室包括:(l)用于所述标准基因组内的聚核苷酸靶标的引物,和(2)与所述引物有关的探针,如果引物结合所述靶标,探针发射浓度依赖性的信号。 In various embodiments, each chamber comprising: (l) for a target polynucleotide primers within the genome of the standard, and (2) associated with the primer probe, if the target primer binding, Probe Launcher concentration-dependent signal.

[0114] 在各种实施方式中,各单元包括用于基因组内的聚核苷酸靶标的引物和与引物有关的探针,如果引物结合所述靶标,探针就发射浓度依赖性的信号。 [0114] In various embodiments, each unit including a probe for the relevant target polynucleotide primer and a primer genome, if the primer binding target, probe concentration-dependent signal is emitted.

[0115] 在另一种实施方式中,芯片的至少一个单元包含包括正向PCR引物、反向PCR引物和至少一个FAM标记的MGB淬火的PCR探针(FAMlabeled MGB quenched PCR probe)的溶液。 [0115] In another embodiment, the chip contains at least one unit comprises a forward PCR primer, a reverse PCR primers and at least a FAM-labeled MGB probes quenched PCR (FAMlabeled MGB quenched PCR probe) was added.

[0116] 在一种实施方式中,将引物对分配到单元中,然后干燥,如通过冷冻。 [0116] In one embodiment, the primer pair assigned to the unit, and then dried, eg by lyophilization. 然后使用者 The user then

可以选择性地分配(如纳米分配(nano-dispense))样品、探针和/或聚合酶,。 It can selectively assign (such as nano allocation (nano-dispense)) sample, the probe and / or polymerase.

[0117] 在本发明的其它实施方式中,纳米孔可以包含干燥形式的任何上述溶液。 [0117] In other embodiments of the present invention, the nano holes may comprise any of the above solution was dried form. 在本实 In the present

施方式中,这种干燥形式可以涂覆到孔上,或引导到孔的底部。 Embodiment, such a dried form may be coated onto the hole, or the guide into the bottom of the hole. 使用者可以在分析前向各个 Before the user can analyze the various

孔加入水和样品的混合物。 Well was added a mixture of water and samples.

[0118] 在本实施方式中,包含干燥(dried down)的反应混合物的芯片可以用衬里(liner)密封,存储或运送到另一个地点。 [01] In the present embodiment, the chip contains dried (dried down) in the reaction mixture can be used lining (liner) sealed, stored or transported to another location. 衬里可整体剥离而不会损害粘合剂的均匀性。 The liner can be peeled off without impairing the uniformity of the overall adhesive. 衬里视觉上不同于盖子,以有助于辨认和易于处理。 Unlike lined lid visually to help identify and easy to handle. 选择衬里的材料以使从粘合剂剥离衬里时静电荷的产生最小化。 Select the liner material so that the electrostatic charge generated when the release liner from the adhesive is minimized. 当使用者准备使用芯片时,破坏密封,除去衬里,并向芯片的单元中加入样品。 When the user is ready to use chips, breaking the seal, remove the liner, the unit is added to the chip samples. 然后,可以密封芯片,并使其接触加热元件。 Then, the chip can be sealed, and allowed to contact with the heating element.

[0119] 在许多应用中,需要密封单元(例如,纳米孔)以防止液体蒸发,并因此在整个热循环过程中保持优选的反应浓度。 [0119] In many applications, you need to be sealed unit (e.g., nanopores) to prevent liquid evaporation, and thus maintaining the preferred reaction concentration throughout thermal cycling.

[0120] 芯片可以用于确定基因型(genotyping)、基因表达或通过PCR进行其它DNA测试。 [0120] chip can be used to determine the genotype (genotyping), gene or other DNA testing by PCR. 在板中进行的分析不限于DNA分析(如Taqman、 Invader、Taqman Gold、SYBR gold和SYBR green),也包括其它分析(如受体结合、酶和其它高通量筛选分析)。 Analysis carried out in the plate is not limited to DNA analysis (e.g. Taqman, Invader, Taqman Gold, SYBR gold and SYBR green), but also include other analysis (e.g., receptor binding, enzyme and other high-throughput screening assays). 在某些实施方式中,使用R0X标记的探针作为内标。 In certain embodiments, the use of labeled probes R0X as an internal standard.

[0121] 本发明也提供了使用包括多个预加载的纳米孔的芯片进行PCR分析的方法,该方法包括:将样品置于纳米孔中以产生反应混合物;用矿物油或另一种密封机构密封芯片的纳米孔;将芯片置于热循环系统;循环该系统;并分析结果。 [0121] The present invention also provides the use of a plurality of nanopores include preloaded chip subjected to PCR analysis, the method comprising: placing the sample in order to produce nanopores reaction mixture; with mineral oil or another sealing mechanism seal the nanopore chip; the chip is placed in a thermal cycle system; circulating the system; and analyze results.

[0122] 根据本发明,芯片的单元包括可操作进行多重PCR的溶液。 [0122] operably multiplex PCR was according to the present invention, the unit comprises a chip. 在优选的实施方式中, 单元能够根据化学性质和包括在溶液中的探针在各个单独的单元中进行多重PCR反应。 In a preferred embodiment, the unit is capable of multiplex PCR reactions each individual unit in accordance with the chemical nature and the probe comprises in solution. "多重PCR"是在同一单元中使用超过一个引物对。 "Multiplex PCR" is to use more than one pair of primers in the same unit. 该方法可以用于相对定量,其中,一个引物对扩增目标,而另一个引物对扩增内部基准(endogenous reference)。 The relative quantification method can be used, wherein a primer pair to amplify the target, while the other primer pairs to amplify the internal reference (endogenous reference). 可以使用包括标准曲线方法或比较Ct方法的各种方法进行多重反应。 Various methods may be used including standard curve method or the comparative Ct method multiplex reaction.

[0123] 可以使用以下探针,如FAM,其是羧基荧光素,具有大约485nm的激发波长和大约510-520nm的发射波长;SYBR Green l,其通常用于RT-PCR,且具有大约488纳米的激发波长和大约510纳米的发射波长;TET,具有大约517纳米到大约538纳米的发射波长;选自HEX、J0E和VIC的探针,其具有525-535nm到大约546_556nm的发射波长;TAMRA,其是羧基四甲基若丹明,且具有大约556纳米到大约580纳米的发射波长;R0X,其是羧基_x_若丹明,具有大约575-585nm到大约605_610nm的发射波长;ALEXA,具有大约350纳米到大约440纳米的发射范围;TEXAS RED,具有大约580_585nm到大约600-610nm的发射波长;Cy3, 具有大约545纳米到大约568纳米的发射波长;Cy5,具有大约635-655nm到大约665_675nm的发射波长;Cy7,具有大约715纳米到大约787纳米的发射波长。 [0123] The following probes can be used, such as FAM, which is carboxyfluorescein, with an excitation wavelength of about 485nm and an emission wavelength of about 510-520nm; SYBR Green l, which is typically used for RT-PCR, and has about 488 nm The excitation and emission wavelengths of about 510 nanometers; TET, having an emission wavelength of about 517 nm to about 538 nm; selected from HEX, J0E and VIC probe, which has 525-535nm emission wavelength to about 546_556nm; TAMRA, which is a carboxy-tetramethyl-rhodamine, and having an emission wavelength of about 556 nanometers to about 580 nanometers; R0X, which is a carboxy rhodamine _x_, of about 575-585nm emission wavelength to about 605_610nm; ALEXA, having transmission range of about 350 nm to about 440 nm; TEXAS RED, having about 580_585nm to the emission wavelength of about 600-610nm; Cy3, having an emission wavelength of about 545 nm to about 568 nm; Cy5, having about 635-655nm to about 665_675nm The emission wavelength; Cy7, having an emission wavelength of about 715 nm to about 787 nm. 优化的干涉滤波器精确地对各个荧光团匹配激发和发射波长,以排除(block out)来自光谱邻近的荧光团的不希望的干扰(cross-talk)。 Interference filter optimized precisely match each fluorophore excitation and emission wavelengths, to exclude (block out) from the spectrum adjacent fluorophore unwanted interference (cross-talk).

[0124] 用于核酸扩增的引物的选择取决于目标核酸序列。 [0124] The choice of nucleic acid amplification primers used depends on the target nucleic acid sequence. 用于本发明的引物一般是寡核苷酸,通常是长度为几个核苷酸的脱氧核糖核苷酸,其可以通过聚合酶链反应以模板特异性的方式延伸。 Primers used in the present invention are generally oligonucleotides, usually the length of deoxyribonucleotides several nucleotides, which can be extended in a template-specific manner by the polymerase chain reaction. 用于扩增目标核酸的合适的引物的设计可以由本领域的技术人员确定。 Suitable for primers designed to amplify the target nucleic acid can be determined by one skilled in the art. [0125] 对于源自PCR或者上文所述的或本领域已知的任何其它核酸扩增反应的扩增核酸的常规检测,可以将引物与可检测的标记偶联。 Routine testing [0125] For PCR, or from any of the above or other art-known nucleic acid amplification reaction of nucleic acid amplification, the primers may be labeled with a detectable conjugate. 适用于本发明中的可检测的标记包括任何可通过光谱、光化学、生物化学、免疫化学、电学、光学或化学手段检测的成分。 Useful in the present invention include any detectable label component can be detected by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. 本领域的技术人员已知许多种合适的可检测标记,其包括发光的标记、酶或其它配体。 It is known to a person skilled in the art of many kinds of suitable detectable label, including luminous label, an enzyme or other ligand. 在优选的实施方式中,技术人员很可能希望采用荧光标记或酶标签,如洋地黄毒苷(digoxigenin)、 P _半乳糖苷酶、脲酶、碱性磷酸酶或过氧化物酶、抗生物素蛋白/生物素复合物。 In a preferred embodiment, it may be desirable to employ skilled fluorescent label or an enzyme tag, such as digoxigenin (digoxigenin), P _-galactosidase, urease, alkaline phosphatase or peroxidase, avidin protein / biotin complex. [0126] 可以通过本领域的技术人员熟知的许多种方法中的任何一种引入标记。 [0126] mark can be introduced by those skilled in the art in many ways well known to any one. 一个方面,在扩增步骤过程中同时引入标记。 One aspect, the amplification step in the process while introducing mark. 因此,例如,采用具有标记的引物或标记的核苷酸的PCR可以提供标记的扩增产物。 Thus, for example, using PCR primers having a nucleotide labeled or tagged amplification product labeled may be provided. 在独立的一个方面中,其中使用标记的核苷酸(例如,荧光素_标记的UTP和/或CTP)或标记的引物将RNA转化为DNA的转录反应将可检测的标记引入转录的核酸中。 In a separate aspect, wherein a labeled nucleotide (e.g., fluorescein-labeled UTP _ and / or CTP) primers or labeled RNA into the DNA of the transcription reaction detectable label introduced nucleic acid transcribed .

[0127] 本发明也提供了适于进行本发明的多核苷酸扩增方法的试剂和试剂盒。 [0127] The present invention is also suitable for the present invention provides a polynucleotide amplification reagents and kits. 这样的试剂和试剂盒可以模仿适于进行常规的PCR、 RT-PCR和其它扩增反应的试剂和试剂盒。 Such reagents and kits can mimic suitable for conventional PCR, RT-PCR and other amplification reaction reagents and kits. 这样的试剂盒包括本发明的芯片和选自扩增试剂、检测试剂及其组合的试剂。 Such a kit of the present invention comprises a chip and selected amplification reagents, detection reagents and reagent combinations. 试剂盒可以包括用于多重扩增产物的下游或后续分析的包装试剂。 The kit may comprise multiple amplification product downstream or subsequent analysis reagent packaging. 包括在单个单元中的引物彼此独立地可以是相同或不同包括多个多重扩增引物的引物组。 Included in a single unit independently of each other primers may be the same or different comprising a plurality of multiplex amplification primer set of the primers.

[0128] 在另一种实施方式中,寡核苷酸探针适于检测单核苷酸多态性,这是本领域中公知的。 [0128] In another embodiment, the oligonucleotide probes suitable for the detection of single nucleotide polymorphisms, which are well known in the art. 这样的探针的具体的例子包括4个寡核苷酸探针的组,除一个核苷酸位置之外,其序列是相同的。 Specific examples of such probes include oligonucleotide probes four groups, except for one nucleotide position, which is the same sequence.

[0129] 4个探针在该位置各包括不同的核苷酸(A, G, C和T/U)。 [0129] In the 4 position of each probe comprises a different nucleotide (A, G, C and T / U). 可以用能够产生可彼此区别开的不同检测信号的标记物标记探针,如能够在不同的、可光谱分辨的波长处发射光的不同荧光团(例如,4种不同颜色的荧光团)。 Markers can be used to produce labeled probes can be distinguished from one another in different detection signals, such as the ability in different spectral resolution can emit light at a wavelength different fluorophores (for example, four different colors of fluorophores).

[0130] 可以通过化学合成、重组体克隆或其组合获得用于本发明的引物对。 [0130] can be produced by chemical synthesis, or a combination of recombinant clones obtained for the primer of the present invention. 本领域中熟知化学多核苷酸合成的方法,因而不需在本文中描述。 It is known in the art of chemical polynucleotide synthesis methods, which need not be described herein. 本领域的技术人员可以通过采用DNA 合成仪或从商业服务定购,使用目标序列获得需要的引物对。 Those skilled in the art by using a DNA synthesizer or ordering from a commercial service, using the target sequence to obtain the desired primer pairs.

[0131] 核酸扩增需要在与用于扩增靶标的酶相容的缓冲液中的目标核酸。 [0131] Nucleic acid amplification required for the amplified target nucleic acid in the target enzyme and compatible buffer. 用于本发明的目标核酸包括任何怀疑包含目标序列的反应样品。 For the target nucleic acid of the present invention include any reaction sample suspected of containing the target sequence. 本发明并不意图限制反应样品的来源或其制备方式。 The present invention is not intended to limit the source of the sample or reaction mode of preparation. 一般地,测试样品可以是生物样品和/或环境样品。 Generally, the test sample may be a biological sample and / or environmental samples. 生物样品可以源自人类、其它动物或植物、体液、实体组织样品、组织培养物或由其得到的细胞及其后代,或从这些来源中的任何一种制备的部分或涂片、或任何其它怀疑包含目标核酸的样品。 Biological samples can be derived from humans, other animals or plants, fluid, solid tissue samples, tissue cultures or cells derived therefrom and their descendants, or from any part of these sources or smear a preparation or any other a sample suspected of containing the target nucleic acid. 优选的生物样品是体液,包括但不限于血液、尿液、脊髓液、脑脊液、滑液(sinovial fluid)、含氨的液体、精液和唾液。 Preferred biological samples are body fluids, including but not limited to blood, urine, spinal fluid, cerebrospinal fluid, synovial fluid (sinovial fluid), ammonia-based liquids, semen and saliva. 其它类型的生物样品可以包括食物制品和如蔬菜、牛奶制品(dairy items)、肉、肉副产品(meat by-products)和废料的成分。 Other types of biological samples may include products and components of vegetables, milk products (dairy items), meat and meat by-products (meat by-products) and waste food such. 环境样品源自环境物质,包括但 Environmental samples from environmental agents, including, but

17不限于土壤、水、污水、化妆品、农业和工业样品。 17 is not limited to soil, water, sewage, cosmetics, agricultural and industrial samples.

[0132] 可以根据本领域中的标准方法或所述的程序进行包含在测试样品中的核酸的制备。 [0132] can be prepared containing the nucleic acid in the test sample according to standard methods in the art or the procedures. 简单地说,可以使用各种溶胞酶或化学溶液根据Sambrook等人(〃 Molecular Cloning :A Laboratory Manual 〃 )提出的程序分离DNA和RNA,或按照生产商提供的说明书通过核酸结合树脂提取DNA和RNA。 Simply put, you can use a variety of lytic enzymes or chemical solutions according to Sambrook et al. (〃 Molecular Cloning: A Laboratory Manual 〃) isolating DNA and RNA procedures proposed, in accordance with the instructions provided by the manufacturer, or a combination of extracted DNA by nucleic acid resin and RNA.

[0133] 反应样品中的核酸可以是cDNA、基因组DNA或病毒DNA。 [0133] The reaction sample nucleic acid may be cDNA, genomic DNA or viral DNA. 但是,本发明也可以使用如mRNA、核糖体RNA、病毒RNA的其它核酸实施。 However, the present invention may also be used such as other nucleic acids mRNA, ribosomal RNA, viral RNA implementation. 这些核酸可以以各种拓扑学结构(topologies)存在。 These nucleic acids can be in a variety of topological structures (topologies) exists. 例如,核酸可以是单链、双链、环状、线性的或串联体(concatamers)的形式。 For example, in the form of single-stranded nucleic acid can be double-stranded, cyclic, linear or series body (concatamers) of. 本领域的技术人员可以认识到:无论核酸的性质如何,只要通过对所用的方法进行合适的且良好确认的改良就可以进行扩增。 Those skilled in the art will recognize that: regardless of the nature of nucleic acids, as long as the method used to make improvements suitable and well acknowledged can be amplified.

[0134] 本发明的一方面,用于进行反应的芯片包括可编址单元的阵列,各个单元设置用于化学反应。 Chip aspect [0134] of the present invention, for carrying out the reaction site comprises an array of programmable cells, each unit is provided for the chemical reaction. 根据用于运行单元中的化学反应的预定温度设置排列芯片的可编址单元。 According to a predetermined temperature for running the chemical reaction unit setting arrangement chip addressable unit. 至少一个单元编址为表明用于进行单元中的化学反应的预定温度。 At least one unit for performing addressed to indicate the predetermined temperature unit in a chemical reaction.

[0135] 在一种实施方式中,多个单元被编址以表明用于进行化学反应的预定温度。 [0135] In one embodiment, a plurality of units is addressed to indicate the predetermined temperature for carrying out chemical reactions. 在另一种实施方式中,各个单元是单独编址以表明用于进行所述单元中的化学反应的预定温度。 In another embodiment, each unit is individually addressed to indicate the predetermined temperature of the unit for carrying out chemical reactions.

[0136] 本发明的芯片的单元的预定温度可以设置为由化学反应产生至少90%的同质产物。 [0136] the predetermined temperature of the chip unit of the invention may be provided by a chemical reaction to produce at least 90% of homogeneous product. 如果优化退火温度,基于预设的反应温度的可编址芯片的配置可以明显地提高反应产物的产量和质量。 If the annealing temperature optimization, configuration addressable chips based on a preset reaction temperature can significantly improve the yield and quality of the reaction products. 当使用者意图用本发明的芯片和装置分析如生物体的全基因组的大量核酸时,这可能是重要的。 When the user intent with chip and apparatus of the present invention is a nucleic acid analysis such as a large genome of the organism, which may be important. 在一种实施方式中,当用作医学诊断仪器时,提高的反应产量在本发明的芯片、装置或系统的应用中发挥重要的作用。 In one embodiment, when used as a medical diagnostic instrument, the reaction yield improved play an important role in the application of the chip, device, or system of the present invention.

[0137] 图5说明本发明的示例芯片500,包括一组较小的芯片510。 [0137] Figure 5 illustrates an example of the present invention, the chip 500 includes a set of smaller chips 510. 这组较小的芯片510, 也称作板500,接触热绝缘框架530。 This set of smaller chips 510, also known as plate 500, contact heat insulating frame 530. 各个较小的芯片510代表本实施例的不同的温度带, 如图5所标记的,带1、2、3、4、5和6。 Different temperature zones on behalf of each of the smaller chip 510 of the present embodiment, as shown in Figure 5 the marks with 1,2,3,4,5 and 6. 在一种实施方式中,温度带对应于用于在芯片500的纳米孔520中进行聚合酶链反应的预定退火温度设置。 In one embodiment, the temperature zone corresponding to a predetermined annealing temperature is set for performing the polymerase chain reaction in the nanopore chip 500. 520. 通过将整体芯片500划分成不同的温度带,使用者可以提高纳米孔520中的反应的特异性。 By the overall chip 500 is divided into different temperature zones, the user can improve the specificity nanopores 520 reactions. 例如,当分析物种的完基因组时, 多个温度带可以允许对基因组进行更准确的测定。 For example, when analyzing the complete genome of the species, multiple temperature zones allows for genome more accurate determination.

[0138] 所述芯片可以包含一个或多个在芯片的底侧蚀刻的沟槽。 [0138] The chip may comprise one or more chips in the bottom side of the trench etching. 一般地,沟槽是允许气体通过的底沟(under-trenches)、开放的通道或路径。 Generally, the trench is to allow the bottom groove (under-trenches), open channel or path through the gas. 沟槽减少芯片的热质量,增加表面积,因而提高芯片的热性能。 Reduce the thermal mass of the chip grooves, increasing the surface area, thereby improving the thermal performance of the chip. 沟槽可以制成任何形状,包括但不限于圆形、椭圆形、卵形、圆锥形、矩形、三角形和多边形。 Grooves may be formed in any shape, including but not limited to circular, elliptical, oval, conical, rectangular, triangular, and polygonal. 沟槽可以进一步被线性或弯曲的壁分割,以形成分隔但邻近的通道,或通过圆形壁分割以形成内和外的环形通道。 Trench may be further divided linear or curved walls to form separate but adjacent channel, or divided by a circular wall with inner and outer annular channel is formed. 沟槽的尺寸规格取决于芯片的总体大小和深度。 The size of the trench depends on the overall size and depth of the chip. 沟槽的深度可以是芯片深度的大约1/10-大约9/10。 Depth of the trench depth can be the chip of about 1/10 to about 9/10. 其它尺寸(即宽度和长度)可能更短、更长或与芯片的相应尺寸相当。 Other dimensions (ie width and length) may be shorter, longer or the chip size is quite appropriate. 尤其,L型的沟槽围绕单元的基底。 In particular, L-type trench around the base unit. 随着空气流过由任一沟槽形成的通路,它通过被动的热逸散从单元的表面除去热量,因而增加热循环的速度。 As the air flows through the passages formed by either a groove, through passive thermal dissipation of heat is removed from the surface of the unit, thus increasing the speed of thermal cycling.

[0139] 几种因素关系到合适的芯片片基的选择。 [0139] Several factors related to the right to select the Chip group. 片基经常是良好的热导体。 Film base is often a good thermal conductor. 良好的热导体一般具有高于lW/m—t1的热传导率,优选高于100W/m—t、更优选高于140W/m—t、尽管材料的热传导率可以是250W/m—1或更高,它通常不超过500W/m—、第二,片基必须是相对惰性和化学稳定的。 Good thermal conductor generally has a higher lW / m-t1 thermal conductivity, preferably higher than 100W / m-t, more preferably greater than 140W / m-t, although the thermal conductivity of the material may be a 250W / m-1 or more high, it is usually not more than 500W / m-, second, the base sheet must be relatively inert and chemically stable. 这样的片基一般显示与用于意图的应用中的反应样品反应的低水平倾向。 Such support generally show low tendency to apply for the intent of the reaction samples Reaction. 另外,也应该根据在其上或其邻近整合热控制元件的能力或可行性选择材料。 In addition, it should be based on it or near capacity integrated thermal control element or the feasibility of the choice of materials. 多种材料满足这些标准。 A variety of materials to meet these criteria. 示例性的材料包括但不限于,类金属或半导体,如硅、硅酸盐、氮化硅、 二氧化硅、磷化镓、砷化镓或其任何组合。 Exemplary materials include, but are not limited to, metals or semiconductors, such as silicon, silicates, silicon nitride, silicon dioxide, gallium phosphide, gallium arsenide, or any combination thereof. 其它可能的材料是玻璃、陶瓷(包括结晶和非结晶的硅酸盐和非硅酸盐基的陶瓷)、金属或合金,包含掺杂剂(例如,提高热导率的氧化铝) 的复合聚合物,或本领域可获得的许多种塑料和有机聚合材料中的任一种。 Other possible materials are glass, ceramics (including crystalline and amorphous silicates and non-silicate-based ceramic), metal or alloy, comprising dopant (e.g., to improve the thermal conductivity of alumina) polymerizable compound material, or available in the art many types of plastic and organic polymeric materials either. 在一种实施方式中,在包括Al或SS-316以及类似的其它物质的这样的片基中制备纳米孔。 In one embodiment, the preparation of nano holes in a sheet such groups include Al or SS-316 and other similar substances. [0140] 在一种实施方式中,使用导热聚合物制造芯片。 [0140] In one embodiment, the use of thermally conductive polymer manufacturing chips. 例如,可以使用聚碳酸酯、聚丙烯或本领域的技术人员已知的任何其它导热聚合物制备芯片。 For example, polycarbonate, polypropylene, or known to those skilled in the art of any other thermally conductive polymers chip.

[0141] 可以使用本领域的技术人员显而易见的任何方法制备芯片。 [0141] You can use this skill in any method apparent chip. 制备本发明的芯片的方法的例子包括但不限于:微型打孔法、放电法、热模压印(hot embossing)和使用从以水作为光导制备的工具的热模压印。 Examples chip method of the present invention include, but are not limited to, preparation: Micro drilling method, discharge method, hot embossing (hot embossing) and use water as a tool from the light guide prepared by hot embossing.

[0142] 或者,可以使用在集成电路(IC)和微机电系统(MEMS)工业中沿用已久的技术制备本发明的芯片。 [0142] Alternatively, you can use the integrated circuit (IC) and micro-electromechanical systems (MEMS) industry-established technique for preparing the present invention chips. 通常通过以下步骤进行制造过程:选择芯片片基,接着使用适当的IC处理方法和/或MEMS微机械加工技术构建和集成各种元件。 Typically the manufacturing process through the following steps: Select the chip-chip-based, building and integrating the various components of the IC and then use the appropriate treatment and / or MEMS micromachining technology.

[0143] 可以根据IC处理和/或MEMS微机械加工的标准技术进行所述芯片的制造。 [0143] The chip can be manufactured in accordance with standard IC process technology and / or MEMS micromachining. 所述芯片可以制造为多层结构。 The chip can be manufactured as a multilayer structure. 该过程一般通过构建底层进行。 The process is generally carried out by constructing the bottom. 然后,采用包括但不限于光刻术、化学气相沉淀或物理气相沉淀、干或湿蚀刻技术的组合来构建位于其上或植入其中的结构。 Then, using, including but not limited to, lithography, chemical vapor deposition or physical vapor deposition, dry or wet etching techniques in combination to build thereon wherein the structure or implant. 例如,气相沉淀能够在其它材料上制造极薄和均一的涂层,而蚀刻容许较大芯片结构的大量生产。 For example, vapor deposition can be made thin and uniform coating on other materials, and allow mass production of larger chips is etched structure. 其它有用的技术,如离子注入、等离子灰化、粘合和电镀,也可以用来改善芯片的表面性质或集成芯片的各种元件。 Other useful techniques, such as ion implantation, plasma ashing, and plating adhesion, can also be used to improve the surface properties of the chip or integrated chip various elements. 下面参考附图中描述的示例性的芯片设计详述制造过程。 Referring to the drawings in the following exemplary description of chip design detailing manufacturing process. 同样的一般过程及其明显的变型适用于制造本文所描述的所述芯片的任一种。 The same general process and obvious variants described herein for the manufacture of any of the chip. [0144] 图4是示例性的芯片设计的一部分的截面图(cross-section view)。 [0144] FIG. 4 is a sectional view (cross-section view) of a portion of an exemplary chip design. 在这种实施方式中,将纳米孔嵌入分别由第一和第二(或底面和顶面)层的片基组成的主体中。 Subject In this embodiment, the nanopores embedded by the first and second (or bottom and top surfaces) film base layer thereof. 该过程以提供一般是耐热材料(如玻璃、耐热玻璃晶片(Pyrex wafer)或本文所述的或本领域已知的任何其它合适的材料的第一层片基开始。下一步是产生形成单元的基础的纳米孔。 纳米孔一般位于通常是硅晶片(参见,例如,图4)的第二层中。硅晶片可以在贴附在第一层上之前经过几个处理步骤。例如,硅晶片可以附在光致抗蚀剂层上,以使得表面在光刻处理的过程中暴露于紫外光之后更易进行化学蚀刻。光致抗蚀剂层通过光掩模的精确对齐限定通过随后的蚀刻步骤形成的纳米孔的大小和位置。然后通过多种本领域已知的方法蚀刻硅晶片以形成孔腔。普遍实用的蚀刻技术包括除去硅晶片以形成需要的形状的化学制品的使用,例如,氢氧化钾(K0H)。 The procedure is generally to provide a heat resistant material (such as glass, heat-resistant glass wafer (Pyrex wafer) or described herein or first layer of the film base known in the art of any other suitable material begins. The next step is to generate form Basic unit nanopores. nanopore usually located usually silicon wafer (see, e.g., FIG. 4) a second layer of silicon wafer can be attached on the first layer until after several processing steps, for example, silicon wafer may be attached to the photo-resist layer on, so that the surface is exposed during the photolithography process after UV light at more chemically etched photoresist layer through a photomask aligned precisely defined by subsequent etching the step of forming the nanopore size and location is then formed by a variety of methods known in the art bore etching a silicon wafer. widespread practical techniques include etching a silicon wafer is removed to form a desired shape using chemicals, e.g., Potassium hydroxide (K0H).

[0145] —旦制造了所述芯片的纳米孔,那么可以改善它们的表面性质以适应特定的应用。 [0145] - Once created nanopores of the chip, you can improve their surface properties to suit a particular application. 在需要大的表面积的地方,可以通过等离子体蚀刻器进一步蚀刻纳米孔的壁以获得非常细的硅枝状晶体(dendrites),其通常称为"黑硅(black silicon)"。 In the large surface area where needed, by plasma etching is further etched nanopores wall to obtain a very fine silicon dendrite (dendrites), which is generally referred to as "black silicon (black silicon)". 黑硅的存在可以急剧增加有效的加热表面积。 The existence of black silicon can dramatically increase the effective heating surface area. 在纳米孔的基部制造的黑硅也可以用作感光(photon-sensing) 设备、温度传感器和其它控制元件的支座(anchor)。 Black silicon at base nanopores can also be used for manufacturing a photosensitive (photon-sensing) device, temperature sensors and other control elements abutment (anchor).

[0146] 如本文所讨论,可以用材料涂覆具有高的内表面积-体积比的纳米孔,以减少其中包含的反应物与孔的内表面相互作用的可能性。 [0146] As discussed herein, can be coated with a material having a high internal surface area - nanopore volume to reduce the possibility of the inner surface of which contains a reactant interaction with the hole. 应用涂层材料的方法的选择取决于所用的涂层材料的类型。 The method of application of the coating material of choice depends on the type of coating material. 一般地,通过直接向纳米孔涂覆材料,接着通过洗去过量的未结合的涂层材料进行涂覆。 Generally, by providing nanoporous coating material, followed by washing off excess unbound coating material coated directly. 某些涂层材料可以经过广泛的加热、辐射和通过化学反应而与表面交联。 Certain coating materials can be subjected to a wide range of heating, radiation and by chemical reaction with the surface cross-linking. 本领域的技术人员了解其它合适的用于在芯片上涂覆制造的纳米孔的方法,或者能够无需过多的实验而确定这些方法。 Those skilled in the art will appreciate other methods suitable for nanoporous coating on the chip manufacturing, or can be determined without undue experimentation methods.

[0147] 样品制备然后包括将PCR反应样品混合物、标记的引物和样品与可以单独地纳米分配以防止蒸发的油滴结合。 [0147] Sample preparation and PCR reaction sample mixture comprising labeled primers and sample can be assigned individually to prevent evaporation of nano droplets binding. 然后样品使用压力分配器和露点(dew point)分配技术分配到单独的纳米孔中。 The sample is then pressure dispenser and dew point (dew point) is assigned to a separate allocation techniques nanopores.

[0148] 为防止水性反应样品的蒸发,可以将样品应用到处于露点或其附近的纳米孔中。 [0148] In order to prevent evaporation of the aqueous reaction of the sample, the sample can be applied to the dew point at or near the nanopores. 如本文所用,"露点"指小滴的大小不会明显变化的温度范围。 As used herein, "dew point" means the droplet size does not significantly change the temperature range. 处于露点时,在水从样品滴蒸发的速度与水从芯片上方的潮湿空气凝结到样品滴上的速度之间达到平衡。 While in the dew point, the water droplets from the sample rate of evaporation and water condensation from moist air to the top of the chip between the sample droplets on the speed of balance. 当实现该平衡时,据说相对于芯片平面的空气达到饱和。 When implementing this balance, it is said with respect to the plane of the chip saturated air. 在一个大气压力下,露点是大约i4t:。 At an atmospheric pressure, dew point is about i4t :. 因此,优选在露点之上不超过大约rc -大约5t:的温度下进行水性反应样品的分配。 Therefore, preferably above the dew point of no more than about rc - about 5t: partitioned sample under aqueous reaction temperature. 本领域的技 Skill in the art

术人员清楚的是:露点温度随外部压力增加而升高。 The art is clear: the dew point temperature increases with increasing external pressure. 因此,在需要时,技术人员可以在加压环境中分配反应样品以防止蒸发。 Thus, when required, the technician may be allocated a pressurized environment in the reaction sample to prevent evaporation.

[0149] 可以通过一系列方法检测所述芯片中存在的扩增的核酸,包括但不限于:(a)例如通过将扩增的产物与可检测的标记结合形成可检测的复合物;和(b)电泳解析扩增的产物与反应物和扩增反应的其它成分。 [0149] through a series of amplified nucleic acid can be detected in the presence of the chip, including but not limited to: (a) e.g. by amplified product with a detectable label to form a detectable complex; and ( b) Electrophoresis Analytical amplified product with the reactants and other components of the amplification reaction.

[0150] 在一定的实施方式中,用如荧光DNA结合染料的可检测标记物使扩增的产物可视化。 [0150] In certain embodiments, such as a fluorescent DNA binding dye with a detectable label so that the amplified product was visualized. 因为插入双链DNA分子的染料的量通常与扩增的DNA产物的量成比例,技术人员可以通过使用本发明的光学系统或本领域的其它合适的设备定量插入染料的荧光,方便地测定扩增的DNA产物的量。 Because the amount of insertion of the double-stranded DNA molecule of dye is generally proportional to the amount of the amplified DNA product, the art by using an optical system of the present invention or other suitable device is inserted in the art of quantitative fluorescent dye, to facilitate expansion measured increasing the amount of DNA product. 适于这一应用的DNA结合染料包括SYBR green、 SYBRblue、 DAPI、碘化丙啶(propidium iodine) 、 Hoeste、 SYBR gold、溴化乙锭(ethidium bromide) 、口丫啶(acridines)、原黄素(prof lavine) 、 口丫啶澄(acridine orange) 、口丫啶黄素(acriflavine) 、 fluorcoumanin、攻瑰树减(ellipticine)、道诺霉素(da皿omycin)、 氯奎(chloroquine)、偏端霉素D (distamycin D)、色霉素(chromomycin) 、 二胺乙基苯菲啶(homidium)、光辉霉素(mithramycin)、钌多卩比啶(rutheniumpolypyridyls)、氨茴霉素(anthr咖ycin)等。 Suitable for the application of DNA binding dyes include SYBR green, SYBRblue, DAPI, propidium iodide (propidium iodine), Hoeste, SYBR gold, ethidium bromide (ethidium bromide), mouth acridine (acridines), proflavine (prof lavine), mouth acridine Cheng (acridine orange), mouth acridine flavin (acriflavine), fluorcoumanin, tree Rose attack minus (ellipticine), daunorubicin (da dish omycin), chloroquine (chloroquine), partial end neomycin D (distamycin D), Chromomycin (chromomycin), the diamine ethylbenzene phenanthridines (homidium), mithramycin (mithramycin), Ruthenium Jie of pyridine (rutheniumpolypyridyls), anthramycin (anthr coffee ycin) and so on.

[0151] 除了各种荧光DNA结合染料外,其它发光的标记(如序列特异性的探针)也可以用于扩增反应,以利于扩增产物的检测和定量。 [0151] In addition to a variety of fluorescent DNA binding dye, other luminescent marker (e.g., sequence-specific probes) may also be used in the amplification reaction, the amplification product to facilitate detection and quantification. 基于探针的定量扩增依赖于所需扩增产物的序列特异性的检测。 Probe-based quantitative amplification relies on the detection of sequence-specific amplification of the desired product. 不同于基于染料的定量方法,它利用发光的靶标特异性的探针(例 Unlike dye-based quantitative methods, which use light-emitting target-specific probes (Example

如,TaqMan⑧探针),导致特异性和灵敏度增加。 Such as, TaqMan⑧ probe), resulting in increased specificity and sensitivity. 本领域中良好地建立了用于进行基于探针的定量扩增的方法,且在美国专利第5, 210, 015号中有记载。 It is well established in the art for a quantitative probe-based methods of amplification, and in U.S. Patent No. 5, 210, No. 015 there is described.

[0152] 在各种实施方式中,芯片另外包括定位特征(alignment feature),其可进行操作以将盖子与芯片对齐或将其附在芯片上,或将芯片与加热元件对齐或将其附在加热元件上。 [0152] In various embodiments, the chip further comprising locating feature (alignment feature), operable to align the cover with the chip or chips attached to on, or the chip is aligned with the heating element attached to or heating element. 在各种实施方式中,这样的特征包括凹形或凸形特征。 In various embodiments, such features include concave or convex features. 这样的特征包括销钉(pins)、隆脊(ridges)、搭锁(snaps)、螺钉(screws)及其组合。 Such features include a pin (pins), ridges (ridges), snap (snaps), screws (screws), and combinations thereof.

[0153] 在各种实施方式中,芯片组件包括温度控制元件,其有助于反应室温度的监测或控制。 [0153] In various embodiments, the chip assembly includes a temperature control element, which help monitor or control the temperature of the reaction chamber. 这样的温度控制元件包括但不限于有助于加热气体或冷却气体流动通过组件的通道或其它结构。 Such temperature control elements include, but are not limited to help heating or cooling gas flowing through the gas passage component or other structure. [0154] 本发明的另一方面公开了进行涉及多个反应样品且需要在至少两个温度水平循环的反应的方法,且该方法包括:提供包含可编址单元的阵列的芯片,各个单元设置为进行化学反应,其中,根据用于进行单元的化学反应的预定温度设置安排单元,使得至少一个单元编址为表明用于进行所述单元内的化学反应的预定温度;根据预定的温度设置将多个反应样品放入芯片的单元中;和控制加热元件以实现至少两个温度水平的循环。 [0154] Another aspect of the present invention discloses a reaction and requires at least two cycles of temperature levels method involves a plurality of reaction samples, and the method comprising: providing an array of addressable units of chips, each unit is provided for the chemical reaction, which, according to the chemical reaction unit for performing a predetermined temperature setting means arranged such that at least one unit for performing addressing to indicate a predetermined temperature of the chemical reaction within the unit; according to a predetermined setting temperature a plurality of chip sample into the reaction unit; and controlling the heating element to effect at least two temperature levels of circulating. 一方面,进行化学反应的方法可以包括提供本文所述的芯片,其中,芯片设置为容纳反应样品。 On the one hand, a chemical reaction method as described herein may include providing a chip, wherein the chip set to receive a reaction sample. 可以提供可相对于芯片定位的加热元件以在至少一个加热元件和芯片之间提供热接触。 Providing a heating element can be positioned relative to the chip to provide at least one heating element and the thermal contact between the chip. 可以通过改变芯片的温度在反应样品中进行化学反应。 By changing the chip temperature chemical reaction in the reaction sample. 例如,可以通过相对于芯片移动至少一个的加热元件使得加热元件与芯片形成热接触来实现改变芯片的温度。 For example, the chip may be moved relative to the at least one heating element such that the heating element is in thermal contact with the chip is formed to achieve the temperature change of the chip.

[0155] 本方法使用本发明的芯片和本发明的热循环装置。 [0155] The method of the present invention of chips and thermal cycling device of the present invention. 典型地,在生物体的完整基因组的分析中,在不同的退火温度下优化不同基因或核酸序列的扩增。 Typically, the analysis of the complete genome of an organism, or to optimize amplification of nucleic acid sequences of different genes at different annealing temperatures. 这些不同的退火温度可以归合到共同的带中以提高分析的灵敏度和特异性。 These different annealing temperatures can be attributed to the common band together in order to improve the sensitivity and specificity of the analysis. 因此本发明的芯片可以按照退火温度带之一进行编址。 Therefore, the chip of the present invention can be addressed in one of the annealing temperature zone.

[0156] 退火温度的选择是对于优化PCR反应的特异性是关键的部分。 [0156] The annealing temperature of choice for optimizing PCR specificity is a key part. 可以以多种方式确定不同核酸序列扩增的退火温度。 It can be determined in a variety of different amplified nucleic acid sequence of the annealing temperature. 一种方法是确定核酸序列的熔点(Tm)。 One method is to determine the melting point of the nucleic acid sequence (Tm). 熔点是一半DNA 双链体解离并变为单链的温度。 Melting point is half duplex DNA dissociation temperature and becomes single stranded. 当设计核酸扩增时,引物长度和序列对于设计成功的扩增的参数是重要的。 When designing nucleic acid amplification, primer length and sequence it is important for successful amplification of design parameters. 例如,DNA的熔点随其长度和鸟嘌呤和胞嘧啶含量的增加而增加,鸟嘌呤和胞嘧啶含量可使用简单公式估计。 For example, DNA replication with its length increases the melting point and guanine and cytosine content increases, guanine and cytosine content can be estimated using a simple formula.

[0157] 选择用于PCR反应的退火温度直接取决于引物的长度和组成。 [0157] Select PCR reaction annealing temperature used depends directly on the primer length and composition. 退火温度(Ta)可以选择为低于所用的引物对的最低Tm大约5C (I皿is和Gelfand, 1990)。 Annealing temperature (Ta) can choose the lowest Tm of primers used for less than about 5 C (I dish is and Gelfand, 1990). 计算退火温度的另一个示例性的方法是: Another exemplary method of calculating the annealing temperature is:

[0158] Ta0pt = 0. 3x (引物Tm) +0. 7x (产物Tm) -25 [0158] Ta0pt = 0. 3x (primer Tm) +0. 7x (product Tm) -25

[0159] 其中,引物Tm是较不稳定的引物模板对的熔点,而产物Tm是PCR产物的的熔点(Rychlik等人,1990)。 [0159] wherein the primer Tm is relatively unstable melting point of the primer template, and the product Tm is the melting point of the PCR product (Rychlik et al., 1990).

[0160] 如果PCR反应的退火温度过低,引物可能与真正的靶标以外的序列退火结合,且可以导致非特异性的扩增和所需要的产物产量的随之减少。 [0160] If the PCR reaction annealing temperature is too low, the primers may bind to true annealing sequences other than the target, and may be reduced leading to non-specific amplification and desired product yields. 过高的退火温度的后果是将产生太少的产物,原因是引物退火的可能性降低。 The consequence of the annealing temperature is too high will produce too little of the product is due to the possibility of primer annealing decreases. 需要考虑的另一方面是具有非常不同的退火温度的引物对可能从不产生适当量的单一产物,也可能无意间导致最有效引发的产物链的不对称的或单链的扩增。 Another aspect to be considered a primer having very different annealing temperatures that may never generate an appropriate amount of a single product, or may inadvertently cause the most effective single-stranded amplification products caused by the asymmetric chain.

[0161] 在PCR热循环中,对于各不同的引物对的最佳Ta和最大Ta在一定范围内变动并可以以连续的方式在不同基因之间变化。 [0161] In the PCR thermal cycling, the optimum and maximum Ta Ta for each different primer pairs and can change in a continuous manner between different genetic variation within a certain range. 典型地,对于给定的引物对产生高质量退火的退火温度是在1_3C的范围内。 Typically, for a given primer annealing to produce high-quality annealing temperature is in the range of 1_3 C. 如果退火温度较高,PCR退火步骤可能慢下来;如果退火温度较低,引物可能在所希望的位置以外的位置结合DNA,或结合其它物质。 If the annealing temperature is higher, PCR annealing step may slow down; if the annealing temperature is low, the primers may be in a position other than the position desired binding DNA, or in combination with other substances.

[0162] —种方法是使基因分析"单元化(bin)";给定分析的温度分配给具有最接近该给定的基因分析的Ta的温度的芯片的可编址部分。 [0162] - a gene analysis method is "a unit (bin)"; given the analysis of the temperature distribution to having the closest to the genetic analysis of a given temperature Ta chip addressable part. 例如,如果装置或芯片具有3个温度带60、 62. 5和65C ,最佳性能Ta = 64C的分析分配在65C的带中。 For example, if a device or chip having three temperature zones 60, 62.5 and 65 C, for optimum performance analysis assigned Ta = 64 C at 65 C in the band.

[0163] PCR反应的退火步骤典型地在40秒或更短时间内发生,部分取决于待扩增的序列的长度。 Annealing step [0163] PCR reactions are typically 40 seconds or more occurs in a short period of time, depending in part on the length of the sequence to be amplified.

[0164] 尽管PCR通常在低于引物熔点(Tm)的计算温度以下5"的温度开始,但最佳温度经常比计算温度高得多。在某些实施方式中,必须根据经验测试退火温度。例如,可以进行具有逐渐升高的温度的多个PCR反应,直至确定最佳退火温度。 [0164] Although the PCR primers generally lower than the melting point (Tm) of calculating a temperature below 5 "start temperature, but the optimum temperature is often much higher than the calculated temperature. In certain embodiments, the annealing temperature must be empirically tested. For example, a plurality of PCR reactions can be carried out with the temperature gradually increased until the optimal annealing temperature is determined.

[0165] 基因组中的许多基因序列可以归合到温度带中。 [0165] Many genes in the genome sequence can be attributed to the combined temperature band. 在一种实施方式中,本发明的芯 In one embodiment, the core of the present invention.

片的单元代表不同的退火温度带,例如,温度带在54-68t:的范围内的增量是2t:。 Unit represents sheet with different annealing temperatures, e.g., temperature zone in 54-68t: incremental range is 2t :. 然后根 Then root

据PCR反应的退火步骤发生的预定温度对单元进行编址。 According to a predetermined temperature annealing step PCR reactions occurring on unit addressing.

[0166] 在根据预定温度对单元编址之后,可以将样品分配到芯片中。 [0166] In accordance with a predetermined temperature after the cell addressing, you can assign the sample to the chip. 可以通过本文所公开的各种方法将样品加到芯片中。 By various methods disclosed herein, the sample was added to the chip.

[0167] 在各种实施方式中,系统或芯片可以另外包括装填设备,其可进行操作以有助于 [0167] In various embodiments, the system may additionally include or chip loading apparatus operable to facilitate

将扩增试剂或样品填充到芯片的可编址单元中。 The amplification reagent or sample is filled into the chip addressable unit. 本文有用的装填设备包括物理和化学器 Loading equipment useful herein include the physical and chemical control

械,其指引、引导、传送或以其它方式填充试剂或样品到可编址的单元中。 Mechanical, its guidelines, guidance, transfer or otherwise fill the reagent or sample to the addressable unit.

[0168] 在各种实施方式中,芯片可以包括凸起的或凹陷的区域,例如,有助于液体在芯片 [0168] In various embodiments, the chip may include raised or recessed areas, e.g., in the chip to help liquid

表面上的分布和流动的特征,如屏障或沟。 Distribution and characteristics of the flow on the surface, such as a barrier or a ditch. 在一种实施方式中,填充系统包括毛细通道。 In one embodiment, the system comprises a capillary channel is filled. This

些特征的尺寸规格是可变化的,取决于一些因素,如避免组件上的气泡及机械上的便利性 These feature sizes can be varied, depending on factors such as the convenience of avoiding bubbles and mechanical assembly on

和可行性。 And feasibility.

[0169] 填充系统包括有助于扩增试剂或样品在芯片表面上放置的任何装置,优选实现这样的试剂或样品在可编址的单元中放置的装置。 [0169] Amplification help filling system comprising means any reagent or sample is placed on the chip surface, it is preferable to achieve such a reagent or sample unit may be placed in addressable device. 本发明可用的这样的装置包括用于将试剂或样品倾注在表面上,以基本上覆盖整个表面的装置。 Such devices of the present invention may be used include for reagents or samples poured onto the surface to substantially cover the entire surface of the device. 在一种实施方式中,填充系统包括用于移液操作、点样或喷射试剂到特定反应室中(例如,通过压电泵)的设备。 In one embodiment, the system comprises means for filling pipetting, spotting or spraying the reagent into the reaction chamber in a specific (e.g., by a piezoelectric pump) device. 填充系统可以是纳米分配器。 The dispenser may be a nano-filled system. 在另一种实施方式中,填充装置包括可操作地填充芯片的反应室的真空泵。 In another embodiment, the filling apparatus comprising a reaction chip operatively filled pump chamber. 填充系统也可以包括用于向芯片施加离心力的设备。 Filling system may also include a device for applying centrifugal force to the chip. 在一种实施方式中,填充系统密切接近芯片中的填充装置或与其形成液体连通。 In one embodiment, the system is filled close proximity chip or filling apparatus in fluid communication therewith. 填充系统可以自动或根据来自计算机的程序运转。 Filling system can operate automatically or according to the program from the computer.

[0170] 驢 [0170] ass

[0171] 本发明的一个方面是设置为容纳小型化芯片的装置的设计,所述小型化芯片设计为用于单个分子的多重分析和/或用于同时进行大量化学反应。 [0171] One aspect of the invention is to design means to accommodate the miniaturization of the chip, the miniaturization of chips designed for multiple analysis of individual molecules and / or for the same time a large number of chemical reactions. 在一种实施方式中,本发明提供了高度自动化、小型化的分析仪器,其允许以多重方式进行具有温度、蒸发、小体积试剂的递送和/或产物检测的精确控制的操作。 In one embodiment, the present invention provides a high degree of automation, miniaturization of the analytical instruments, which allows multiple ways with temperature, evaporated to a small volume of precisely controlled delivery of reagents and / or products of the detected operation.

[0172] 本发明一个方面的装置包括至少一个用于加热或冷却芯片的加热元件。 Means [0172] with one aspect of the present invention comprises at least one heating element for heating or cooling chip. 可以理解:在加热元件设置为降低芯片的温度的情况中,该部件基本上作为冷却元件发挥功能。 Will be understood: the heating element is set to reduce the temperature of the chip case, the member functions substantially as a cooling element. [0173] 在一种实施方式中,所述装置包括基座,具有位于基座上的至少一个加热元件。 [0173] In one embodiment, the apparatus comprises a base, having at least one heating element is located on the base. 加热元件可以提供可用温度的范围。 The heating element may be provided usable temperature range. 例如,加热元件可以设置为按照需要提供大约-2(TC-大约12(TC范围内的温度。 For example, the heating element may be set to provide approximately -2 (TC- about 12 (a temperature within the range of TC as required.

[0174] 加热元件可以设置为容纳芯片。 [0174] The heating element may be provided to accommodate the chip. 在各种实施方式中,加热元件可以位于芯片下面或芯片上面。 In various embodiments, the heating element may be located below the chip or chips above. 在只存在一个加热元件的特定实施方式中,加热元件可以相对于芯片移动。 There is only one heating element in a particular embodiment, the heating element may be moved relative to the chip. 需要时,芯片可以是静止的。 If desired, the chip can be stationary.

[0175] 图6是所述装置一种实施方式的示意图的非限制性的例子。 [0175] FIG. 6 is an embodiment of the apparatus of non-limiting example of a schematic diagram. 图6描述基座600,具有至少一个位于基座600上的加热元件602。 Figure 6 depicts the base 600, located on the base having at least one heating element 600 602. 加热元件602适合容纳芯片650,这样的芯片能够在芯片650上的一个纳米孔652中包含并限制反应样品。 Heating element 602 is adapted to receive chip 650, such a chip on the chip can be a nanopore 652 650 and limit response contains a sample. 装置的加热元件602可以相对于芯片650在两个位置间移动。 Heating element 602 with respect to the device chip 650 to move between two positions. [0176] 图6中所示的加热元件602可以分为图7中所示的不同的温度带。 The heating element 602 shown in [0176] FIG. 6 can be divided into different temperature zones 7 in Fig. 图7是位于芯片700下面的用于控制和/或改变芯片温度的加热元件的示意图。 7 is located below the chip 700 for controlling and / or changing the temperature of the heating element chip FIG. 在一种实施方式中, 加热元件的温度范围对于变性温度可以是9(TC _951:的范围,对于引物退火阶段可以是52C -65〇的范围,和对于引物依赖性延伸阶段可以是681: -75〇的范围。加热元件700的非限制性的例子如图7所示。这个例子中的加热元件700是具有分别用于PCR的变性、引物退火阶段和引物依赖性延伸阶段的95t: (702)、52C (704)和72C (706)的温度带的加热元件。加热元件700的各个温度带的尺寸规格可以发生大小变化。在图7描述的一种实施方式中,位于芯片实例下面的加热元件是大约9英寸长X9英寸宽。 [0177] 然后将适合包含和限制反应样品的芯片放在适合容纳芯片的装置的加热元件700 上。使用位于图7的芯片下面的加热元件700,然后物理移动加热元件使得芯片接触95C 的块,接着是52t:的块,然后是72t:的块。由于加热元件上的较大表面积,加热元件相对于芯片的移动使得关键的斜面下降速度快得多。由于芯片在温度之间的转移,较大的表面积也使得能够从芯片背侧冷却。该技术也使得芯片以比传统的在循环之间简单地冷却的方式快得多的速率加热和冷却。 In one embodiment, the temperature range for denaturation temperature of the heating element may be 9 (TC _951: range, the primer annealing stage may be a range of 52 C -65 square, the extension and the primer-dependent phase can be 681: range -75 billion non-limiting examples of the heating element 700 is shown in Figure 7 in this example is a heating element 700, respectively, for PCR denaturation, primer annealing and primer-dependent extension phase stage. 95t:. heating element (702), 52 C (704) and 72 C (706) temperature with a heating element for each temperature zone sizes 700 size variations may occur to one embodiment described in FIG. 7. , the heating element is located below the chip instance about nine inches long X9 inches wide. [0177] Then for the reaction of the sample containing the chip and limitations placed on the heating element 700 for receiving the chip device. Use of the chip of FIG. 7 following the heating element 700, and then physically moving the heating element such that the chip contact block 95 C, followed 52t: block, then 72t:. Due to the large surface area of the block on the heating element, the heating element is movable relative to the chip so critical slant decline much faster. Since the chip transferred between temperature, the larger the surface area also enables the chip backside cooling. This technology also allows the chip to between cycles than the traditional way of simply cooling faster heating and cooling rates.

[0178] 另一种用于控制和/或改变芯片800的温度的方法如图8所述。 [0178] Another method for controlling and / or changing the chip temperature of a method 800 in FIG. 8. 图8是位于芯片800上面的加热元件850的非限制性的例子。 Figure 8 is a non-limiting example the heating element 800 is located above the chip 850. 如图8所示,加热元件可以是位于芯片800上面的一系列开/关电弧卤素灯852。 8, the heating element 800 may be located above a series of chips on / off the halogen lamp 852 arc. 热反射器854位于各卤素灯852周围,以确保由加热灯852获得均一的热源。 Heat reflector 854 is located around each of the halogen lamp 852, in order to ensure by the heating lamp 852 to obtain a uniform heat. 电弧灯852的高输出功率和输出与硅的光谱匹配提供了电弧灯852 的非常快的"斜面上升(ramp up)"速度。 High output power and spectral match to silicon output arc lamp 852 arc lamp 852 provides very fast "ramp rise (ramp up)" speed. 灯是电弧灯的事实意味着电弧灯852的关闭实际上是瞬时的。 The fact that the arc lamp arc lamp lights off means 852 virtually instantaneous. 图8也显示芯片800也可以用辐射固化粘合剂802密封,以帮助防止反应样品的蒸发。 Figure 8 also shows the chip 800 can also be used radiation-curable adhesive 802 seals to help prevent evaporation of the reaction of the sample. 在反应循环之后,在一个非限制性的实施例中,芯片随后用扫描仪860扫描,例如,高光谱(hyper spectral)或CCD扫描器。 After the reaction cycle, in one non-limiting embodiment, the chip 860 is then scanned with a scanner, e.g., hyperspectral (hyper spectral) or CCD scanner. 在某些方面,这样的光耦合系统在扩增过程中在多个时间点将激发光束传送到包含反应样品的孔中,并在循环之间的多个时间点中的各时间点监测来自纳米孔的光学信号。 In certain aspects, such an optical coupling system during amplification excitation beam at a plurality of time points to transfer the reaction sample contained in the hole, and at various time points to monitor a plurality of time points in the cycle from between nano aperture optical signal.

[0179] 图9图示地显示开/关电弧灯以摄氏度计的温度(y-轴)相对于以秒计的时间(x-轴)的变化。 [0179] FIG. 9 graphically display on / off arc lamp temperature in degrees Celsius (y- axis) versus time in seconds (x- axis) changes. 从图9可见,电弧灯的卤素灯斜面上升速度对于95+/_0.51:的温度是在少于大约2秒的时间内20C -IO(TC /秒,并且同样少于大约2秒的时间内对于65+/-0. 5C 的温度具有20C -IO(TC的斜面下降速度。电弧灯的温度在PCR反应过程中从大约65t:变换到大约95t:。 Seen from FIG. 9, the halogen lamp arc bevel rising speed for 95 + / _ 0.51: The temperature in less than about two seconds 20 C -IO (TC / sec, and also less than about 2 seconds . The time for the temperature 65 +/- 0 5 C having 20 C -IO (TC temperature ramp rate of decline in the arc lamp during the PCR reaction from about 65t:. Transformation to about 95t :.

[0180] 需要时,所述设备设计为使脉冲热量进入可选择芯片中由铝/硅制造的纳米孔中的移动最小化。 [0180] When needed, the device is designed to make the heat pulse into the nanopore chip selectively manufactured from aluminum / silicon mobile minimized. 一种使脉冲热量最小化的方式是减少Al/Si在最高温度下花费的时间,从而减少"热预算"。 A heat pulse is minimized to reduce time manner Al / Si at a maximum temperature it takes to reduce "thermal budget." "斜面上升"和"冷却"速度的加速和提供从加热到冷却的最快可能的转换,或者因此"转向"是重要的。 Acceleration "ramp up" and "cooling" and provides the fastest speed possible transition from heating to cooling, or so "to" is important. 这种加热和冷却芯片的方法也确保芯片加热的均匀性。 This heating and cooling chip method also ensures the uniformity of chip heating. 这样的均匀性防止由于可能导致纳米孔性能的变化的几度的温度差异产生的应力。 Such uniformity may lead to prevent changes in properties of the nanopore stress generated by the temperature difference of a few degrees. [0181] 从图IO可见,有几种电弧灯能够传送的温度输出分布。 [0181] can be seen from Figure IO, with a temperature output arc lamp can be transmitted in several distribution. 沿(a)行的曲线图显示温度(y-轴)相对于时间变化(x-轴)的变化。 Along the curve (a) with respect to the display line time (x- axis) of temperature change (y- axis). 相应地,沿(b)行的曲线图显示芯片对于(a) 行中所述的快速热处理(RTP)分布的响应。 Accordingly, along the curve (b) shows the response line chip for rapid thermal processing (RTP) (a) of the distribution line. 响应于温度随时间的较缓慢的变化,如尖峰或脉冲的光学加热器分布中,芯片温度在芯片的整个深度保持恒定。 In response to temperature changes more slowly with time, such as spikes or pulses of the optical distribution heater chip temperature is kept constant throughout the depth of the chip. 响应于温度随时间的较快速的变化,如激光或闪光辅助的加热器的分布中,芯片温度随芯片相对于热源的深度而变化。 With more rapid changes over time, such as in response to the temperature distribution of the laser or flash-assisted heater, the chip temperature with the chip relative to the depth of the heat source and change.

[0182] 非受具体理论的限制,图IO举例说明许多对应于本发明的温度输出分布。 [0182] Non-restricted particular theory, Figure IO illustrates many output corresponding to the temperature distribution of the present invention. 如图10(a) (1)所示,特征在于圆形的热分布的尖峰加热器分布产生如图10(b) (1)所示的类似于热源温度的芯片响应温度。 FIG. 10 (a) (1), the heat distribution, characterized in that the circular spike heater profile generator in FIG. 10 (b) (1) similar to the chip in response to the temperature of the heat source temperature as shown. 在特征在于成峰的热分布的脉冲加热器分布(图10(a) (2)) 中,热源的温度快于对热源作出响应的芯片的温度,但芯片的温度在芯片相对于距热源的距离的整个深度上仍然保持相对均一(图10(b) (2))。 It characterized by a peak in the heat distribution of the heater pulse distribution (Figure 10 (a) (2)), the temperature of the heat source to the heat source to make the chip faster than the temperature response, but the temperature of the chip in the chip away from the heat source with respect to It remained relatively uniform (FIG. 10 (b) (2)) over the entire depth of the distance. 对于如图10(a) (3)所示的特征在于锐利尖峰分布的激光加热器分布,热源比芯片作用快得多。 For the FIG. 10 (a) (3) characterized in that the sharp peak distribution shown in the distribution of a laser heater, the heat source is much faster than the chip effect. 因此,仅芯片的表面层被加热(图10(b) (3))。 Therefore, only the surface layer of the chip is heated (FIG. 10 (b) (3)). 芯片的深度越深,温度分布越低。 The deeper the depth of the chip, the lower the temperature distribution. 最后,对于特征在于初始进行大部加热, 接着是对表面退火的闪光处理的闪光辅助分布(图10(a) (4)),芯片的响应类似于芯片对激光分布的响应(图10(b) (4))。 Finally, characterized in that the conduct most initial heating, followed by a response to the flash surface annealing process of flash-assisted distribution (Figure 10 (a) (4)), the chip's response is similar to the distribution of the laser chip (Figure 10 (b ) (4)).

[0183] 本发明的另一方面,描述了用于进行需要在至少两个温度水平循环的化学反应的 [0183] Another aspect of the present invention, describes the need for at least two temperature levels in the chemical reaction cycles

装置,包括:用于进行反应的包括可编址单元的阵列的芯片,各个单元设置为进行化学反应,其中,单元根据用于进行单元中的化学反应的预定温度设置进行排列,使得至少一个单 Apparatus, comprising: means for reacting comprises an array of addressable units of chips, respective units to carry out a chemical reaction, wherein means are arranged according to a predetermined temperature for performing the chemical reaction unit is provided, such that the at least one single-

元编址为表明用于进行所述单元中的化学反应的预定温度;和热接触芯片的加热元件。 Yuan addressed to indicate the predetermined temperature for carrying out chemical reactions in the cell; and a heating element in thermal contact with the chip. [0184] 在一种优选的实施方式中,芯片的可编址单元设置为进行核酸扩增反应,包括但不限于实时PCR。 [0184] In a preferred embodiment, the chip addressable unit is set to carry out a nucleic acid amplification reaction, including but not limited to real-time PCR. 单元可以是包括试剂、探针、引物、dNTP或其组合的纳米孔。 Units may comprise reagents, probes, primers, dNTP, or a combination of nanopores. 预定温度可以是用于进行PCR反应的不同的退火温度。 The predetermined temperature may be different annealing temperatures for the PCR reaction.

[0185] 加热元件可以是简单的加热器,如包括电阻加热器或热电加热器的盘,或精细的热循环装置。 [0185] The heating element may be a simple heater such as a resistance heater or a thermoelectric disc comprising a heater, or fine thermal cycle apparatus. 在某些实施例中,加热元件热接触散热器,以在芯片热接触加热元件时使得芯片能够发生快速温度变化。 In certain embodiments, the heat sink in thermal contact with the heating element, when the chip is in thermal contact with the heating element so that the chip temperature changes can occur rapidly. 风扇也可以与加热元件结合,以提供对于快速热循环的更多的控制。 Fans may also be combined with the heating element, to provide for more rapid thermal cycling control. 加热元件的其它例子包括薄膜加热器,其可以通过传导快速加热或具有加入到膜中的电磁加热器。 Other examples of the heating element comprises a thin film heater, which can be quickly heated by conduction or added to the film having an electromagnetic heater. 尤其适于制造薄膜加热器的材料是铟锡氧化物(IT0) 。 In particular, materials suitable for manufacturing the thin film heater is indium tin oxide (IT0). ITO是具有非常高的导电率的透明陶瓷材料。 ITO is a transparent ceramic material having a very high conductivity. 因为ITO可以大量或以薄层的形式制备,它作为集成的或外部的加热元件尤其有用。 Because ITO or can be prepared in the form of a large number of thin layers, it is particularly useful as an integrated or external heating element.

[0186] 在另一种实施方式中,加热元件在大小和构型方面与芯片相容。 [0186] In another embodiment, the heating element in terms of size and configuration compatible with the chip. 在一种实施方式中,所述装置进一步包括多个加热元件。 In one embodiment, the apparatus further comprises a plurality of heating elements. 可以将加热元件作为可拆分的单元邻近芯片放置、 放置在其底部和/或在其顶部。 The heating element can be used as a detachable unit placed near the chip, placed at the bottom and / or at the top. 在优选的实施方式中,为了最小化加热元件边缘的边缘效应,加热元件的面积明显大于芯片面积。 In a preferred embodiment, in order to minimize the edge effect heating element edge area of the heating element is significantly greater than the chip area.

[0187] 在图lla-c中说明了加热元件结构和设置的一些实施例。 [0187] illustrate some embodiments of the heating element structure and set in the lla-c in Fig. 在这些实施例中,加热元件1100结合散热器1110和风扇1120,以改善对于温度变化和热循环的斜面上升时间的控制。 In these embodiments, the heating elements 1110 and 1100 combine the heat sink fan 1120, in order to improve control of the temperature change and the thermal cycling of the rise time of the ramp. 可以在加热元件1110和样品(或芯片)1140之间使用金属热块1130,其与元件1100 和芯片1140形成密切的热接触。 1110 may be in the heating element and the sample (or chip) using a metal heat block between 1130 1140, 1140 formed its close thermal contact with the element 1100 and chips. 即使芯片1140不具有高的热导率,块1130也可以具有高的热导率,以产生具有重现性的温度变化。 Even chip 1140 does not have a high thermal conductivity, block 1130 may also have a high thermal conductivity, temperature change to generate reproducibly. 如果多个加热元件用来限定一个温度带或多个温度带,任何小的热干扰(例如,微风)可能产生温度的明显变化。 If the heating element is used to define a plurality of temperature zones or a plurality of temperature zones, any small thermal interference (e.g., wind) may produce significant changes in temperature. 如果使用热块使阻抗降低,不同带之间的热转移会变得平稳。 If you use a heat block impedance reduce heat transfer between different band will become stable. 另外,金属热块可以在顶面上容纳温度传感器。 The metal thermal block can hold a temperature sensor on the top surface. 温度传感器可以对应于各个带,或可以是芯片本身的一部分。 Temperature sensors may correspond to each band, or may be part of the chip itself. 热块也可以提供真空通道,以利用真空保持芯片与块和/或加热元件的紧密热接触。 Heat block can also provide a vacuum channel, the vacuum holding the chip in order to take advantage of the block and / or heating element in close thermal contact. 如图lla-c所示,加热元件1100可以包括一个或多个热电加热元件1150或电阻加热元件1160或二者的组合。 As shown in Figure lla-c, the heating element 1100 may comprise a combination of one or more thermoelectric heating element or resistive heating elements 1150 1160, or both. [0188] 加热元件可以提供温度梯度。 [0188] The heating elements can provide a temperature gradient. 本文的温度梯度可以是在单一加热元件上一部分的温度高于另一部分的温度。 Temperature gradient herein may be in a single part of the heating element temperature is higher than the temperature of the other portion. 例如,电阻加热器或热电加热器可以设置为在加热元件上产生热梯度。 For example, a resistance heater or a thermoelectric heater can be set to produce a thermal gradient in the heating element. 热梯度也可以定义为芯片上一部分的温度高于另一部分的温度。 Thermal gradients may also be defined as part of the on-chip temperature higher than the temperature of the other portion. 例如,如果使用多个加热元件,一个加热元件可以向芯片的一侧或一端传送较高的温度,而第二加热元件可以向芯片的一侧或一端传送不同的温度,从而产生热梯度。 For example, if a plurality of heating elements, one element can be heated to the chip side or end of transmission higher temperature, while the second heating element may transmit different temperatures or at one end to one side of the chip, resulting in a thermal gradient.

[0189] 所述装置可以进一步包括可操作地偶联芯片的光学系统,其中,光学系统检测来自单元的光学信号。 Means [0189] may further include a chip operatively coupled optical system, wherein the optical signal from the detecting unit of the optical system. 在一种示例性的实施方式中,芯片、加热元件和光学系统组成本发明的系统。 In one exemplary embodiment, a chip, a heating element and an optical system consisting of system of the invention.

[0190] —方面,本发明的公开包括用于进行涉及至少两个温度水平的循环的化学反应的装置,其包括:设置为容纳用于容纳化学反应的样品座的主体;包含多个温度带的第一加热器;和提供均一温度的第二加热器,其中,第一加热器和第二加热器设置为可在第一方位和第二方位之间移动,并且其中第一方位使得加热器热接触样品座,而第二方位不使得加热器热接触样品座。 [0190] - aspect, the present invention discloses an apparatus comprises at least two chemical reactions involved in the cycle of temperature levels, comprising: a body configured to receive a sample holder for accommodating a chemical reaction; comprises a plurality of temperature zones The first heater; and to provide a uniform temperature of the second heater, wherein the first and second heaters arranged to be movable between a first position and a second position, and wherein the first orientation so that the heater thermal contact with the sample holder, and the second orientation does not make contact with the sample heater hot seat.

[0191] 样品座可以是用于进行反应的芯片,其包括可编址单元的阵列,各个单元设置为进行化学反应,其中,单元根据用于进行单元的化学反应的预定温度设置排列,使得至少一个单元编址为表明用于进行所述单元内的化学反应的预定温度。 [0191] sample holder may be used for the reaction of the chip, which comprises an array of addressable units, each unit is set to carry out a chemical reaction, wherein means are arranged in accordance with a predetermined temperature setting unit for carrying out a chemical reaction, so that at least Addressing to indicate a unit for performing a predetermined temperature chemical reactions within the cell. 在一种实施方式中,多个温度带对应于单元排列所依据的预定温度设置。 In one embodiment, a plurality of temperature zones corresponding to the predetermined temperature is set to a cell arrangement is based.

[0192] 在装置和样品座设置为进行PCR反应的实施方式中,预定的温度带对应于可以进行多重PCR反应的不同退火温度。 [0192] In the apparatus and sample holder to carry out PCR reactions embodiment, the predetermined temperature band corresponding to the multiplex PCR reaction can be performed in different annealing temperatures. 在进一步的实施方式中,为了扩增基因组的大部分,如果不是全部的话,装置和样品座能够进行一系列PCR反应。 In a further embodiment, in order to amplify the genome of most, if not all, and a sample holder device capable of a range of PCR reactions. 在本实施例中,整个基因组需要多种退火温度以获得需要的反应特异性。 In the present embodiment, the whole genome requires multiple annealing temperature to obtain a desired reaction specificity. 例如,根据不同核苷酸序列的退火温度,这些温度可以归合成2t:的温度带。 For example, different nucleotide sequences according to the annealing temperature, which temperature may be normalized synthesized 2t: the temperature zone. 样品座的单元可以根据进行单元中的反应所处的温度带进行编址。 Sample holder unit can be addressed with according to unit temperature at which the reaction. 在一种实施方式中,装置容纳样品座,其包括具有根据与所述装置的第一加热器上的6 个不同温度带对应的6种不同的退火温度带编址的单元的芯片。 In one embodiment, the means for receiving a sample holder, which comprises a chip according to six different temperatures of the first heater means on the tape corresponding to 6 different annealing temperatures with addressing unit.

[0193] 在优选的实施方式中,第一和第二加热器根据方案在第一和第二方位之间移动。 [0193] In a preferred embodiment, the first and second heater under the program moving between the first and second orientation.

所述装置可以进一步包括用于在第一和第二方位之间移动第一加热器和第二加热器的电 The apparatus may further comprise power for moving between the first and second orientation of the first and second heaters

动机。 Motivation. 加热器可以通过任何本领域的技术人员显而易见的方法来移动。 The heater may be moved by any apparent to those skilled in this art methods.

[0194] 第一方位使加热器热接触样品座。 [0194] The first orientation of the heater in thermal contact with the sample holder. 在一种实施方式中,样品座用来进行PCR反应。 In one embodiment, the sample holder used for PCR reactions.

第一加热器具有多个温度带,且当第一加热器热接触样品座时,第一加热器可以用来提供 A first heater having a plurality of temperature zones, and when the first heater is in thermal contact with the sample holder, the first heater may be used to provide

PCR反应退火步骤所需的温度。 PCR reaction temperature required for the annealing step. 在一种实施方式中,当第二加热器处于第一方位且热接触样 In one embodiment, when in a first orientation and a second heater in thermal contact with the sample

品座时,第二加热器提供PCR反应过程中核酸的延伸或变性所需的温度。 When the product base, the second heater is required during PCR reaction extension or nucleic acid denaturation temperature.

[0195] 在本发明的装置和方法的实施例中,移动第二加热器到第一方位以与PCR反应的 [0195] In an embodiment of the apparatus and method of the present invention, the movement of the second heater to the first orientation of the reaction with PCR

样品座热接触。 In thermal contact with the sample holder. 为了使包含在样品座的样品中的核酸变性,第二加热器传递大约95t:的温 In order to be included in the sample nucleic acid denaturation in the sample holder, the second heater transfer approximately 95t: temperature

度。 Degrees. 在变性步骤之后,移动第二加热器到第二方位,并移动第一加热器到第一方位以热接触 After the denaturation step, moving the second heater to a second orientation, and to move to the first heater in thermal contact with a first orientation

样品座。 Sample holder. 第一加热器向样品座提供用于使引物与样品的核酸序列退火的温度。 The first heater temperature to provide a nucleic acid sequence of primer annealing the sample to the sample holder. 进行变性步 Denaturation step

骤接着退火步骤的过程可以重复或循环,直至获得需要的扩增产物。 Next step annealing step process can be repeated or cycle until the amplification product needs.

[0196] 图12说明本发明的热循环装置1200的示例性的实施方式。 [0196] FIG. 12 illustrates an exemplary embodiment of the thermal cycling device of the present invention 1200. 在本实施例中,装置1200包括具有对应于芯片1250的6种不同可编址预定温度的6个不同温度带的第一加热器。 In the present embodiment, the apparatus 1200 includes six different temperatures corresponding to the chip 1250 having six different addressable with a first predetermined temperature heater. 可以移动第一加热器1210以与芯片1250热接触或脱离热接触,如图12中的顶视图所示。 You can move the first heater in thermal contact with the chip 1210 to 1250 or from the thermal contact in the top view shown in Figure 12. 为了加热芯片1250的6个不同的可编址预定温度区域到相同的温度,第二加热器1220在整个加热器上提供均一的温度。 To heat the chip 1250. 6 different addressable predetermined temperature zones to the same temperature, the second heater 1220 provides a uniform temperature throughout the heater. 例如,第二加热器1220可以向本发明的芯片提供用于所进行PCR的变性和延伸步骤的均一温度,而第一加热器1210可以提供多种退火温度以提高进行大量反应的特异性。 For example, the second heater 1220 may provide a uniform temperature for PCR denaturation and extension steps carried out by the chip of the present invention, and the first heater 1210 may provide a variety of annealing temperatures to increase the specificity of the reaction a lot. 用于分析该许多反应的成像源1260可以位于与加热器的第一方位相对的芯片的对侧。 Many of the reactions for analysis of the imaging source 1260 may be positioned relative to the first heater chip orientation opposite side.

[0197] 图13显示了当加热器1210处于与具有可编址单元的芯片1250热接触的第一方位时,来自图12的实施例的第一加热器1210的侧视图。 [0197] Figure 13 shows in a first orientation when the heater 1210 and 1250 chip heat addressable unit has contact, the first embodiment of the heater from the side view of FIG. 12 1210. 例如,加热器的各个温度带1212、1214可以由不同的热电加热元件1216、1218提供,且某些实施例的细节如图13所示。 For example, the temperature of each heater band 1212,1214 1216,1218 may be provided by different thermoelectric heating element, and certain details of the embodiments shown in Figure 13. [0198] 在一种实施方式中,第一加热器可以提供温度梯度。 [0198] In one embodiment, a first heater temperature gradient can be provided. 用于第一加热器和第二加热器的加热器类型的例子包括,但不限于电阻加热器和热电加热器。 Examples of the type of heater is used for the first and second heaters include, but are not limited to resistive heater and a thermoelectric heater.

[0199] 在进一步的实施方式中,所述装置包括热接触第一加热器、第二加热器或这两者的散热器。 [0199] In a further embodiment, the apparatus comprises a heater in thermal contact with a first, second, or both heater radiator.

[0200] 为了监测温度,所述装置也可以包括多个温度传感器。 [0200] In order to monitor the temperature, the apparatus may also include a plurality of temperature sensors. 在一种实施方式中,多个温度传感器具有至少一个指定为测量装置的各个温度带的温度的温度传感器。 In one embodiment, a plurality of temperature sensors each having at least one designated as the temperature measuring device with a temperature of the temperature sensor. 温度传感器可以是热电偶或本领域可以获得的任何其它的传感器。 The temperature sensor may be a thermocouple or any other sensor available in the art.

[0201] 加热元件可以经过电引线连接到电源,电源在元件上提供电压并实现随后的单元加热。 [0201] The heating element can be connected to a power source via electrical leads, the power supply voltage and to achieve the subsequent unit in the heating element. 加热元件也可以与监测并调节单元温度的温度传感器偶联。 The heating element can also be monitored and adjusted with a temperature sensor coupled unit. 温度传感器可以控制单元阵列的温度,并因此控制单元阵列的热分布。 Temperature sensors can control the temperature of the cell array, the cell array and thus control heat distribution. 将芯片和/或第一加热器分成不同的温度带为需要不同的热循环分布的化学反应的平行进行提供了额外的灵活性。 The chips and / or first heater into different temperatures provides additional flexibility with parallel distribution of thermal cycles require different chemical reaction. 或者,温度传感器可以与单个单元或带偶联,使得各个单元或带的温度可独立地控制。 Alternatively, the temperature sensor may be a single unit or with a coupling, so that the temperature of each unit or tape may be independently controlled. 温度传感器可以作为邻近单元基部或处于单元基部的可分离的单元而被包括。 Temperature sensors can be used as a base or in the adjacent cell unit detachable base unit is included. 它也可以整合到单元的内表面或外表面。 It can also be integrated into the inner surface or the outer surface of the unit. 更进一步,可以制造温度传感器作为加热元件的组成部分。 Furthermore, the temperature sensor can be manufactured as an integral part of the heating element.

[0202] 由本发明的热循环装置提供的温度分布如图14所示。 [0202] temperature thermal cycling apparatus provided by the present invention, the distribution shown in Figure 14. 在本实施例中,第一加热器的6个不同温度带由比第二加热器提供的95t:的均一温度更低的独立温度所代表。 In this embodiment, six different temperatures of the first heater band from the 95t than a second heater provided: uniform colder temperatures represent independent. 例如,当用本发明的芯片、系统和装置扩增整个人类基因组时,为了扩增基因组的每个基因(大约30, 000),可以在少于大约15分钟、10分钟或甚至5分钟的时间内进行40个循环。 For example, when using the chip, system and apparatus of the present invention amplify the entire human genome, in order to amplify each gene genome (about 30, 000), may be less than about 15 minutes, 10 minutes or even 5 minutes within 40 cycles. [0203] 用于提供至少两个温度水平的循环的装置的主体设置为容纳样品座。 [0203] for providing at least two temperature levels of the main circulating means is provided to receive the sample holder. 通过包括真空力的各种方法,样品座可以保持在主体中的正确位置。 By various methods including a vacuum force, the sample holder may remain in the body in the correct position. 在一种实施方式中,主体包括用于将样品座稳固地保持在原位的真空吸盘,使得加热器热接触样品座。 In one embodiment, the body includes means for holding the specimen holder firmly in place in the vacuum chuck, so that the heater in thermal contact with the sample holder. 对于本领域的技术人员显而易见的,夹子、销钉、粘合剂、狭槽或任何其它固定方法可以用于设置为容纳样品座的主体。 For this apparent to those skilled in the art, clips, pins, adhesive, slots or any other fixing methods may be used to accommodate the sample holder body.

[0204] 所述装置也可以进一步包括测定加热元件相对样品座的第一和第二方位的位置的传感器。 [0204] The apparatus may further comprise a heating element relative to the sample holder measuring the position of the first sensor and the second orientation.

[0205] 本发明的另一方面,用于进行涉及至少两个温度水平的循环的反应的装置,包括:设置为容纳芯片的主体,芯片包括多个用于容纳化学反应物的纳米孔;和提供第一温度的第一加热器和提供第二温度的第二加热器,其中,第一加热器和第二加热器设置为可在第一和第二方位之间移动,且其中,第一方位使加热器热接触样品座,和第二方位不使加热器热接触样品座。 [0205] Another aspect of the present invention, for the at least two temperature levels involving circulating reaction apparatus, comprising: a main body disposed to receive the chip, the chip includes a plurality of nano-holes for receiving the chemical reactants; and providing a first temperature of a first heater and a second heater provided a second temperature, wherein the first and second heaters to be disposed between the first and the second position of the mobile, and wherein a first orientation of the heater in thermal contact with the sample holder, and a second orientation without the heater in thermal contact with the sample holder.

[0206] 在一种实施方式中,多个纳米孔是可编址的,其中,纳米孔根据预定温度设置排列,使得至少一个纳米孔编址为表明用于进行所述纳米孔内的化学反应的预定温度。 [0206] In one embodiment, a plurality of nanopores is addressable, in which the nanopores are arranged according to a predetermined temperature is set such that at least one nanopore addressed as an indication for the chemical reaction of the nano-hole The predetermined temperature. [0207] 在另一种实施方式中,第一加热器包括多个温度带。 [0207] In another embodiment, the first heater comprises a plurality of temperature zones. 多个温度带可以对应于预定的温度设置,单元根据该温度设置形成阵列。 It may correspond to a plurality of temperature with a predetermined temperature setting, form an array of cells in accordance with the temperature setting.

[0208] 第一加热器和第二加热器可以根据方案在第一和第二方位之间移动。 [0208] The first and second heaters can be moved between the first and second orientation under the program. 在一种实施方式中,所述装置可以进一步包括用于在第一和第二方位之间移动第一加热器和第二加热器的电动机。 In one embodiment, the apparatus may further comprise means for moving between the first and second orientation of the first and second heaters motor.

[0209] 在一种实施方式中,第一加热器可以提供温度梯度。 [0209] In one embodiment, a first heater temperature gradient can be provided. 用于第一加热器和第二加热器的加热器类型的例子包括,但不限于电阻加热器和热电加热器。 Examples of the type of heater is used for the first and second heaters include, but are not limited to resistive heater and a thermoelectric heater.

[0210] 在进一步的实施方式中,所述装置包括热接触第一加热器、第二加热器或这两者的散热器。 [0210] In a further embodiment, the apparatus comprises a heater in thermal contact with a first, second, or both heater radiator.

[0211] 为了监测温度,所述装置也可以包括多个温度传感器。 [0211] In order to monitor the temperature, the apparatus may also include a plurality of temperature sensors. 在一种实施方式中,多个温 In one embodiment, a plurality of temperature

度传感器具有至少一个指定为测量装置的各温度带的温度的温度传感器。 Having at least one sensor measuring apparatus designated as the temperature at each temperature with a temperature sensor.

[0212] 本发明的示例性装置和系统1500如图15所示。 [0212] Examples of the apparatus and system of the present invention is shown in Figure 15. 1500. 芯片1510可加载于用于进行涉 1510 chip can be loaded to be used for involving

及至少两个温度水平的循环的化学反应的装置中。 And at least two chemical reactions means the temperature level of the cycle. 芯片1510可以通过能够保持它们定位 1510 chip can be able to maintain their positioning

的任何方法,如真空或夹子,设置定位。 Any method, such as vacuum or clip, set positioning. 芯片也可以通过将芯片滑入紧靠使芯片对准合适位 The chip can also slide against the chip so that the chip at a suitable position

置的制动器或壁的位置而放置在装置中。 Position opposing brake or walls placed in the device. 芯片可以放置在能够上下移动的样品座1520上。 Chip can be placed on a sample holder capable of moving up and down 1520.

样品座可以设置为与装置1500的加热器1530偶联。 Sample holder may be provided with a heater means 1500 to 1530 G.

[0213] 在一种实施方式中,芯片1510和/或芯片的孔通过薄塑料盖1512密封,例如,用于覆盖芯片或纳米孔板的标准PCR带。 [0213] In one embodiment, the chip 1510 and / or hole chip sealed by a thin plastic cover 1512, for example, for covering the chip or nano-well plates with standard PCR. PCR带可以由如聚乙烯的透明材料制成,且是可移动的和有时是可更换的。 PCR band may be made of a transparent material such as polyethylene, and is removable and replaceable sometimes. 透明的油也可以用于覆盖芯片1510的孔和/或表面。 Transparent oil can also be used to cover the hole chip 1510 and / or surface. 在一种实施方式中,芯片1510和/或孔被油和盖子覆盖。 In one embodiment, the chip 1510 and / or holes are covered with oil and cover.

[0214] 在一种实施方式中,在将芯片置于本发明的热循环装置中之前,不除去覆盖芯片1510的盖子1512。 Before [0214] In one embodiment, the chip is placed in the thermal cycling device of the present invention, without removing the cover 1512 covering 1510 chips. 在芯片在装置中入位之后,加热器1530可以向芯片1510提供使芯片1510与顶端盖片1540形成光学接触或热接触的力。 After the chip into place in the device, the heater 1530 may provide the chip 1510 and the top cover plate 1540 form an optical contact or thermal contact force to chip 1510. 顶端盖片1540可以由透明材料或任何允许光学器件观察芯片的反应的材料制成。 The top flap 1540 may be made of a transparent material, or any optical device allowing observation of the reaction chip material. 可以用作顶端盖片的材料包括,但不限于玻璃、硅石、硅和本领域的技术人员明显了解的聚合物或塑料。 May be used as the front cover sheet materials include, but are not limited to, glass, silica, silicon and apparent to a person skilled in the art to understand the polymer or plastic.

[0215] 顶端盖片1540也可以包括加热器1542,如铟锡氧化物(ITO)加热器,其可以加热芯片1510的顶部或芯片1512和/或孔的盖子。 [0215] the top cover sheet 1540 may also include a heater 1542, such as indium tin oxide (ITO) heater, which can heat the top of the chip or chips 1510 1512 and / or the cover hole. 顶端盖片的加热器1542可以用于平衡芯片表面的温度,使得不会在芯片上的单元的表面或开口处发生冷凝。 Top end cap 1542 may be used for the heater plate temperature equilibrium surface of the chip so that the chip is not in the opening or the surface of the condensation unit. 例如,当进行PCR反应时,试剂和/或样品的液体组分可能加热到它们在反应单元或反应孔的表面或盖子上冷凝的点。 For example, when the PCR reaction, the liquid component reagents and / or samples may be heated to their reaction condensed on the surface of cells or wells or cover point. 冷凝可以干扰用于监测单元中的反应的光学系统。 Condensation may interfere with the optical system unit for monitoring reaction. 在本发明的实施方式中,如图15所示,装置的加热器1530可以提供使芯片1510与包括加热器1542的顶端盖片1540形成热接触的力,加热器1542可以平衡芯片1510表面的温度以防止冷凝。 In an embodiment of the present invention, shown in Figure 15, the heater means 1530 may be provided so that the top of the heater chip 1510 and 1542 include a cover sheet 1540 is formed in thermal contact force, heating temperature of 1542 to balance the surface of the chip 1510 to prevent condensation. 也如图15所示,顶端盖片1540可以通过一对弹簧或压縮装置1552与装置的桥体1550连接。 Also shown in Figure 15, the top cover sheet 1540 by a pair of springs or compression means 1552 and 1550 to connect the bridge device. 弹簧或其它装置1552可以释放顶端盖片1540上由加热器1530对抗芯片1510的力产生的部分压力,从而使得装置和系统更加稳固。 A spring or other means to the top cover portion 1552 can be released pressure from the on-chip 1540 1510 1530 against the force of the chip generated by the heater so that the apparatus and the system more stable. 任何用于压力缓冲的方法或装置可以用来偶联顶端盖片与装置的桥体。 Any method or apparatus for pressure buffer can be used to couple the top cover plate and bridge devices.

[0216] 在图15的示例性的系统和装置中,可以通过热循环装置1500的侧面卸载或加载芯片1510。 [0216] In an exemplary system and apparatus of Figure 15, you can unload or load side 1500 1510 chip by thermal cycling device. 然后可以移动装置的加热器1530到与芯片1510形成热接触的方位,加热器1530也提供了使芯片热接触顶端盖片1540的力。 You can then move the heater means 1530 to 1510 formed the orientation of the chip in thermal contact with the heater 1530 also provides the chip in thermal contact with the top cover sheet 1540 of force. 在图15中,芯片具有覆盖孔的塑料盖子1512。 In FIG. 15, covering the chip with a plastic cover aperture 1512. 也在图15中,顶端盖片1540包括熔融Si(V石英材料,还包括ITO加热器1542。 IT0加热器1542通过如图15所示连接到ITO加热器上的电引线1544操作。顶端盖片1540然后通过在系统中提供应力释放的压縮弹簧1522连接装置的桥体1550。弹簧1522也可以发挥作用以改善芯片1510与装置的加热器1530和顶端盖片1540的加热器的热接触。[0217] 实践中,可以通过处理储存在与加热元件可操作地连接的计算机可读介质上的预先设定的算法来实现加热元件控制并进而控制反应样品的温度。加热元件的移动也可以受方案或算法的控制,其可以由计算机提供或存储在计算机可读介质上。其它方面,控制步骤可以包括基于储存在可读介质上的方案处理从可操作地连接样品座或芯片的单元的温度传感器元件得到的温度或运动传感信号。这可以通过采用可以处理模拟信号或数字信号的用于温度控制的常规电子元件来实现。优选地,在反馈控制电路上运行电子元件。它们可以控制一个单元的温度,但更经常控制共同形成一个温度带的多个单元的温度或温度带自身的温度。在一定的实施方式中,分别控制不同带的温度。热循环分布和持续时间取决于采用所述芯片的特定的应用。[0218] Also in FIG. 15, the top cover sheet 1540 comprises melting Si (V quartz material, further comprising a heater 1542. IT0 ITO heater 1542 shown in FIG. 15 is connected by electrical leads to the heater 1544 ITO operation top cover sheet 1540 and then providing stress release in the system of the compression spring 1522 bridge connecting means spring 1550. 1522 may also play a role to improve the thermal contact between the heater chip device 1510 and 1530 and the top cover sheet heater 1540. [ 0217] In practice, the processing can be stored in a computer operatively connected with the heating element readable medium on predetermined algorithms to achieve control of the heating element and thus control the temperature of the reaction sample. moving the heating element may be affected by the program or control algorithm, which can provide or stored on a computer readable medium. Other aspects, including those based on program control step can be stored on a readable medium processing unit connected to the sample holder or chips from a temperature sensor operatively computer temperature or motion sensing element signal obtained. This can be through the use of an analog signal or a digital signal processing routine for temperature control of electronic components is achieved. Preferably, the electronic component is running on a feedback control circuit which can control a unit temperatures, but more often together forming temperature or a temperature control unit of a plurality of temperature zones with the temperature itself. In certain embodiments, each controlling a different temperature zone heat cycle duration depends on the distribution and use of the application specific chip. [0218]

[0219] 可以向所述芯片提供能够检测和/或监测在芯片中发生的化学反应的结果或进程的光学系统。 [0219] can provide the ability to detect and / or monitor the occurrence of chemical reactions in the chip outcome or process of the optical system to the chip. 这样的光学系统通过首先光学激发反应物,接着收集和分析来自芯片的反应物的光学信号来实现这些功能。 Such an optical system through the first optical excitation of reactants, followed by collecting and analyzing the optical signal from the chip of the reactants to achieve these functions. 可应用于本发明的光学系统包括3种元件,即光学激发元件、光学传输元件和感光元件。 The present invention can be applied to an optical system comprising three kinds of elements, i.e., the optical excitation element, the optical transmission element and the photosensitive element. 光学系统也可以任选地包括光学选择元件。 The optical system may also optionally comprise optical selection element. [0220] 图16是显示实验设置中的设备的典型实例的方块图。 [0220] FIG. 16 is a block diagram shows the experimental setup of equipment typical example. 图16显示连接作为光学激发元件的典型实例的激光器1610的计算机系统(或数字设备)1600。 Figure 16 shows the connection of the typical examples of the optical excitation element of a computer system (or digital device) laser 1610 1600. 光学激发元件作为用于光学激发包含在纳米孔中的反应物的激发光束而发挥作用。 Excitation optical element functions as a light beam for optical excitation is contained in the excitation of nanopores reactants. 该元件包括产生不同波长、强度和/或相干性质的光束的多种光源。 The element includes generating a different wavelength, intensity and / or the nature of the various sources of coherent light beam. 这样的光学激发源的典型实例包括,但不限于激光器、发光二极管(LED)、紫外灯泡和/或白光源。 Typical examples of such optical excitation sources include, but are not limited to a laser, light emitting diode (LED), an ultraviolet lamp and / or white light source.

[0221] 用于本发明的光学传输元件发挥两个功能。 [0221] For the optical transmission element of the present invention performs two functions. 第一,它向芯片纳米孔中的反应物汇集和/或引导光学激发源。 First, it brings together the chip nanopores reactants and / or guiding the optical excitation source. 第二,它向感光元件传输和/或引导由芯片纳米孔中的反应物发射的光学信号。 Second, it is transmitted to the photosensitive element and / or guiding the optical signal from the chip nanopores reactants emitted. 适用于本发明的光学传输元件包括各种引导光从一个位点到另一个位点的光学装置。 Useful in the present invention, an optical transmission element includes a variety of points of light guide optical device from one position to another site. 这样的光学传输装置的非限制性的例子包括光纤、光多路复用器(MUX)和解复用器(DE-MUX)、衍射光栅、阵列波导光栅(AWG)、光学开关、反射镜、透镜、准直仪(co 11 imator)和任何其它弓|导光传输通过适当的折射指数和几何形状的设备。 Non-limiting examples of such optical transmission apparatus comprising an optical fiber, an optical multiplexer (MUX) and demultiplexer (DE-MUX), diffraction gratings, arrayed waveguide grating (AWG), optical switches, mirrors, lenses collimator (co 11 imator), and any other bow | photoconductive transmission through appropriate refractive index and geometry of the device. [0222] 感光元件分析来自纳米孔中的反应物的光学信号的光谱。 [0222] Analysis of the photosensitive member from the spectrum of the optical signal nanopore reactants. 合适的感光元件可以在给定的波长检测光学信号的强度,且优选可以同时测量整个波长范围的光学信号的强度。 Suitable photosensitive member can detect the intensity of the optical signal at a given wavelength, and preferably can simultaneously measure the intensity of the entire wavelength range of the optical signal. 优选元件也可以提供光谱数据分析,以显示光谱峰的波长、光谱峰的宽度和背景光谱噪音测量值。 Preferably element can also provide spectral data analysis, to show the spectrum peak wavelength, the spectral width of the peak and the background noise spectral measurements. 用于本发明的合适的感光元件的典型实例是雪崩光二极管(APD)、电荷耦合装置(CCD)、电子倍增电荷耦合装置(EMCCD)、光电倍增管(PMT)、光电倍增阵列、门敏感FET、纳米管FET和PIN二极管。 Typical examples of suitable photosensitive member of the present invention is an avalanche photodiode (APD), a charge coupled device (CCD), electron multiplying charge-coupled device (EMCCD), photomultiplier tube (PMT), photomultiplier array, gate sensitive FET nanotube FET and PIN diodes. 如本文所用,CCD包括常规CCD、电子倍增电荷耦合装置(EMCCD)和其它形式的强化的CCD。 As used herein, CCD including conventional CCD, electron multiplying charge-coupled device (EMCCD), and other forms of enhanced CCD.

[0223] 尽管可以使用各种元件的许多组合装配所述光学系统,用于分析激发的反应物的光谱的有用组件包括光学传输元件和感光元件。 [0223] Although the various elements can be used in many combinations of assembling the optical system for the analysis of the excitation spectrum of reactants useful components include optical transmission member and the photosensitive member. 这样的组件本文也称为"光谱分析器"。 Such components are herein referred to as "spectral analyzer." [0224] 需要时,本发明的光学系统可以包括光学选择元件。 [0224] If desired, the optical system of the present invention may include an optical selection element. 在激发光到达包含在纳米孔中的反应物之前,该元件选择和/或改进它们的光学性质。 Before the excitation light reaches the reactants contained in the nanopore, the component selection and / or improve their optical properties. 光学选择元件也可以用于在信号到达感光元件之前选择和/或改进来自纳米孔中的反应物的光学信号。 Optical selection element may also be used before the signal reaches the photosensitive member selection and / or improvements in the optical signal from the nanopores reactants. 合适的光学选择元件可以选择并改变许多的光学性质,包括,但不限于偏振、光强度、波长、多种光线之间的相差、多种光线之间的时间延迟。 Suitable optical selection element can select and change a lot of optical properties, including, but not limited to between the polarizer, the light intensity, the wavelength, a difference of a variety of light, the time delay between the multiple light. 这样的光学选择元件的典型实例是偏振滤波器、光学衰减器、波长滤波器(低通、带通或高通)、波板和延迟线。 Typical examples of such optical selection element is a polarizing filter, an optical attenuator, wavelength filter (low-pass, band-pass or high-pass), the wave plate and the delay line.

[0225] 前述的光学元件可以采取各种构型。 [0225] The foregoing optical element may take various configurations. 它们可以形成所述芯片的组成部分或保持为单独的单元。 They may form part of the chip or kept as a separate unit. 所有这些元件是可商购的。 All of these components are commercially available. 因此,在一种实施方式中,本发明提供了其中光学传输元件和感光元件制成芯片片基的芯片。 Thus, in one embodiment, the present invention provides a chip in which the optical transmission element and the photosensitive element is made of chip base sheet. 一方面,感光元件整合到待监测芯片上的各个纳米孔中。 On the one hand, the photosensitive element to be integrated into the monitoring of each chip nanopores. 另一方面,超过一种类型的感光元件整合到纳米孔中以提高检测能力或效率。 On the other hand, more than one type of photosensitive element integrated into the nanopores in order to improve the detection capability or efficiency. 另一方面,可以沿纳米孔侧面或在纳米孔的基部制造感光元件,或者制造感光元件成为纳米孔盖子的部分。 On the other hand, at the base of nanopore sensor fabrication, or manufacture of photographic elements become part of the nanopores can cover the sides or along nanopores. 适合这样的构型的感光元件包括,但不限于雪崩光二极管、电荷耦合装置(包括常规CCD、电子倍增电荷耦合装置(EMCCD)和其它形式的强化CCD)、门敏感的FET、纳米管FET、 PIN 二极管。 Such a configuration suitable photosensitive element including, but not limited to the avalanche photodiode, charge coupled device (including a conventional CCD, electron multiplying charge-coupled device (EMCCD), and other forms of reinforced CCD), gate sensitive FET, nanotube FET, PIN diodes. 尤其优选雪崩光二极管,因为它允许通过电子转移的雪崩过程扩增信号而检测单光子。 Especially preferably avalanche diode light, as it allows the amplification of the avalanche process of electron transfer and detection of single-photon signal. 使用本文所述的或本领域已知的标准IC处理技术可以制造这些元件与支持电路一起作为所述芯片的部分。 Use described herein or known in the art of standard IC processing techniques can make these elements, together with the support circuitry as part of the chip.

[0226] 在另一种实施方式中,本发明提供了其中芯片和光学系统保持为单独的单元的装置。 [0226] In another embodiment, the present invention provides a system in which the chip and the optical holding means is a separate unit. 本实施方式的一个方面包括用于进行需要在至少两个温度水平循环多个循环周期的化学反应或生物反应的装置。 One aspect of the present embodiment includes means for performing the required chemical reaction or biological reaction cycle a plurality of temperature levels of at least two cycles. 所述装置包括本发明的芯片和可操作地连接芯片并检测来自纳米孔的光学信号的光学系统。 The apparatus of the present invention comprises a chip and a chip operatively coupled and detecting optical system of the optical signal from the nanopores. 优选地,检测的光学信号与在纳米孔中发生的化学反应的产物量相关。 Preferably, the optical signal is detected and the amount of the product of the chemical reaction occurring in the related nanopores.

[0227] 图16说明这方面的示例性的光学系统。 [0227] FIG. 16 illustrates an exemplary optical system in this regard. 在示例性的实施方式中,该系统包括受计算机或其它数字设备1600控制的光学传输元件,如可调谐激光器1610或氙气灯。 In an exemplary embodiment, the system includes an optical transmission elements of a computer or other digital device controlled by 1600, 1610 or as a tunable laser xenon lamp. 然后使用Powell透镜1620、望远镜1630和/或线性聚焦激光器1640进一步聚焦激光以提供在所有纳米孔上的均一分布。 Then use the 1620 Powell lens, binoculars 1630 and / or 1640 linear laser focus to further focus the laser to provide uniform distribution of all nanopore. 来自芯片上的纳米孔的光学信号通过透镜1690(如管透镜(tubelen))校准,并经过可调滤波器1690(低通或高通)或陷波滤波器传送到电荷耦合装置(CCD) 1615用于光谱分析。 The optical signal from the nanopore chip 1690 through the lens (such as a tube lens (tubelen)) calibrated and tunable filters through 1690 (low-pass or high-pass) or notch filter is transferred to a charge-coupled device (CCD) 1615 by for spectroscopy. 这种特定的实施方式提供了用于监测在芯片上制造的纳米孔中发生的化学反应的进程和/或结果的低成本解决方案。 This particular embodiment provides a process for monitoring chemical reactions on the chip nanopores in the manufacture and / or the results of cost-effective solutions.

[0228] 在进一步的实施方式中,光学传输元件可移动以放置于如图16的选项1所示的XY阶段。 [0228] In a further embodiment, the optical transmission member is movable to be placed on the XY stage 16 in FIG options shown. 在可选择的实施方式中,芯片放置于如图16的选项2所示的XY阶段。 In an alternative embodiment, the chip 16 is placed on the option of XY stage 2 as shown in FIG. [0229] 在进一步的实施方式中,提供反馈控制或自我学习(self learning)以在片基的特定纳米孔实现优化的化学反应。 [0229] In a further embodiment, there is provided a feedback control or self-learning (self learning) to specific nanopores in the film base to optimize the chemical reaction. 例如,使用包括纳米孔阵列的固定位置的片基与可移动的光学传输元件和感光元件结合,在用一个或多个光学传输单元加热(或提供一系列加热和/或冷却步骤)之后,感光元件可以检测特定纳米孔中希望的化学反应的结果。 For example, using a fixed location comprising an array of nanopores of the film base and the movable optical transmission member and the photosensitive member in combination, with one or more optical transmission units heating (or a range of heating and / or cooling step), the photosensitive elements can be detected in the results of a particular nanopores desired chemical reactions. 在基于检测步骤分析给定的纳米孔中的各化学反应后,可以在随后的加热和/或冷却的循环轮次中实现合适的校正(例如提高或降低反应温度或反应循环的持续时间)以按照需要优化化学反应。 After analyzing a given nanopores of each chemical reaction based on the detection step, appropriate correction can be achieved in the subsequent heating and / or cooling cycles rounds (for example, increase or decrease the duration of the reaction temperature or reaction cycles) to according to the need to optimize the chemical reaction. 可以使传输元件和敏感元件重复通过在各种纳米孔位置来重复进行该过程,直至在各个纳米孔中检测到化学反应的需要的结果。 And the transmission element can be repeated by a variety of sensor position nanopores This process is repeated until a result of chemical reaction is detected in the respective desired nanopores. 以这种方式,可以顺序监测和操作给定的纳米孔的化学反应,以提供用于给定的纳米孔的优化的化学反应。 In this way, you can monitor and operate a chemical reaction sequence given nanopores to provide optimized chemical reaction given for the nanopores. 基于个别反应的独特的性质(例如,在PCR反应中使用不同的引物和模板),在不同的纳米孔位置可能需要不同的条件。 Based on the unique nature of the individual reactions (for example, using different primers and templates in PCR reactions), in different locations may require different nanopores conditions. 因此,可以结合指向中个纳米孔位置的反馈回路过程使用本发明的装置,以优化片基上的各个位置的化学反应。 Thus, the binding point position of a nanopore using a feedback loop process apparatus of the present invention, in order to optimize the chemical reaction at various locations on the base sheet.

[0230] 在示例性的实施方式中,可以使用Chungara系列CCD照相机。 [0230] In an exemplary embodiment, the series can be used Chungara CCD camera. Chungara CCD 照相机可以用于大多数要求低噪音和长暴露成像的应用,如在天文学和光谱学的领域。 Chungara CCD camera can be used in the most demanding low noise and long exposure to imaging applications, such as in the field of astronomy and spectroscopy. Chungara CCD控制器能够阅读多种CCD,因为它基于模块性和共同的硬件结构。 Chungara CCD controller can read a variety of CCD, because it is based on modularity and common hardware architecture. 例如,可以使用的CCD的例子范围从1536x1024 Kodak KAF-1602CCD到4096x4096 KodakKAF-16801 CCD。 For example, an example of the scope of the CCD can be used from 1536x1024 Kodak KAF-1602CCD to 4096x4096 KodakKAF-16801 CCD. 照相机可以经过以太网或无线连接来连接主机,从而允许计算机和CCD照相机之间存在大的距离。 The camera can be connected via Ethernet or wireless connection to the host, allowing the presence of a large distance between the computer and the CCD camera.

[0231] 在另一种实施方式中,本发明提供了其中芯片、装置和光学系统是单独的单元的系统。 [0231] In another embodiment, the present invention provides a chip in which the device and the optical system is a separate unit of the system. 在一种实施方式中,本发明的装置、系统、芯片,本发明的热循环装置或加热元件,及可操作地与芯片偶联并检测来自芯片的可编址单元的光学信号的光学系统。 In one embodiment, the present invention is an apparatus, system, chip, thermal cycling device or heating element of the present invention, and operably coupled with the chip and detecting optical system of the optical signal from the chip addressable unit. 优选地,检测的光学信号与在单元中发生的化学反应的产物量相关。 Preferably, the optical signal is detected and the amount of the product of the chemical reaction occurring in the cell associated.

[0232] 在另一种优选的实施方式中,通过可操作地与芯片偶联的所述光学系统检测所述芯片中的扩增的核酸。 [0232] In another preferred embodiment, through an optical system operatively associated with said detector coupled to the chip in the chip amplified nucleic acid. 光学系统能够向扩增反应中的反应物传输合适的激发光,收集并分析来自反应物的发射的光学信号。 The optical system is capable of transmitting to the amplification reaction the reaction of the appropriate excitation light, the collection and analysis of the optical signal transmitted from the reactants. 优选地,检测的光学信号表示多循环期间在扩增反应中扩增的核酸的量。 Preferably, the optical signal detected indicates the amount of a nucleic acid amplification reaction during multiple cycles of amplification. 在某些方面,光学系统在扩增过程中的多个时间点传输激发光到包含反应样品的孔中,并在各个时间点监测来自纳米孔的光学信号。 In certain aspects, an optical transmission system, a plurality of time points during amplification excitation light to the wells containing the reaction sample, and the optical signal from the surveillance nanopores in each time. 通过分析光学信号的相对强度,优选在多循环期间,技术人员可以定量地监测扩增反应的进程。 By analyzing the relative intensity of the optical signal, preferably in a multi-cycle period, technicians can quantitatively monitor amplification reaction process. 典型地,被监测的光学信号是荧光信号。 Typically, the optical signal is monitored fluorescence signals. 一旦开始扩增,可以进行检测和/或监测扩增产物而无需打开纳米孔。 Once amplified, can be detected and / or monitoring of the amplification product without opening the nanopores. [0233] 图17说明具有热循环装置1710和具有基于预定温度的可编址单元阵列的芯片1720的本发明的示例性的系统1700,所述热循环装置1710具有可以在第一和第二方位之间移动的第一加热器1712和第二加热器1714。 [0233] FIG. 17 illustrates a heat cycle device 1710 and having a first and a second orientation may be based on exemplary system chip 1720 predetermined temperature addressable cell array of the present invention, 1700, the 1710 has a thermal cycling device movement between the first and second heater heater 1712 1714. 在本实施例中,光学系统1730可操作地与芯片1720偶联,且装置1710是Ch皿gara CCD照相机1732。 In the present embodiment, the optical system 1730 is operably coupled with the chip 1720, and 1710 is a device dish gara CCD camera 1732 Ch. 如图17中的侧视图所示,加热器1712、1714可以独立地移动,并热接触本发明的芯片1720以提供如整个基因组的PCR扩增反应的大量化学反应的快速热循环。 The side view shown in FIG. 17, the heater 1712,1714 can move independently, and in thermal contact with the chip 1720 of the present invention to provide a whole genome such as PCR amplification reaction a large number of chemical reactions rapid thermal cycling.

[0234] 图18说明本发明的示例系统1800,包括光学系统1810、加热装置1820和用于进行化学反应的芯片1830。 [0234] Figure 18 illustrates an example system 1800 of the present invention, includes an optical system 1810, 1820 and a heating means for carrying out a chemical reaction chip 1830. 系统的加热装置1820可以是用于进行PCR反应的热循环设备。 The heating device 1820 may be a system for carrying out PCR thermocycling reaction apparatus. 如图18所示,加热装置可以包括用于进行PCR的变性块1822和退火块1824。 As illustrated, heating means 18 may comprise a block for performing PCR denaturation and annealing blocks 1822 1824. 加热装置1820 也可以如图18中的箭头所示沿水平和垂直方向移动。 Heating means 1820 may be moved as shown in FIG horizontal and vertical directions of arrow 18. 芯片1830插在可以垂直移动的样品座1840上。 1830 chip can be inserted in the vertical movement of the sample holder 1840. 芯片1830和/或芯片的纳米孔可以被塑料盖1832覆盖或不覆盖。 Chip 1830 and / or nanopore chip may be covered with a plastic cover or not to cover 1832. 加热器1820可以迫使样品座1840热接触包括薄膜ITO加热器1852的顶端盖片1850。 Heater 1820 can be forced in thermal contact with the sample holder 1840 including top film ITO heater cover sheet 1850 1852. 顶端盖片1850通过压縮弹簧1854连接系统1800,以允许系统的样品座1840和加热器1820、 1852之间形成更好的热接触。 Top cover sheet by a compression spring 1854 1850 1800 connection system, to allow the system of sample holder heater 1840 and 1820, the formation of better thermal contact between the 1852. 系统1800也可以包括死制动块(dead stop) 1856,以防止顶端盖片1850压入光学系统1810中。 System 1800 can also include brake pads dead (dead stop) 1856, in order to prevent the top cover sheet 1850 is pressed into the optical system 1810.

[0235] 在图18中,光学系统1810包括CCD照相机1812、激发光源1814、光学器件1816和光学滤波器1818。 [0235] In Figure 18, the optical system includes a CCD camera 1810 1812 1814 excitation light source, optical devices, optical filters 1816 and 1818. 系统可以包括多个光学系统。 The system can include a plurality of optical systems. 在另一种实施方式中,系统包括多个CCD 照相机、激发光源、光学器件或光学滤波器。 In another embodiment, the system comprises a plurality of CCD cameras, the excitation light source, an optical device or an optical filter. 当芯片1830热接触加热装置1820时,光学系统1810可以发挥接收来自在芯片1830中发生的化学反应的信息的功能。 When the chip is in thermal contact with the heating device 1830 1820 1810 optical system can play a function information received from a chemical reaction occurring in the chip 1830. [0236] 图19是显示逻辑设备的典型实例的方块图,通过所述逻辑设备可以实现检查或分析与本发明相关的数据。 [0236] FIG. 19 is a block diagram showing a typical example of the logic device, it can carry out the inspection or analysis of data related to the present invention, by the logic device. 这样的数据可能与个体的基因型、遗传组成或疾病、障碍或状态有关。 Such data may be composed or disease, disorder or condition related to an individual's genotype, genetic. 图19显示连接装置1920的计算机系统(或数字设备)1900,其与装置1924 —起使用以例如产生结果。 Figure 19 shows the connection means 1920 computer system (or digital device) 1900, with the device 1924-- from the use of, for example, produce a result. 计算机系统1900可以理解为可以从介质1911和/或网络端口1905读取指令的逻辑装置,网络端口1905可以任选地连接具有固定介质1912的服务器1909。 Computer system 1900 may be understood as the medium from 1911 and / or network port read instruction logic means 1905, a network port 1905 may optionally be connected to server 1912 having fixed media 1909. 如图19显示的系统包括CPU 1901、磁盘驱动1903、如键盘1915和/或鼠标1916的任选输入设备和任选的监视器1907。 19 The system displays, including CPU 1901, 1903 disk drive, such as a keyboard 1915 and optional input device and optional monitor / mouse 1916 or 1907. 可以通过指明的通讯介质实现与本地或远程服务器1909的数据通信。 Data communication can be achieved with local or remote server 1909 through the communication media indicated. 通讯介质可以包括任何传输和/或接收数据的手段。 Communication medium may include any means to transmit and / or receive data. 例如,通讯介质可以是网络连接、无线连接或因特网连接。 For example, the communication medium can be a network connection, a wireless connection or Internet connection. 这样的连接提供环球网上的通讯。 Such a connection provides global online communications. 可以设想:与本发明相关的数据可以通过这样的网络或连接传输以由某一方1922接收和/或检查。 It is contemplated: the data related to the present invention can be transmitted to a network or connected by a party with 1922 received and / or check. 接收方或使用者1922可以是患者、卫生保健提供者或卫生保健管理者。 1922 recipient or user may be patients, health care providers or health care manager. 在一种实施方式中,计算机可读介质包括适于传输生物样品的分析结果的介质。 In one embodiment, a computer-readable medium suitable for transmission includes a biological sample analysis result media. 该介质可以包括关于受试者的基因型、遗传组成或疾病条件或状态的结果,其中,这样的结果使用本文所述的方法得到。 The medium may include genotype on the subject, the results of genetic makeup or disease condition or state in which, using the methods described herein such results. [0237] 輛„ [0237] vehicles "

[0238] 用于热循环的所述芯片和装置在需要可控制的温度的化学和生物学应用中具有广泛的用途。 [0238] The chips and devices for thermal cycling with a wide range of uses in chemical and biological applications may require temperature controlled. 优选本发明的方法、芯片和装置与有助于将该方法的一个或多个步骤结合的设备一起执行,所述步骤包括芯片的操作、热循环和成像。 The method of a preferred method of the present invention, the chip and the means to assist or more of the steps performed by the device together with the binding, comprising the step of operating the chip, thermal cycling and imaging. 因此,本发明提供了用于相对于所述物种的标准基因组的物种同时测定从单个成员获得的生物样品的遗传表达谱的系统。 Accordingly, the present invention provides a method for the simultaneous determination of genetic biological sample obtained from the individual members of the system relative to the expression profile of the species standard species genome. [0239] 在一种实施方式中,本发明可以用于改变和/或维持反应样品的温度。 [0239] In one embodiment, the present invention can be used to alter and / or maintain the reaction temperature of the sample. 在多种情况下需要改变和/或维持反应样品的温度,包括但不限于鉴别蛋白质-蛋白质相互作用、检查DNA或RNA杂交和进行酶促反应。 In many cases the need to change and / or maintain the reaction temperature of the sample, including but not limited to identification of protein - protein interactions, DNA or RNA hybridization and check the enzymatic reaction. 该方法包括将反应样品置于在与加热元件热接触的芯片中制造的纳米孔中,并向加热元件施加电压。 The method comprises the reaction sample is placed in a nanopore in thermal contact with the heating element in the manufacture of chips, a voltage is applied to the heating element.

[0240] 在另一种实施方式中,用于热循环的所述芯片装置用来进行涉及多个反应样品并需要至少两个温度水平的循环的化学反应。 [0240] In another embodiment, the chip device used for thermal cycling reaction involving a plurality of samples and requires at least two temperature levels of cyclic chemical reactions. 该方法包括:(a)提供包含本文所述的单元阵列的芯片;(b)将多个反应样品置于芯片的单元中;和(c)控制加热元件以实现至少两个温度水平的循环。 The method comprises: (a) providing a chip comprising cell arrays as described herein; (b) a plurality of chip sample in the reaction cell; and (c) controlling the heating element in order to achieve the level of at least two temperature cycles.

[0241] 实施所述方法一般通过将反应样品置于热接触加热元件的所述芯片的纳米孔中进行。 The method described in [0241] Usually the reaction by heating the sample in thermal contact with the nanopore chip component carried. 需要时,可以通过可操作地连接所述芯片的分配系统应用反应样品。 When necessary, by operably connecting the chip application response distribution system samples. 从手动操作的移液管到自动化的机器人系统的各种分配器具都是本领域可获得的。 From pipetting manually operated appliance pipe to a variety of automated robotic dispensing system it is available in the art. 优选的分配器具包括压电纳米分配器。 Preferred dispensing apparatus comprises a piezoelectric nanodispenser.

[0242] 所述芯片和装置尤其适于进行定量的核酸扩增。 [0242] The chip and the device is particularly suitable for quantification of nucleic acid amplification. 因此,本发明提供了用于监测核酸扩增反应产物的形成的方法,优选实时监测。 Accordingly, the present invention provides a method for monitoring nucleic acid amplification reaction products formed, preferably real-time monitoring. 在一定的优选实施方式中,通过整合到芯片中的感光元件直接监测包含的扩增核酸。 In certain preferred embodiments, the nucleic acid amplification by integration into the direct monitoring sensor chip contains. 感光元件记录反映扩增反应过程中任何检查时间的扩增核酸的量的光学信号强度。 The photosensitive recording element to reflect the optical signal intensity of the amplified nucleic acid amplification reaction the amount of time during any inspection. 光学信号可以是在用合适的入射光激发标记的反应物时发射的任何种类的荧光信号。 Any kind of fluorescent signal when the optical signal can be excited by incident light with suitable reactants labeled emission.

[0243] 扩增并检测目标核酸的所述方法例如,在药物筛选、疾病诊断、系统发生分类、确定个体基因型和亲子鉴定中具有广泛用途。 [0243] The method of the target nucleic acid amplification and detection, for example, occur classification in drug screening, disease diagnosis, system to determine individual genotypes and paternity has wide range of uses.

[0244] 在一种实施方式中,本发明的系统、芯片、装置或方法可以用于发现治疗相关的生物标志物。 [0244] In one embodiment, the system, chip, device or method of the present invention can be used to find treatment-related biomarkers. 例如,本发明可以用来鉴定用于慢性阻塞性肺病(COPD)和肺癌的生物标志物。 For example, the present invention can be used for the identification of chronic obstructive pulmonary disease (COPD) and lung cancer biomarkers. [0245] 在更基础的水平时,目标核酸的扩增和检测可以用于鉴定和定量患病和正常的组织之间、在不同类型的组织和细胞之间、处于不同发育阶段或不同的细胞周期点的细胞之 [0245] at a more basic level, the amplification and detection of target nucleic acid can be used to identify and quantify between disease and normal tissue, between different types of tissues and cells, in different stages of development or different cell The point of the cell cycle

间和受到各种不同环境剌激或先导药物影响的细胞之间的差别基因表达。 And by differences in gene expression between the various environmental stimuli or leading the influence of drugs between cells.

[0246] 在本发明的各种配置中,公开了提供用于获得结构基因组信息(如一种或多种单 [0246] In the various configurations of the present invention is disclosed for obtaining structural genomics provide information (such as one or more mono

核苷酸多态性(SNP)的存在与否)和功能性基因组信息(如一个或多个基因的表达或表达 Nucleotide polymorphism (SNP) in the presence or absence) and functional genomics information (such as expression or expression of one or more genes

量)的消费者分析的方法。 Amount) methods consumer analysis. 如此,该分析可以设置为检测生物样品中的遗传物质的存在或 Thus, the analysis may be provided to detect the presence of biological sample or genetic material

表达。 Expression. 该方法包括提供设置为用于接收批量分析(stock assay)的命令的使用者界面,提 The method comprises providing to a user interface for receiving bulk analysis (stock assay) command, mention

供设置为用于接收对于惯常分析的设计要求和用于指令所述分析的使用者界面,和响应于 For setting user interface for receiving the usual analysis of design requirements and instructions for the analysis of, and response to

消费者的惯常分析或批量分析的指令向消费者传送至少一个惯常分析或批量分析。 Transfer instructions habitual consumer analysis or batch analysis to the consumer at least one of the usual analysis or batch analysis. 在特定 In particular

的其它方面,本发明包括系统、装置、芯片和方法,用于构建为消费者提供设置为检测遗传 The other aspect, the present invention includes a system, device, chip and method for constructing the consumers to detect genetic

物质的存在或表达的分析方法的系统。 System analysis of the presence or expression of substance. 在一种实施方式中,可以根据使用者的需要定制芯片。 In one embodiment, can be customized according to the user's chip.

[0247] 本发明提供了用于容纳并处理少量液体的设备和方法,包括用于在含有多核苷酸的液体样品上进行扩增反应的设备和方法。 [0247] The present invention provides an apparatus for receiving and processing a small amount of liquid and methods, including apparatus and method for performing amplification reactions in the liquid sample containing polynucleotide. 本发明的实施方式包括用于在液体样品中进行包括多核苷酸的热循环扩增反应的化学反应的芯片。 Embodiment of the present invention include a polynucleotide comprising for performing thermal cycling amplification reaction chemical reaction in the liquid sample chip.

[0248] 所述芯片和其它设备在需要可控制的温度的许多其它化学和生物学应用中具有 [0248] The chips and other devices with many other chemical and biological applications require a controlled temperature of

实用性。 Practicality. 这样的应用包括各种反应,如氧化还原反应、水解、磷酸化和聚合反应。 Such applications include a variety of reactions such as oxidation-reduction reaction, hydrolysis, phosphorylation and polymerization. 另外的应 Also to be

用涉及鉴别包括生物分子(如蛋白质、糖蛋白、核酸和脂质)以及无机化学品或其组合的相 Identification by including biological molecules involved (such as proteins, glycoproteins, nucleic acids and lipids) or combinations thereof, and inorganic chemicals phase

互作用。 Interaction. 化学反应也可以包括核酸分子之间、核酸和蛋白质之间、蛋白质和小分子之间的相 Chemical reaction between the phases may also comprise nucleic acid molecules, between nucleic acids and proteins, proteins and small molecules

互作用。 Interaction. 化学反应可以在引入所述芯片的纳米孔中的细胞外或细胞内发生。 Chemical reactions can take place outside of the intracellular introduction of the chip in a cell or nanopores.

[0249] 在检测特定的蛋白质-蛋白质相互作用的存在方面的应用具有特别的意义。 [0249] In the detection of specific proteins - have a special meaning application presence aspect of protein interactions. 这样 This

的应用一般采用置于所述芯片的单元中的蛋白质探针和靶标蛋白质。 The applications are generally placed on the use of cell protein chip probe and target protein.

[0250] 在本实施方式的一个方面,蛋白质-蛋白质相互作用是在靶标蛋白质(例如,抗原)和对于该靶标特异性的抗体之间。 [0250] In one aspect of this embodiment, protein - protein interaction is between a target protein (e.g., an antigen) and for the target specific antibody. 另一方面,蛋白质-蛋白质相互作用是在细胞表面受体和其相应配体之间。 On the other hand, protein - protein interactions between cell surface receptors and their respective ligands. 在更另外的一方面,蛋白质-蛋白质相互作用涉及细胞表面受体和免疫脂质体或免疫毒素;在其它方面,蛋白质-蛋白质相互作用可以涉及胞质蛋白质、核蛋白质、伴随蛋白质或锚定于其它细胞内膜结构上的蛋白质。 In a more another aspect, protein - protein interaction involves a cell surface receptor and immunoliposome or an immunotoxin; In other aspects, protein - protein interaction may involve cytoplasmic proteins, nuclear proteins, or proteins anchored to accompany Other cell membrane protein structure.

[0251] 用于细胞蛋白质的术语"膜"、"胞质"、"核"和"分泌的"指胞蛋白质大部、主要或优选定位的细胞外和/或亚细胞位置。 [0251] For cell protein, the term "film", "cytosolic", "nuclear" and "secreted" protein refers to cells most, or preferably positioned mainly extracellular and / or subcellular location.

[0252]"细胞表面受体"代表能够结合其各自的配体的膜蛋白质的亚群。 [0252] "cell surface receptor" means capable of binding their respective ligands membrane proteins subpopulations. 细胞表面受体是 Cell surface receptors

锚定在细胞质膜上或插入其中的分子。 Anchored in the cytoplasmic membrane or molecule inserted therein. 它们构成蛋白质、糖蛋白、多糖和脂质的大家族,其 They constitute a large family of proteins, glycoproteins, polysaccharides and lipids, which

不仅作为质膜的结构组成,也作为控制各种生物功能的调节元件发挥作用。 Not only as membrane structures, but also control various biological functions as regulatory elements play a role.

[0253] 通常通过在允许蛋白质探针和靶标之间形成复合物的条件下将探针与靶标蛋白 Under conditions [0253] typically by allowing the probe and the target protein complex formed between the probe and the target protein

质接触进行反应。 Contacting the reaction mass. 如反应温度、反应持续时间、缓冲液条件等的条件取决于待研究的特定的 As the reaction temperature, duration of the reaction conditions, buffer conditions and the like to be studied depends on the particular

相互作用。 Interactions. 一般地,优选在生理相关的温度和缓冲液条件下进行反应。 Generally, the reaction is preferably carried out at physiologically relevant temperatures and buffer conditions. 生理相关的温度范 Physiologically relevant temperature range

围是从大致室温到大约37t:。 Wai is from approximately room temperature to about 37t :. 这可以通过调节所述芯片的加热元件来实现。 This is accomplished by adjusting the heating element of the chip to achieve. 典型地,生理 Typically, physiological

缓冲液包含生理浓度的盐,并调节到范围是大约6. 5-大约7. 8、优选大约7. 0-大约7. 5的 Salt buffer containing physiological concentrations, and adjusted to the range of from about 5 to about 6. 7.8, preferably from about 7. 0 to about 7.5 in

中性pH。 Neutral pH. 在Sambrook等人(1989),同上中列出各种生理缓冲液,本文不再详述。 In Sambrook et al. (1989), supra, listing various physiological buffer, paper will not elaborate.

[0254] 根据本领域的标准方法或本文所述的方法可以直接或间接检测复合物的形成。 Either directly or indirectly detect the formation of complexes [0254] The method according to standard methods in the art or described herein. In

直接检测方法中,与可检测的标记一起提供探针,且当复合物形成时,探针发射与未反应的探针的光学信号明显不同的光学信号。 Direct detection method, and provide a detectable label probes together, and when the complex is formed, the optical signal transmission and unreacted probe probe distinctly different optical signals. 理想的探针一般不会干扰靶标结合或干扰产生的靶标-探针复合物的稳定性。 Usually the probe does not interfere with the desired target binding or interference target - probe complex stability. 如上所述,本领域的技术人员已知各种适于这样的应用的标记, 其中大部分是荧光探针。 As described above, those skilled in the art suitable for such markers known to various applications, most of which are fluorescent probes. 可以通过标准的定量分析或使用上述的光学系统的定量方法定量在结合反应过程中形成的探针_靶标复合物的量。 Quantitative methods can quantify the amount of standard quantitative analysis or use of such an optical system formed during the binding reaction _ probe target complexes.

[0255] 本文所述的实施例和其它实施方式是示例性的,且不打算限于描述本发明的装置、系统、组合物、材料和方法的全部范围。 [0255] The examples and other embodiments described herein are exemplary and not intended to be limited to describe the devices, systems, compositions, materials and methods of the present invention the entire range. 可以在本发明的范围内进行具体实施方式、装置、系统、组合物、材料和方法的等价改变、修饰、变化而具有基本上类似的结果。 Can be carried out within the scope of the present invention is equivalent to changing the specific embodiments, devices, systems, compositions, materials and methods, modified, changes have substantially similar results. 这样的改变、修饰或变化不认为是背离本发明的精神和范围。 Such changes, modifications or variations are not regarded as a departure from the spirit and scope of the invention.

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Classifications
International ClassificationB01J19/08, C08F2/01
Cooperative ClassificationB01L2200/10, B01L2300/0654, B01L2300/0829, B01L7/5255, B01L7/525, B01L2300/1894, C12Q1/686, B01L2300/0819, B01L2200/147, B01L2300/1827, B01L2300/0893, B01L2300/1844, B01L2300/1805, G01N21/253, B01L7/54, G01N21/0332
European ClassificationB01L7/5255, G01N21/25B2
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