WO2003100098A1

WO2003100098A1 - Analyte microdetector and methods for use

Info

Publication number: WO2003100098A1
Application number: PCT/US2003/016182
Authority: WO
Inventors: Christopher J. Savoie; Satoru Kuhara
Original assignee: Gene Networks, Inc.
Priority date: 2002-05-23
Filing date: 2003-05-22
Publication date: 2003-12-04
Also published as: AU2003239569A1

Abstract

This invention includes arrays for detecting nucleic acids, proteins and small molecules for use in studies in the fields of genomics, proteomics, medicine, pharmaceutical sciences, physiology, metabolics and biochemistry. Systems are provided for rapid, high throughput screening of molecules using microlenses and electromagnetic radiation. Electromagnetic radiation passes through a labeled sample on a substrate and detection signals arising from an interaction of that electromagnetic radiation and a labeled sample are focused using microlenses, thereby decreasing the effects of parasite light. By reducing parasite light, the number of false positive results can be decreased , and the size of a sample can be decreased accordingly. Decreasing the size of a sample needed for detection can permit increases in the numbers of samples detected per array. Signal to noise ratio can be increased in other embodiments by providing reflective layers on arrays, thereby permitting multiple interactions between incident electromagnetic radiation and a sample.

Description

ANALYTE MICRODETECTOR AND METHODS FOR USE

Related Application:

This application claims priority to United States Provisional Patent Application Serial No: 60/382,669, filed May 23, 2002. The above application is herein incorporated fully by reference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to the screening and analysis of analytes using microarrays. Specifically, this invention relates to microdetectors and methods for analyte detection use arrays of microlenses and optical fibers that reduce parasite light from contaminating signals received from labeled nucleic acids during fluorescence detection. More particularly, microdetectors and methods are provided that are used for DNA analysis.

Description of Related Art

Detection of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger RNA (mRNA) are increasingly used to analyze and characterize genetic information from a variety of animal and plant sources. In diagnostics, it can be desirable to detect the presence of regions of nucleic acids that are related to disease conditions, such as cancer, or to genetic information that can reflect a predisposition to a disease. Currently, numerous nucleic acid based diagnostic methods and devices are available and in use. In drug development, target genes are desirably identified and then targeted for manufacture of factors that can regulate the expression of certain genes, including genes related to disease or health. For example, single nucleotide polymorphisms (SNPs) can provide important information about genetic variations that might predispose an individual to a gene-based disease, or can be useful to track variations in genes among individuals or among populations of individuals. Fundamental understanding the functional relationships between different genes within an organism is lacking. In vivo, expression of genes is rarely an isolated, individual event. Rather, multiple genes within a family of genes may be expressed in a coordinated fashion. In some cases, the expression of one gene or group of genes may be associated with the decreased expression of a different gene or group of genes. The recent availability of genetic information from human and other sources has dramatically increased, and correlation of gene sequence and gene function are very important areas of genomics, proteomics and diagnostics.

Current methods for screening genetic information include the use of DNA arrays, which are devices having a number of pre-manufactured areas having specific DNA placed thereon. The DNA in these areas can be selected to be complementary to, and therefore bind to DNA under study. DNA is made of long strands of nucleotides bound together by covalent bonds. There are four nucleotides found in DNA. Each nucleotide consists of a deoxyribose sugar residue, a phosphate group, and one of four nucleotide bases, adenine (A), thymine (T), cytosine (C) and guanine (G). One commonly viewed mechanism for such complementary binding includes the known tendency of the four nucleotide bases in DNA to bind with each other. It is known that an adenine moiety in a DNA sequence can become associated with a thymine moiety on another strand of DNA. Similarly, a cytosine moiety can become associated with a guanine moiety on another strand.

It is also well known that DNA in humans and other organisms is arranged in two strands, known as "complementary" strands, in which the sequences of nucleotides in one strand match the corresponding sequence in the other strand, with A in one strand apposed to T in the other, and C in one strand apposed to G in the other. The structural basis for such DNA- DNA binding was popularized by Watson and Crick, and many advances in genetics and molecular biology have been derived from the widespread understanding of DNA complementary binding. In fact the genomics industry is, to a large part, based upon the knowledge of complementary binding of nucleic acids and the detection of DNA having specific nucleotide sequences using DNA arrays. The use of DNA arrays involves the application of a sample containing DNA of interest to a DNA array. If a sequence of DNA in the sample are complementary to a sequence on the DNA array, that DNA in the sample may bind to the complementary DNA in the array. Currently, methods for detecting sample DNA bound to an array include fluorescent- or dye-labeling of the sample DNA prior to its binding to the array. Thus, when a labeled sample of DNA is bound to an array, the unbound DNA can be removed from the array by washing, and only the labeled complementary DNA remains bound to the array.

Fluorescently labeled, bound DNA can be detected using a fluorescence detector placed over the array. For example, a fluorescence scanning instrument can have a probe that is aligned with an area or "spot" on the array having DNA with a particular sequence of bound DNA attached thereto. A photon of incident light of a certain wavelength interacts with the fluorescent label on the sample DNA producing a photon having a different wavelength (λ; i.e., a fluorescent signal), which is then detected by a probe. The probe captures the emitted fluorescence signal and transmits it to a detector, such as a charge coupled detector (CCD) or other optical instrument, and the presence of such fluorescence indicates the presence in the sample of the DNA of interest.

DNA can also be labeled using a dye that has characteristic absorption at certain wavelengths. When dye-labeled DNA is placed in a suitable detector and a beam of light having an appropriate wavelength is directed through the sample, a portion of the light passing though the dye- labeled sample is absorbed. The change in intensity thus represents a detection signal. The change in absorption of light is thus another useful principle upon which DNA detection can be carried out. The term "detection signal" as used herein includes electromagnetic signals, and/or changes in electromagnetic signals, whose presence indicates that the sample contains an analyte of interest. In a single human genome, there are about 3 x 10⁹ base pairs, which represent between 30,000 and about 100,000 different genes. Each gene may have thousands of individual bases, and gene groups may have millions of bases. Therefore, genetics and genomics researchers have the daunting task of identifying and analyzing huge numbers of different DNA sequences. Because of the huge numbers of bases which are subject to analysis, methods for rapid screening of genetic information is highly desired. Currently, substrates having DNA in arrays ("DNA chips") may have a large number of areas thereon (e.g., 10,000 or more), each of which has a desired sequence of DNA applied thereto. It is apparent that providing smaller areas on a chip can permit the use of more areas on the array. Thus, DNA chips having large number of small spots are termed "DNA microarrays."

One currently limiting feature of fluorescent and dye-labeling methods is related to the size of the detecting probe and the size of the area or spot on the array. If the probe is smaller than the spot, and is relatively close to the spot, then the light captured by the probe reflects primarily the light emitted by that spot on the array. However, as the sizes of spots decreases, the probe may not be sufficiently isolated from other nearby spots, and light captured by a probe can represent signals generated by the desired spot and also by nearby spots. This type of signal is referred to herein as "parasite light." If the DNA sequences on nearby spots are different from the spot under analysis, the parasite light can contaminate the signal, and the true identity of the DNA in the sample can be difficult to determine.

Figure 1 depicts a situation for prior art devices. DNA array 100 has a substrate 104 having areas 108, 109 and 110 thereon, comprising DNA "receptors" having labeled analytes 112, 113 and 114, respectively. Three probes 116, 120 and 124 are shown above the receptors 112, 113 and 114, respectively. An incident light beam 128 produces signals from the labeled analytes 113. Some of the signal emitted by analyte 113 is captured by probe 120. However some parasite light (curved upward arrows) is captured by probes 116 and 124 thereby producing spurious signals.

Thus, the current art is limited by the interference, by parasite light, of signals arising from labeled DNA and other analytes on microarrays. Parasite light therefore becomes limiting as the size of a spot decreases and the density of spots on DNA microarrays increases.

SUMMARY OF THE INVENTION

Therefore, one object of this invention is the development of devices to reduce the capture of parasite light from DNA-, other oligonucleotide-containing microarrays protein-containing arrays and other arrays.

Another object of this invention is the development of methods to detect signals arising from labeled DNA or other oligonucleotides without interference from signals generated by nearby areas on a DNA microarray.

A further object of this invention is the development of arrays that can increase the sensitivity of detection of analytes.

To address these and other objects, this invention includes the use of planar, highly reflective surfaces on arrays to increase the likelihood of a beam of light interacting with the analyte of interest.

In other embodiments of this invention, "microlenses" which can capture light arising from small areas on a DNA or other oligonucleotide- containing microarray. In some embodiments, light corresponding to a single spot on a DNA microarray can be captured by a microlens having a cross-section exhibiting concentric "rings" or areas of different densities of the microlens between the center and the periphery. Light signals can be focused by the microlens, thus reducing the amount of parasite light reaching nearby detectors. Signals reaching the probe from directly under the probe can be captured more efficiently than signals reaching the probe from more laterally displaced locations.

In some embodiments, fluorescence or absorbance detection can be carried out using incident light directed toward a spot from above the surface of the oligonucleotide or DNA microarray. In such situations, it can be desirable to provide the DNA on a substrate having a very smooth, reflecting surface, which can reduce the scattering of light. Thus, in these embodiments, incident light can have two opportunities to interact with a label, one while the beam is passing "down" through the DNA sample, and another while the reflected beam is passing "up" through the sample. Moreover, signals generated by such an incident beam can be detected if they pass "up" from the spot to be captured by a microlens, or if they pass "down" to the surface of the substrate and are reflected back "up" through the sample and then to the microlens.

In other embodiments, dye-labeled oligonucleotides or DNA can be detected on the surface of a reflective layer, in which an incident beam passes through the sample on the DNA microarray and is absorbed by the dye in the sample. As with fluorescence detection, there can be two opportunities for light absorption to occur, one while the light beam is passing down through the sample, and another while the reflected beam is passing up through the sample. As with fluorescence, it can be desirable to provide a very flat, smooth reflective surface to decrease side scattering and to also decrease loss of signal by light passing into the substrate and being lost. In either case, the difference in energy between an incident beam and the collected light represents a detection signal, indicating the presence of an analyte in the sample.

In certain other embodiments, two dyes can be used, which provides "dual label" detection. Currently, green dye (e.g., Sci-3) and red dye (Sci-5) are used, although the principles embodied in this invention can be used for other labels.

In other embodiments, dye- or fluorescently labeled oligonucleotides and/or other DNA can be detected by a beam of light passing through the substrate, through the sample where fluorescence or absorption occurs and either the fluorescence or absorption signal is captured by a microlens on the other side of the substrate. In these embodiments, because there is little or no reflection, contamination of a signal by parasite light can be further reduced.

In yet further embodiments, RNA, protein or small molecule analytes can be detected using similar microlenses in ways similar to those suitable for detecting nucleic acids.

Other embodiments of this invention include DNA microarrays having known DNA nucleotide sequences thereon to hybridize with complementary DNA or other nucleic acid for detection.

Still further embodiments include visualization tools and systems suitable for rapid detection of samples on microarrays.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described with reference to particular embodiments thereof. Other objects, features, and advantages of the invention will become apparent with reference to the specification and drawings in which:

Figure 1 depicts a cross-section view of a prior art microarray in which parasite light can contaminate a signal from a spot on the array.

Figure 2 depicts a cross-section view of a microlens used in detectors of this invention.

Figure 3 depicts a cross-section view of an embodiment of a DNA microarray of this invention having a reflective surfaces to amplify the amount of light detected.

Figure 4 depicts a cross-section view of an embodiment of this invention in which a microlens focuses light before passing through a sample.

Figure 5 depicts a cross-section view of an alternative embodiment of this invention using a microlens to focus light after passing through a sample.

Figure 6a depicts a top view of the relationship between a DNA spot and a microlens of one embodiment of this invention. Figure 6b depicts a top view of the relationship between a DNA spot and a microlens of a different embodiment of this invention than depicted in Figure 6a.

Figure 7 depicts a cross-section view of an array of microlenses on an array.

Figure 8 depicts an oblique view of a two-dimensional array of samples placed on an array of microlenses.

Figure 9 depicts a cross-section view of a system for analyzing an array of samples placed on an array of microlenses as shown in Figure 8.

DETAILED DESCRIPTION

Embodiments of this invention comprise a substrate having one or more areas thereon ("spots"), each area typically having one type of analyte receptor thereon. Receptors may be DNA, RNA or other molecules that can bind specifically with analytes of interest. The number and distribution of these areas is quite variable, with the only limiting factors being the number of receptors necessary to bind a sufficient number of labeled analyte molecules to produce a signal having a suitable magnitude. Typically, such a substrate can have up to about 100,000 different spots, and in some embodiments, can have 1,000,000 different spots thereon. The spots can be arranged together closely as necessary to provide the desired number of different spots on the substrate, given the resolution of a labeled analyte attached to the receptors on that spot. Such substrates having a plurality of spots is herein termed an "microarray."

Microarrays useful for genetic or genomic screening can use DNA sequences complementary to a nucleic acid of interest, or alternatively can be an antibody directed against a specific DNA or RNA sequence. For many purposes, DNA receptors are desirable, providing sufficiently tight binding to analytes under known conditions to enable a user to determine the specificity of binding and therefore the identity of the analyte. DNA receptors can be used to detect cDNA, RNAs including mRNA or genomic DNA. Prior art methods involving the use of conventional detectors suffers from parasite light. Detection of analytes can be substantially improved by using a novel combination of microarrays, highly planar, reflective surfaces, providing small spot sizes or the use of microlenses to capture signals arising from a specific location corresponding to a specific analyte.

In certain embodiments of this invention, increases in signal to noise (S/N) ratio can be obtained by the use of reflective layers on an array substrate. In prior art devices without reflective layers, incident light is provided with a single opportunity to interact with a labeled analyte on the substrate. If the incident light interacts with the labeled analyte, a change in absorption or fluorescence can be observed. However, if the incident beam does not interact with the labeled analyte, no detection signal is produced, and the sample is considered not to have the analyte. However, by the use of reflective surfaces, if an incident beam fails to interact with the labeled analyte on the first pass, the beam can be reflected back, upwards through the sample, providing an additional opportunity for interaction to occur. If the surface of the substrate is planar and a layer of reflective material is also planar, then there can be two surfaces that can reflect the light, and the opportunity to detect a labeled analyte can increase.

It can be desirable to provide surfaces having high planarity and even surfaces. In some embodiments of this invention, films of gold, aluminum or platinum can be used, and in alternative embodiments, diamond (carbon) can be used. Diamond surfaces can be desirable in situations in which the resonance of the surface is desirably small.

Figure 2 depicts a portion of a prior art microlens 200 comprising a substrate 204 and a series of concentric areas or "rings" 208, 212, 216 and 220, each having a different density compared to adjacent rings. Microlenses can act by capturing light signals and permitting them to be transmitted through rings 208, 212, 216 and 220. During passage from one are to another, the signals can be focused by refraction to a beam having decreased diameter. Microlenses are commercially available and can be obtained from Nippon Sheet Glass Co. Microlenses in two-dimensional arrays are also available from Nippon Sheet Glass Co, and are suitable for the devices and methods for array detection of this invention.

Microlenses can be made to have any desired number of rings, and having any desired dimensions. For example, the lens shown in Figure 2 has a radius of about 100 μm. It can be appreciated that the radial dimension of any particular ring should be sufficiently large to permit the passage of light. Thus, if one is detecting light having a wavelength of 100 nm, the rings should have a size capable of transmitting the light. Alternatively, if one is using longer wavelengths, e.g, 1000 nm, the dimensions of the rings can be correspondingly larger. Similarly, one can appreciate that in waveguides having the same overall diameter, by reducing the number of rings, each ring may have a larger radial dimension. Thus the user can select appropriate combinations of ring diameter and ring number. Microlenses can have any suitable diameters, for example, from about 100 μm to about 1000 μm. In other embodiments, the diameter of a microlens can be in the range of about 100 μm to about 500 μm, and in still other embodiments, in the range of about 200 μm to about 300 μm, and in yet further embodiments, about 250 μm in diameter.

Microlenses can also be provided that, instead of having discrete rings, has gradients of density. Moreover, any type of microlens that focuses or concentrates light can be used according to the devices and methods of this invention.

EXAMPLES

Example 1: Microarray Substrates Having Increased Reflectivity

Figure 3 a depicts a schematic diagram of a DNA microarray 300 under observation conditions. A substrate 304 has a layer of gold 308 thereon, which has a smooth surfaces 312 and 313. Spots 316 and 324 are present on layer 308 and samples containing DNA of interest are shown as 320 and 328. Waveguides 332 and 336 are placed over spots 316 and 328, respectively, and receive light that has passed through spots 316 and 324, respectively. In two-color embodiments, one color can be used to label material in spot 316 and another color can be used to label material in sport 324. In certain other embodiments, two dyes can be used, which provides "dual label" detection. Currently, green dye (e.g., Sci-3) and red dye (Sci-5) are used. However, it can be readily appreciated that other types of dyes and their combinations can be used according to this invention.

Figure 3 a also depicts how smooth surfaces 312 and 313 can increase the amount of light received by reflection. One incident beam 340 has two portions, 340a, which passes through sample 316 and is reflected by surface 312 upwards to detector 332. Another portion of incident beam, 340b, passes through sample 316 and is reflected by surface 313. Thus, there are two opportunities for a label in sample 320 to interact with the incident beam 328. Similarly, other incident beams 344a and 344b pass through sample 324 and is reflected by either surface 213 or surface 313 Reflected beam 329 is captured by ring 332 of microlens 324 and reflected beam 333 is captured by ring 328 of microlens 324.

Example 2: Microlens Analyte Detector I

In certain embodiments of this invention, equipment can be provided in which light passes through a substrate containing a microlens and then into the microlens, then through a sample to produce a detection signal, and then to a detector.

Figure 4 depicts an embodiment of this invention 400 comprising a substrate 404, a microlens 408 a sample 409 having labeled molecules 410 in the sample 409. Three beams of incident light from a light source 414, namely 416, 420 and 424 pass through substrate 404 and is focused by microlens 408. Focused light passes through sample 409, which produces a detection signal, which is collected by detector 412 for analysis. Example 3 : Microlens Analyte Detector II

It can be appreciated that microlenses can focus light passing through the microlens in the opposite direction to that shown in Figure 4. In certain embodiments, the incident light can first pass through a sample containing labeled molecules, thereby producing a detection signal, which then passes into a substrate having a microlens, then through the microlens, then through the substrate, and then to a detector.

Figure 5 depicts an alternative embodiment 500 in which a light source 518 produces incident beams 522a, 522b and 522c, which pass through sample 512 having labeled molecules 514 therein. The light then passes through microlens 508 where it is focused into three beams 516a, 526b and 526c. The focused light is then collected by detector array 530 having three detectors 534, 538 and 542. The focused light is depicted being captured by detector 538 on areas 538a, 538b and 538c.

It can be appreciated that the relationship between the size of a microlens and the size of a sample can vary. Figures 6a and 6b depict two embodiments of this invention. Figure 6a depicts an embodiment 600 in which a substrate 604 having microlens 608 therein has a sample 612 that is larger in diameter than microlens 608. Figure 6b depicts a different embodiment 602 in which substrate 604 has microlens 608 and sample 614 that is smaller in size than microlens 608.

Example 4: Microlens Array I

Figure 7 depicts an embodiment of this invention 700 in which a substrate 704 has a plurality of microlenses 708, 712 and 716. Samples 720, 724 and 728, respectively, are shown over microlenses 708, 712 and 716. It can be readily appreciated that numerous other embodiments in which a plurality of microlenses having samples are provided.

Figure 8 depicts another embodiment 800 of this invention in perspective view, showing a plurality of locations, labeled by columns 1, 2, 3 and 4, and by rows A and B, thereby defining a 2 x 2 matrix having 8 elements 808. Each element can have similar or different labeled analytes thereon. Embodiment 808 can be made having any desired number of elements 808 thereon. It can be appreciated that once a desired size is found, elements 808 can be placed in an array using square (as shown) or hexagonal packing. It can also be appreciated that other types of arrangements are possible.

To use microlens arrays of this invention, one need merely provide an array of microlenses, for example, as provided by Nippon Sheet Glass Co., and place onto one or more microlenses, a sample containing a molecule of interest. In certain embodiments for genetic analysis, samples of DNA "receptors" can be attached to the surface of the glass using methods known in the art. DNA receptors can be chosen to have nucleotide sequences complementary to those to be analyzed. A sample containing DNA for analysis is provided and labeled DNA is prepared using methods known in the art, for example, a dual label method using dyes. One also can detect analytes after binding them to a substrate using an analyte-specific receptor, for example, antibodies, which can be used to bind proteins, peptides and small molecule analytes. Monoclonal antibodies can be especially useful because they can be made to specifically recognize and bind to nearly any desired analyte, including nucleic acids, proteins, and small molecules.

Example 5: Microlens Array Reader System

Figure 9 depicts an example 900 of a microlens arrays reader system of this invention. Light source 908 provides uniform illumination of an array of sources 904, which direct incident beams of light through waveguides 912. Array 904 is positioned in relationship with an array 800 of microlenses 808 with samples having labeled analytes 806 thereon. Array 800 is positioned relative to detector array 1000, which has a plurality of detector elements 1004 therein, each having an output for transmitting signals to signal analyzer 1009.

Depending on the type of analysis to be carried out, a user can select an array 800 to have any desired number and configuration of analyte receptors thereon. The user then can expose the array to samples containing analytes to be detected, and then can place the array 800 in the detector system, comprising source array 904 and detector array 1000. Once the analysis is completed, array 800 can be removed from the system 900 and another array 800 can be inserted into the reader. Thus, multiple analyses can be accomplished in a high-throughput system for acquisition of large amounts of data.

As applied to genomics, proteomics, and detection of other analytes, such systems can be of great advantage because the number of replicates can be reduced by a factor reflecting the decreases in "false positives" resulting from parasite light collected using prior art systems.

INDUSTRIAL APPLICABILITY The arrays and systems of this invention are useful for detecting and analyzing analytes present from a variety of different sources. The analytes include nucleic acids, proteins and small molecules, and can be useful for genetic, physiological, medical, pharmacological and metabolic studies in the fields of genomics, proteomics, medicine and biochemistry.

Claims

1. An array for detection of a labeled analyte, comprising: a substrate; at least one area on said substrate having a receptor capable of binding to said labeled analyte; and at least one reflective layer between said substrate and said receptor.

2. The array of claim 1, wherein said reflective layer is selected from the group consisting of gold, aluminum, platinum and synthetic diamond.

3. An array for detection of a labeled analyte, comprising: a substrate having at least one microlens therein; and at least one analyte receptor on said microlens.

4. An array for detection of a labeled analyte, comprising: a substrate having a plurality of microlenses therein; and a plurality of analyte receptors, each of said plurality of analyte receptors having selectivity for an analyte, and said plurality of analyte receptors being associated with at least one of said plurality of microlenses.

5. The array of claim 3, further comprising a labeled analyte associated with said at least one receptor.

6. The array of claim 3, wherein said at least one microlens has a diameter in the range of about 100 μm to about 1000 μm.

7. A system for analyte detection, comprising: a light source; an array comprising at least one microlens; and a detector.

8. The system of claim 7, wherein said array comprises at least one analyte receptor associated with said at least one microlens.

9. The system of claim 7, further comprising a signal analyzer.

10. The array of claim 3, wherein said substrate is substantially transparent to a wavelength of light used for detection of said analyte.

11. A method for detecting a labeled analyte, comprising the steps of:

(a) providing an array comprising at least one microlens and having a receptor for said analyte associated with said microlens;

(b) providing a sample containing a labeled analyte which can associate with said receptor;

(c) permitting said labeled analyte to associate with said receptor;

(d) exposing said labeled analyte to electromagnetic radiation; and

(e) detecting a signal produced by an interaction of said labeled analyte and said electromagnetic radiation.

12. A method for detecting a labeled analyte, comprising the steps of:

(a) providing a substrate having a reflective layer thereon and at least one area having receptors that can associate with said receptor;

(b) applying a sample of labeled analyte on said receptor;

(c) permitting said labeled analyte to associate with said receptor;

(d) exposing said labeled analyte to electromagnetic radiation; and