WO2006011346A1

WO2006011346A1 - Microarray reader

Info

Publication number: WO2006011346A1
Application number: PCT/JP2005/012607
Authority: WO
Inventors: Tadao Sugiura; Kotaro Minato; Tetsuo Sato; Takashi Yoshida
Original assignee: National University Corporation NARA Institute of Science and Technology
Priority date: 2004-07-30
Filing date: 2005-07-07
Publication date: 2006-02-02
Also published as: JP2006038816A

Abstract

A microarray reader (100) for detecting a specific intercalation between a probe DNA and a target DNA when a sample containing a fluorescent material and the target DNA is brought into contact with a microarray substrate (70) on which the probe DNA is immobilized. The microarray reader (100) comprises a laser light source (11), an objective lens (14) for directing the light beam projected from the laser light source (11) to a microarray substrate (70) so that an evanescent field may be produced on the surface of the microarray substrate (70) where the probe DNA is immobilized, and a photodetector (17) for detecting fluorescent light emitted from the fluorescent material contained in the sample and excited by the evanescent field. With this microarry reader, higher sensitivity is achieved and the detection time is shortened.

Description

Specification

Microarray reader

Technical field

[0001] The present invention relates to a microarray reading apparatus capable of detecting a microarray with high sensitivity and high speed.

Background art

Since being reported in 1995, DNA microarrays have been widely used in drug discovery and medical applications only in the fields of cell biology and basic medicine as a method that can comprehensively detect mRNAs working in cells and tissues. It is becoming used in the field. DNA microarrays allow you to investigate a large amount of information about each gene at once, such as what gene is transcribed in the nucleus of the cell and how much mRNA is transcribed in the cell. It has become established as the main research method for transcriptome analysis.

[0003] By comprehensively detecting mRNA, it is possible to determine the role of genes in cells and to detect genes that work at specific stages of development. Cryptome can also investigate cellular responses to stress, the relationship between diseases and genes. For this reason, this microarray technology is expected to bring more useful information in the future, and is actively researched and developed in Japan and overseas.

[0004] The principle of DNA microarray will be briefly described. In general DNA microarrays, in order to detect mRNA of each gene, DNA having a sequence complementary to the base sequence of each mRNA (referred to as probe DNA) is preliminarily fixed on a substrate, and the cells are fixed. MRNA of each gene is detected by hybridizing cDNA (called target DNA) generated by reverse transcription of mRNA extracted from tissue samples and tissue samples.

[0005] In the hybridization, if the target DNA that is also single-stranded has a complementary sequence to the probe DNA that has been heat-treated into single-stranded DNA, it can be re-stable stably. Combined (no, By using the fact that the binding becomes unstable if the sequences are different, the DNA of each sequence is identified. Usually, the probe DNA is fixed on the substrate as a circular spot with a diameter of about 100 to 200 μm. Probe DNAs with different base sequences are arranged in an array on the substrate, and the base position depends on the spot position. Detect if there is a DN A in the sequence. If a fluorescent dye is attached to the target DNA, the amount of target DNA that is hybridized with the intensity of fluorescence emission on the substrate can also be measured.

[0006] However, in order for DNA microarrays to be used in actual medical diagnosis, inspection at agricultural test sites, food inspection, etc., further shortening of inspection time, higher detection sensitivity, and reproducibility of inspection results There is a need for improvement. Therefore, in recent years, a completely new concept of DNA microarray detection method has been proposed by our research group.

[0007] For example, in Non-Patent Document 1, the target DNA is hybridized to the probe DNA, and the V, process is observed in time series, and how much DNA of each base sequence is estimated from the time variation of the amount of hybridization. By doing this, the concept of DNA microarray reading is disclosed.

[0008] In recent years, a technique has been developed in which an evanescent field is generated in the vicinity of a DNA microarray substrate, and the amount of DNA hybridized to the probe DNA is measured in real time by exciting fluorescent molecules with illumination by the evanescent field and observing the fluorescence. Has been developed (for example, see Non-Patent Document 2). As shown in FIG. 9, the evanescent field 501 is a light field that is generated when light is incident from the glass substrate 500 side at an incident angle greater than the total reflection angle. It can be present at a thickness of about ~ 200 nm. In the illumination by the evanescent field 501, the incident light energy is totally reflected and returns to the glass substrate 500 side. However, if the fluorescent molecule 502 exists in the evanescent field 501, it can be excited to emit fluorescence. When the fluorescent molecule 502 is excited by illumination with the evanescent field 501, only the fluorescent molecule 502 existing in the vicinity of the surface of the glass substrate 500 can be fluorescently excited. Therefore, if used in the hybridization process of the DNA microarray, the probe DNA Only the molecules hybridized to can be detected by selective fluorescence excitation. [0009] In addition, when fluorescence excitation is performed by illumination with an evanescent field, even if fluorescent molecules are present in the solution used for flowing the target DNA, the excitation light is hardly applied and the fluorescence excitation is not performed. The knock ground at the time is small!

[Non-Patent Document 1]

Kaori Haruna, Tadao Sugiura, Tetsuhiro Sato, Keito Tabata, Kotaro Taki, “Real-time detection of hybridization processes in DNA microarrays”, Biomedical Engineering 41 (Supp 0: 148,2003

[Non-Patent Document 2]

Carolin Peter er al., Optical DNA— sensor chip for real-time detection of nybndization events ”Fresenius J Anal Chem (2001) 371: 120—127

[0011] As described above, there is a need for further shortening of inspection time, higher detection sensitivity, and improved reproducibility of inspection results with respect to the conventional DNA microarray technology. In particular, in DNA microarrays, the ability to detect DNA with a specific sequence through a hybridization process. This hybridization takes about 8 to 16 hours, and the work involved in detection When the starting force was included until the end of detection, a considerable amount of time was required. This means that it takes time to hopelessness for medical applications and high-throughput analysis purposes.

[0012] As one means for solving the above problem, the research group of the present inventors discloses the concept of a real-time observation type DNA microarray detection technique in Non-Patent Document 1.

[0013] In spite of that, Non-Patent Document 1, which is powerful, discloses an abstract concept for a detection method of a real-time observation type DNA microarray, but at the stage when this Non-Patent Document 1 was issued. , The power that has not been embodied in its concrete configuration. That is, Non-Patent Document 1 does not disclose a real-time observation type DNA microarray detection method to a practical extent.

[0014] Further, Non-Patent Document 2 discloses a real-time DNA microarray reader using an evanescent field. This device is also considered to be unbearable for actual use. That is, the DNA microarray reader disclosed in Non-Patent Document 2 is Since the probe DNA is spotted on the surface of the DNA array, there is a problem that it lacks versatility compared to general DNA microarray substrates. In addition, in this device, the fluorescence intensity at the spot is detected by a light detector by moving the shutter, but in this system, imaging cannot be performed and high-precision DNA microarray detection is performed. I can't. Furthermore, since this apparatus does not consider the scanning mechanism at all, it cannot be detected while scanning, and it cannot cope with a microarray having more spots. is there.

[0015] Therefore, there has been a strong demand for the development of a microarray reader that can realize high sensitivity and a microarray reader that can shorten the detection time so that it can withstand actual use.

Disclosure of the invention

The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a microarray reading apparatus capable of realizing high sensitivity and further a microarray reading apparatus capable of reducing detection time. It is in.

[0017] As a result of intensive studies to solve the above-mentioned problems, the present inventor firstly arranged a means for exciting fluorescence and a means for detecting fluorescence to reduce the time required for detection of the microarray. It was found that higher accuracy can be achieved by arranging them on the same side. In addition, paying attention to the hybridization process and observing the hybridization process in real time, the test result can be obtained without waiting until the noise hybridization is completely completed, and the detection time can be reduced. I found that I could shorten it. Furthermore, it was found that the viewing angle can be expanded by irradiating the excitation excitation laser beam with the external force of the objective lens, a large number of spots can be detected at once, and the detection time can be shortened. . The present inventor has completed the present invention based on these new findings. That is, the present invention includes the following inventions (1) to (15).

[0018] (1) When the sample containing at least the fluorescent substance and the target substance is brought into contact with the microarray substrate on which the probe substance is immobilized, the probe substance and the target substance are specific. A microarray reader for detecting an interaction, comprising: a light irradiation means for irradiating light; and a probe substance on the microarray substrate. A light incident means for causing the light irradiated by the light irradiation means to enter the microarray substrate so as to generate an evanescent field on a fixed surface; and a sample excited by the evanescent field. A light detecting means for detecting fluorescence emitted from the fluorescent material contained therein, the light detecting means having an optical lens functioning as an objective lens, and the optical lens A microarray reader that functions as an incident means.

[0019] (2) When the sample containing at least the fluorescent substance and the target substance is brought into contact with the microarray substrate on which the probe substance is immobilized, the probe substance and the target substance are specific. A microarray reader for detecting an interaction, wherein the light irradiation means for irradiating light and the probe material on the microarray substrate are fixed to generate an evanescent field on the surface. Light incident means for making the light irradiated by the means incident on the microarray substrate, and light for detecting the fluorescence emitted from the fluorescent material contained in the sample excited by the evanescent field Detecting means, and the light detecting means has an optical lens functioning as an objective lens, and the light incident means is the light illuminating means. A microarray reader, wherein the light from the projecting means is incident on the microarray substrate without passing through the optical lens.

[0020] (3) The light incident means causes the light irradiated by the light irradiation means to be incident at an incident angle equal to or greater than an angle at which the light is totally reflected on the surface of the microarray substrate on which the probe substance is fixed. The microarray reader according to (1) or (2).

(4) The microarray reading apparatus according to (2), wherein the light incident means is disposed between the microarray substrate and the optical lens.

[0022] (5) The light incident means has at least one reflecting surface for reflecting the light irradiated by the light irradiating means, and the reflecting surface is a reflected light force by the reflecting surface. The microarray reader according to (2), which is provided so as to be incident at an incident angle greater than an angle at which the probe substance on the microarray substrate is fixed.

(6) The refractive index of the light incident means is substantially the same as the refractive index of the microarray substrate. The microarray reader according to (2).

[0024] (7) The light irradiation means and the light detection means are arranged on the back side of the surface of the microarray substrate on which the probe substance is fixed, according to any one of (1) to (6). Microarray reader.

(8) An oil layer is provided between the microarray substrate and the light incident means (

The microarray reader according to any one of 1) to (7).

[0026] (9) The microarray reading device according to any one of (1) to (8), further comprising temperature adjusting means for adjusting the temperature of the sample containing the target material on the microarray substrate. .

[0027] (10) The microarray reader according to any one of (1) to (9), wherein the specific interaction between the probe substance and the target substance is detected in real time.

[0028] (11) The microarray reading device according to any one of (1) to (10), further comprising position changing means for changing a relative position between the microarray substrate and the light incident means.

[0029] (12) Further, the amount of the target substance that interacts with the probe substance is estimated using detection data obtained by observing the process in which the target substance interacts with the probe substance in time series. Computation processing means for performing computation processing to perform (1) to

(11) The microarray reader according to any one of the above.

[0030] (13) The microarray reader according to any one of (1) to (12), wherein the probe substance and the target substance are single-stranded DNAs.

[0031] (14) The microarray reader according to any one of (1) to (13), wherein the fluorescent substance is bonded to the target substance.

[0032] (15) The fluorescent substance specifically binds to a substance in a state where the probe substance and the target substance interact with each other in any one of (1) to (14) The microarray reader according to the description.

[0033] In this case, the arithmetic processing means may be realized by a computer. In this case, the arithmetic processing means controls the arithmetic processing means to be realized by a computer by causing the computer to operate as the respective means. · Calculation program and computer that recorded it A computer-readable recording medium also falls within the scope of the present invention.

Brief Description of Drawings

FIG. 1 is a diagram schematically showing a configuration of a microarray reader according to an embodiment of the present invention.

FIG. 2 (a) is a view of an example of the configuration of a hybridization cell according to an embodiment of the present invention as viewed from above.

FIG. 2 (b) is a view showing a cross section cut along the line aa ′ in FIG. 2 (a).

FIG. 3 is a diagram schematically illustrating a state when light is incident on the microarray substrate according to the embodiment of the present invention.

FIG. 4 is a diagram schematically illustrating a state where light is incident on a microarray substrate according to another embodiment of the present invention.

FIG. 5 is a diagram schematically showing a configuration of a microarray reader according to another embodiment of the present invention.

6 is a perspective view schematically showing a part of the configuration of the microarray reader shown in FIG. 5 upside down.

FIG. 7 (a) is a diagram showing the result of a hybridization experiment in the example according to the present invention, and shows the result at t = 0 when the microarray reader according to the present example is used. It is.

FIG. 7 (b) is a diagram showing the result of a hybridization experiment in the example according to the present invention, and the result at t = l (min) when the microarray reader according to the example is used. FIG.

FIG. 7 (c) is a diagram showing the result of a hybridization experiment in the example according to the present invention, and the result at t = 5 (min) when the microarray reader according to the example is used. FIG.

FIG. 7 (d) is a diagram showing the result of a hybridization experiment in the example according to the present invention, and shows the result at t = 20 (min) when using the microarray reader according to the present example. FIG.

[FIG. 7 (e)] is a diagram showing the results of an experiment on the nodularization in the example according to the present invention. FIG. 6 is a diagram showing the results at t = o of a control experiment.

FIG. 7 (£) is a diagram showing a result of a hybridization experiment in an example according to the present invention, and a diagram showing a result at t = 1 (min) of a control experiment.

FIG. 7 (g) is a diagram showing the results of an experiment on the nodification in the example according to the present invention, and shows the results at t = 5 (min) of the control experiment.

FIG. 7 (h) is a diagram showing a result of a hybridization experiment in an example according to the present invention, and a diagram showing a result at t = 20 (min) of a control experiment.

FIG. 8 is a graph showing the results of FIGS. 7 (a) to 7 (h).

FIG. 9 is a diagram schematically illustrating a phenomenon in which light is incident on a glass substrate to generate an evanescent field.

BEST MODE FOR CARRYING OUT THE INVENTION

[Embodiment 1]

An embodiment of the present invention will be described below with reference to FIGS.

[0036] <Microarray substrate and inspection object>

First, the microarray reader according to the present invention provides the probe substance and the target substance when the sample containing at least the fluorescent substance and the target substance is brought into contact with the microarray substrate on which the probe substance is immobilized. This is to detect a specific interaction with.

[0037] Therefore, first, a microarray substrate to be read and a detection target (sample) to be detected using the microarray substrate will be described. Note that the term “read” in this specification is used synonymously with detection, measurement, measurement, and the like.

[0038] When the microarray substrate used in the present invention comes into contact with a sample containing a target substance, it specifically interacts with the target substance contained in the sample (for example, binding, hybridization, etc.), and the target As long as the probe substance for detecting the substance is fixed, the specific configuration such as the type, number, amount, spot size, etc. of the probe substance is not particularly limited. However, in order to take full advantage of the microarray feature of simultaneously measuring a large number of samples at the same time, it is preferable that there are multiple types of probe substances. [0039] Further, as will be described later, since the fluorescence generated on the microarray substrate is detected through the microarray substrate, the microarray substrate is also configured to have at least a material strength for transmitting light. For example, as a microarray substrate, a substrate made of a material having a high light transmittance such as a glass substrate, a polycarbonate, a resin made of PMMA, or the like can be suitably used, but the refractive index changes due to heat. Considering the rate, a glass substrate is most suitable. According to the microarray reader according to the present invention, a conventionally known microarray substrate having the above-mentioned conditions can be read, and there is an advantage that the application range is wide.

[0040] Specific examples of the probe substance immobilized on the microarray substrate include nucleic acids (including polynucleotides and oligonucleotides), polypeptides (including proteins, oligopeptides, and antibodies), Examples include low molecular weight substances such as molecular substances (hormones, etc.) and environmental hormones. In addition, the target substance is not particularly limited as long as it interacts with the probe substance. However, nucleic acids (including polynucleotides and oligonucleotides), polynucleotides, and the like interact with the probe substance. Peptides (including proteins and oligopeptides), in vivo low molecular weight substances (hormones etc.), low molecular weight substances such as environmental hormones, etc. are suitable. Of these, single-stranded DNA is particularly suitable as the probe substance and the target substance. Moreover, single-stranded RNA is also preferably used as the target substance.

[0041] Further, as will be described later, the microarray reader according to the present invention generates an evanescent field and excites a fluorescent substance contained in the sample by the evanescent field to cause mutual interaction between the probe substance and the target substance. The action is detected. For this reason, the sample solution distributed on the microarray substrate 70 needs to contain! It is preferable that the strong fluorescent substance is bonded to the target substance, but this method is not limited to this method.For example, the probe substance alone or the target substance exists, and in this case, it binds to these substances. However, when the probe substance and the target substance interact with each other (for example, binding, hybridization, etc.), the probe substance and the target substance interact with each other in a specific state. It may be a fluorescent substance that binds to each other. [0042] For example, when single-stranded DNA is used as a probe substance and a target substance, a fluorescent functional group (eg, Cy3, Cy5, etc.) is bound to the 5 'end of the target DNA, Alternatively, the target DNA can be incorporated into the target DNA, and the target DNA is not added with a fluorescent substance. For example, POPO-3 (Molecular Probes, Inc)) can be used.

[0043] Further, the sample solution to be inspected is not particularly limited as long as it contains at least the above-mentioned target substance and fluorescent substance. It is not limited. For example, the base of the solution is preferably an aqueous solution, but is not limited thereto, and may be a solution containing an organic solvent or the like. For example, when a biopolymer such as DNA protein is to be detected, a buffer solution (buffer) suitable for the substance can be preferably used.

[0044] Furthermore, the specific configuration and conditions are not particularly limited as long as the sample solution to be inspected comes into contact with the probe substance on the microarray substrate. . More preferably, it is preferable to distribute or flow the sample solution to be inspected on the microarray substrate.

[0045] As described above, in the microarray substrate to be read by the microarray reader according to the present invention, it is preferable to use single-stranded DNA as the probe substance and the target substance. For this reason, the case where single-stranded DNA is used as the probe substance and the target substance, that is, the case where the so-called DNA microarray detection result is read will be described below as an example.

In the present embodiment, a microarray reading apparatus 100 configured using an inverted microscope as a base and using an objective lens as a light incident means will be described. In other words, since the microarray reading apparatus 100 according to the present embodiment is an observation apparatus based on an inverted microscope, illumination by the evanescent field is performed through an objective lens included in the microscope optical system, and fluorescence is transmitted through the microscope optical system. It is configured so that it can be detected. An example of an inverted microscope that can be used as a base is Examples include TE-2000 made by Nikon. The microarray reading apparatus according to the present invention is not limited to the one configured based on the inverted microscope, and has the configuration of the present invention as long as the effect of the present invention can be obtained. It is a matter of course that it is included in the technical scope of.

FIG. 1 is a diagram schematically showing the configuration of the microarray reading apparatus according to the present embodiment. As shown in the figure, the microarray reader 100 according to the present embodiment includes a laser light source 11, a first lens 12, a reflecting surface 13, an objective lens 14, an excitation light cut filter 15, an imaging lens 16, and a light detection. 17, a temperature control unit 18, an arithmetic processing unit 19, and a position changing unit 21. Further, the microarray reader 100 uses the microarray substrate 70 as a reading target.

[0048] The laser light source 11 functions as light irradiation means for irradiating light. As will be described later, the laser light source 11 may be any other specific configuration or condition as long as it can emit light for generating an evanescent field on the microarray substrate 70 ( For example, the wavelength, intensity, type, etc. of the irradiated light are not particularly limited. For example, a laser light source for irradiating laser light of a 2 co Nd: YAG laser (wavelength: 532 nm), an LED, a mercury lamp, or the like can be suitably used as the fluorescence excitation laser.

[0049] The first lens 12 may be another specific configuration (for example, a material) as long as it is an optical lens for condensing the laser light emitted from the laser light source 11 and guiding it to the reflecting surface 13. , Size, thickness, etc.) are not particularly limited. The reflecting surface 13 may be a reflecting means used in a conventionally known optical system device as long as it reflects the laser light from the first lens 12 to the objective lens 14. In the present embodiment, the reflecting surface 13 converts the laser light from the laser light source 11 that reaches via the first lens 12 with light parallel to the optical axis of the microscope optical system (for example, the objective lens 14). Thus, the light is reflected to the objective lens 14 as light offset by a certain distance from the optical axis.

The objective lens 14 is an optical lens that functions as an objective lens for fluorescing generated on the microarray substrate 70. Further, in the present embodiment, the objective lens 14 is a laser beam irradiated by the laser light source 11 so as to generate an evanescent field on the surface of the microarray substrate 70 on which the probe substance is fixed. It also functions as a light incident means for making the light incident on the microarray substrate 70. The objective lens 14 is not particularly limited as long as it can execute the above-described functions. For example, as shown in an example described later, as the objective lens 14, an optical lens having a numerical aperture NA = 1.45 made by Nikon, an objective for TIRF can be used.

In addition, an oil layer 20 is provided between the objective lens 14 and the microarray substrate 70. The oil layer 20 is for matching the refractive index of the objective lens 14 and the refractive index of the microarray substrate 70 to reduce reflection on the boundary surface. Without the oil layer 20, the light is totally reflected on the upper surface of the objective lens 14! /, And the evanescent field cannot be generated on the surface of the microarray substrate 70. This is also a microarray substrate

Even if the plate thickness of 70 is different, the objective lens 14 force is also used to adjust the incident position of the light incident on the microarray substrate 70 so as to focus on the microarray substrate 70. .

The excitation light cut filter 15 may be an optical filter that cuts (removes) short-wavelength light (excitation light) and transmits only the fluorescence generated on the microarray substrate 70. What is called a so-called SC filter can be preferably used. By using this filter, fluorescence can be detected with higher accuracy.

The imaging lens 16 is an optical lens for causing the light transmitted through the excitation light cut filter 15 to be imaged by the photodetector 17, and an imaging lens used in a conventionally known optical device is preferably used. The specific configuration (for example, material, size, thickness, etc.) is not particularly limited. The objective lens 14, the excitation light cut filter 15, and the imaging lens 16 constitute a microscope optical system (optical system mechanism) in an inverted microscope as a base.

The photodetector 17 is a photodetector that can be used in a conventionally known optical device as long as it functions as a photodetector for detecting the light imaged by the imaging lens 16. It can be suitably used. For example, as shown in the examples described later, the force that can use a cooled CCD force camera (Hamamatsu Photonicas, ORCA-ER) is not limited to this. When a CCD camera is used, the CCD control that controls the operation of the CCD camera 17 is performed. A control unit or a computing device (such as a PC) for displaying or processing image data detected by the CCD camera may be provided. In the microarray reader 100 according to the present embodiment, the objective lens 14, the excitation light cut filter 15, the imaging lens 16, and the photodetection device that function as the above-described microscope optical system (optical system mechanism). The photo detector 17 is collectively referred to as photo detection means.

[0055] The temperature adjusting unit 18 may be any one for adjusting the temperature of the sample solution that exists on the microarray substrate 70 described later (more preferably, it exists by flowing or flowing on the microarray substrate 70). Conventionally known temperature adjusting means can be preferably used. For example, by using a temperature control means consisting of a ceramic heater (50 x 50mm), a thermocouple temperature sensor (TC), and a heater control unit, PID control of the heater and TC is performed by the heater control unit. The temperature of the sample on the microarray substrate 70 can be adjusted optimally. For example, when the microarray substrate 70 is a DNA microarray, the temperature control unit 18 converts the temperature of the sample solution flowing on the microarray substrate 70 to a temperature suitable for performing hybridization (for example, as will be described later). 65 ° C). For this reason, when detecting a DNA microarray, the microarray reader 100 has a configuration necessary for evaluating the hybridization process in real time. However, in the case of a microarray for detecting proteins (for example, a protein chip), since the binding may be detected in real time at room temperature, the temperature control unit 18 is not necessarily required.

[0056] The arithmetic processing unit 19 uses the detection data obtained by observing the process of the target DNA hybridizing to the probe DNA in time series, and the target DNA (target DNA In the case of multiple types, a calculation process is performed to estimate how much the DNA of each base sequence has been accumulated. Details of the arithmetic processing performed by the arithmetic processing unit 19 will be described later.

The position changing unit 21 functions as position changing means for changing the relative position between the microarray substrate 70 and the objective lens 14. As the position changing unit 21 to be applied, a moving stage or the like that is conventionally used in optical devices such as a known microscope can be suitably used. The position changing unit 21 provides a po- sition provided on the microarray substrate 70. Even if there are multiple probes (regions where a large number of probe DNAs are immobilized), by changing the relative positions of the microarray substrate 70 and the objective lens 14, hybrids in different DNA spots can be obtained. The session can be easily detected. Further, the position changing unit 21 may be a mechanism capable of uniaxial scanning, but more preferably a mechanism capable of two-axis scanning capable of two-dimensional scanning. preferable.

In this embodiment, the force by which the microarray substrate 70 is moved to change the relative position with the objective lens 14 is not limited to this method. For example, the microarray substrate 70 is fixed. In addition, even if the optical system mechanism such as the objective lens 14 and the imaging lens 15 and the position of the photodetector 17 are moved, it does not matter.

[0059] <Hybridization 'Cell>

As shown in FIG. 1, the microarray substrate 70 to be read is set as a hybridization cell 50 with respect to the microarray reader 100. The hybridization cell 50 according to the present embodiment is configured to have a temperature adjustment function for observing the hybridization in real time by combining with the temperature adjustment unit 18 described above. ing. In other words, in order to detect hybridization in real time, (0 temperature adjustment is possible, the sample solution containing the target DNA can be circulated inside the GO cell, and its flow rate is Therefore, it is preferable to use a powerful hybridization cell, and the specific configuration of the hybridization cell 50 will be described below.

[0060] FIG. 2 (a) is a diagram showing an example of the configuration of the hybridization cell, as viewed from above.

FIG. 2B is a diagram showing a cross section cut along the line aa ′ in FIG. As shown in FIG. 2 (b), the hybridization cell 50 according to the present embodiment includes a microarray substrate 70, a slide glass 71, a spacer 72, and a fixing device 73.

In FIG. 2 (b), the hybridization cell 50 has a slide glass 71 on the upper surface and a microarray substrate 70 on the lower surface, and the microarray substrate 70 also functions as a cover glass. The probe DNA is disposed (immobilized) on the surface of the microarray substrate 70 on the side facing (opposing) the slide glass 71. The probe DNA is arranged in a pot format as shown in Fig. 2 (a). [0062] The reason for spotting the probe DNA on the side that functions as the cover glass is that, in the subsequent processing, when detecting hybridization between the probe DNA and the target DNA by illumination with an evanescent field, Fig. 2 (b) This is because laser light from the microarray reader 100 enters from the side opposite to the surface of the microarray substrate 70 on which the probe DNA is immobilized. That is, as shown in FIG. 2 (b), the objective lens 14 of the microarray reader 100 is also close to the surface side force opposite to the surface on which the probe DNA is fixed on the microarray substrate 70, and the laser beam is incident thereon. Will do. The microarray substrate 70 is made of a cover glass suitable for the objective lens 14 in the microarray reader 100.

A spacer 72 having a predetermined thickness is inserted between the microarray substrate 70 and the slide glass 71. The interval between the microarray substrate 70 and the slide glass 71 is not particularly limited as long as the interval is such that the sample solution containing the target DNA can be circulated. For example, if the interval of 100 m is provided, the sample solution can be circulated appropriately. In this case, the thickness of the spacer 72 is 100 m. Further, the material of the spacer 72 is not particularly limited, and a known spacer can be suitably used. For example, a packing made of Teflon (registered trademark) can be used.

[0064] A spacer 72 having a predetermined thickness is inserted between the microarray substrate 70 and the slide glass 71, and the vertical force is also pressed by the fixing device 73, so that the distance between the microarray substrate 70 and the slide glass 71 is 100 m. Hold at intervals. Then, the sample solution is poured into this gap. As the fixing device 73, a conventionally known fixing device can be used, and its specific configuration is not limited. For example, a stainless steel frame can be used.

[0065] Further, the slide glass 71 is provided with two holes 74 · 74 (for example, a diameter lmm). As shown in Fig. 2 (a) and Fig. 2 (b), the sample solution is pressed by the stainless frame through one of the holes 74 to make contact with the probe DNA immobilized on the surface of the microarray substrate 70. Then, the sample solution is made to flow on the probe DNA by being taken out from the other hole 74. The sample solution is not shown The sample can be distributed through the gap between the microarray substrate 70 and the slide glass 71 by the sample transport device. As the sample transport device, for example, a peristaltic pump can be cited. By applying a pressure with this peristaltic pump, the hybridizing senor 50 can be circulated.

[0066] Furthermore, the fixing device 73 is configured such that the temperature adjusting unit 18 can be provided on the slide glass 71 side, for example. For example, a ceramic heater is attached to the slide glass 71 side, a thermocouple temperature sensor (TC) is installed on the microarray substrate 70 side, and the temperature is measured. The temperature of the solution can be kept constant. In particular, in the hybridization treatment, it is preferable to perform the noise hybridization while the temperature of the sample solution is kept at 65 ° C.

[0067] In addition, the specific conditions of the yarn and the concentration, temperature, etc. of the buffer used as a sample solution to be circulated in the hybridization cell are not particularly limited. Buffers and conditions used for known DNA microarray experiments can be suitably used. For example, TE buffer (10 mM Tris-HC1 (pH 7.5), ImM EDTA, etc.), SSC buffer (4 X SSC S 0.2% SDS, 20 X Denhart), etc. are suitable as shown in the examples described later. Can be used.

[0068] <Description of operation of microarray reader>

Next! The operation of the microarray reader 100 will be described by taking as an example the case of performing time series observation of the hybridization process using the hybridization cell 50 including the microarray substrate 70 described above. To do.

[0069] In the hybridization experiment according to the present embodiment, first, a predetermined cDNA is spotted as a probe DNA! /, A sequence complementary to this cDNA on the microarray substrate 70. The sample solution containing the target DNA (having a fluorescent functional group bound thereto) having a flow is passed through the hybridization cell 50 to which the sample solution is brought into contact. Next, the microarray reader 100 detects whether or not the target DNA is hybridized with the probe DNA (DNA spot) on the microarray substrate 70. Specifically, this is done as follows.

First, as shown in FIG. 1, laser light is emitted from a laser light source 11. Irradiated The light is collected by the first lens 12 and subsequently reflected by the reflecting surface 13. The reflection surface 13 is a beam parallel to the optical axis of a microscope optical system (for example, the objective lens 14), which is a laser beam from the laser light source 11 that arrives via the first lens 12. The light is reflected off the objective lens 14 as light offset by a certain distance from the optical axis.

The laser light reflected by the reflecting surface 13 enters the objective lens 14 in a direction in which the optical axial force of the objective lens 14 is also offset. Then, the light incident on the objective lens 14 becomes light incident on the microarray substrate 70 with a large incident angle at the exit of the objective lens 14 by the function of the objective lens 14 while being a parallel ray. Illuminate the area where the probe DNA is immobilized. That is, it can be said that the objective lens 14 functions as a light incident means for causing the laser light to be incident at an incident angle that is greater than the angle at which the laser light is totally reflected on the surface of the microarray substrate 70 on which the probe DNA is fixed.

[0072] Specifically, the sample solution containing the target DNA is an aqueous solution, and the microarray substrate 70 is described as a glass substrate. The refractive index of water is 1.33, and the glass having a refractive index of 1.5. The refractive index is lower than For this reason, as shown in FIG. 3, the objective lens 14 causes the incident angle Θ of the laser light to the microarray substrate 70 to be larger than the total reflection angle Θ (62 °) at the interface between water and glass. The laser beam is microarrayed by adjusting

Fully reflected on the lower surface side of the surface 70a where the probe DNA80 is immobilized on the substrate 70. At this time, a thin evanescent field 60 is generated on the upper surface side of the surface 70a of the microarray substrate 70.

[0073] As shown in FIG. 3, the evanescent field 60 generated on the surface 70a of the microarray substrate 70 exists only from the surface 70a of the microarray substrate 70 to a region of about the wavelength (region of about 100 to 200 nm). Absent. For this reason, only the fluorescent functional group 8 la of the target DNA 81 specifically hybridized with the single-stranded probe DNA 80 is fluorescently excited, and the fluorescent functional group 82 a of the free target DNA 82 is not excited. Therefore, only the target DNA 81 hybridized to the probe DNA 80 can be selectively detected in the hybridization process of the DNA microarray.

That is, in the present embodiment, the process on the microarray substrate 70 side under the total reflection condition. The evanescent field 60 generated when light enters from the back side of the surface 70a on which the DNA is immobilized is used to excite the fluorescent material added to the target DNA. Since the evanescent field 60 is a light field localized in a region having a thickness of about a wavelength from the microarray substrate 70, when laser light is incident under total reflection conditions, the incident light is incident on the surface 70a of the microarray substrate 70. The light is totally reflected on the lower surface side and returns to the inside of the microarray substrate 70. However, if a fluorescent material is present in the region of a thickness of about a wavelength from the surface 70a of the microarray substrate 70, the fluorescent material absorbs light and causes fluorescence to be emitted. Can do. In addition, since a light field exists only in the immediate vicinity from the surface 70a of the microarray substrate 70, only the fluorescent material possessed by the target DNA hybridized with the probe DNA immobilized on the microarray substrate 70 is excited by fluorescence to emit light. Can be made.

The generated fluorescence is collected by the objective lens 14, imaged again through the excitation light cut filter 15, the imaging lens 16 t, and the mechanism of the microscope optical system, and detected by the photodetector 17. In particular, in the present embodiment, the temperature control unit 18 is used to distribute the sample solution containing the target DNA in a state where the temperature of the sample solution is kept constant at 65 ° C. Is in contact with. Therefore, the hybridization process can be evaluated in real time by observing the temporal change of the fluorescence image.

[0076] That is, when an evanescent field is generated by incident light and the microarray is observed, the fluorescence intensity emitted from the substrate surface increases as the target DNA hybridizes to the probe DNA on the substrate and is immobilized. By detecting the intensity, the increase in target DNA can be monitored. Therefore, according to the microarray reading apparatus 100, the time required for hybridization can be shortened, so that the detection time can be greatly shortened as compared with the conventional apparatus.

Further, as shown in FIG. 1, in the microarray reading apparatus 100, a laser light source 11 that functions as a light irradiation means, an objective lens 14 that functions as an optical system mechanism, an excitation light cut filter 15, and an imaging lens 16 The photodetector 17 functioning as a photodetector is disposed on the back surface side of the surface 70a of the microarray substrate 70 on which the probe DNA is fixed. This is because the laser light source 11 as the light irradiation means and the objective lens 14 as the light incident means are connected to the surface 70a of the microarray substrate 70 on which the probe DNA is fixed. In order to generate a cent field, the back surface side force of the front surface 70a of the microarray substrate 70 also needs to be irradiated with laser light.

[0078] When fluorescence emission occurs in the vicinity of the surface 70a of the microarray substrate 70, much of the emitted fluorescence energy is radiated to the side of the microarray substrate 70 having a higher refractive index than that in the sample solution. For this reason, in order to efficiently detect the light emitted to the microarray substrate 70 side, it is necessary to provide an optical system mechanism for detecting fluorescence and the photodetector 17 on the back side of the surface 70a of the microarray substrate 70. This is because it is preferable.

[0079] Further, by providing an incident optical system for fluorescence excitation and an optical system for observation on the side of the microarray substrate 70, a surface of the slide glass 71 opposite to the surface facing the microarray substrate 70 is provided. Can be in a free state. The surface of the slide glass 71 that has become free is made of an opaque material such as a temperature control unit 18 or a metal block for controlling the temperature of the protein chip, etc. Can be installed.

In addition, the microarray reader 100 is provided with a position changing unit 21. For this reason, if necessary, the relative position between the microarray substrate 70 and the objective lens 14 and the like can be appropriately changed by the position changing unit 21, and detection can be performed while scanning. It is also possible to correspond to a microarray having many spots.

Furthermore, in the present embodiment, microarray reading apparatus 100 observes in real time how the target DNA is hybridized to probe DNA and immobilized on the surface of microarray substrate 70. The number X of target DNA molecules immobilized on the surface of the microarray substrate 70 at this time is predicted to increase as shown in the following formula (1).

[0082] X = C (l -exp (-t / a)) · · · (1)

Here, C is the number of molecules of the target DNA contained in the sample, and is a value that is desired to be obtained by detection of the microarray substrate 70. a is a constant that determines the specific force between the reaction rate and the desorption rate when the hybridization reaction occurs. In the detection using a conventional DNA microarray reader, all genes are read after hybridization, so the value of t should be set long enough to eliminate the effect of a. It was necessary to measure the coefficient C related to the number. Therefore, take a sufficiently long time for α It was necessary to perform hybridization. For example, with a conventional DNA microarray reader, the hybridization process alone required approximately 8-12 hours or more, and the entire detection process required additional time.

On the other hand, the microarray reader 100 according to the present embodiment capable of real-time observation observes and detects the progress of the hybridization. For this reason, the arithmetic processing unit 19 uses the data obtained for the time series change of hybridization to fit the amount of hybridization to the above equation (1), so that the value of α in each spot is obtained. And the value of C. Specifically, the arithmetic processing unit 19 first integrates the fluorescence intensity with respect to each spot (DNA spot) extracted by image processing to obtain a hybridizing amount Xr for the spot. Then, C and α are determined so that the square error (Xr−X) ² between the hybridized amount Xr and the number of molecules X in the equation (1) is minimized. To determine C and α, for example, the steepest descent method can be used.

Therefore, according to the microarray reader 100 according to the present embodiment, it is not always necessary to wait until the hybridization is completed, and the final amount during the progress of the hybridization. Can be estimated.

[0085] In addition, the merit of this method is that, in the case of a conventional DNA microarray substrate, when the amount of target DNA immobilized on the substrate is larger than the total amount of probe DNA, the probe DNA runs short on the way. If the real-time observation type is used, the number of molecules can be estimated from the rising speed at the rising part, so this problem can be avoided. For this reason, even if the pot is small and probe DNA is deficient during hybridization, the target DNA concentration can be measured accurately, even with a small spot DNA microarray.

[0086] Therefore, according to the microarray reader according to the present embodiment, the target DN is hybridized to the probe DNA, the process of the hybridization is observed in time series, and the time change power of the amount of hybridization is also the DNA of each base sequence. It is possible to estimate how long it was. For this reason, the time required for the hybridization can be shortened to shorten the inspection time. Furthermore, the detection sensitivity is improved by arranging the optical system mechanism for generating the evanescent field and the optical system mechanism for detecting fluorescence on the same side. I'll do it with you.

[0087] In addition, microarray reading apparatus 100 does not directly spot DNA on the prism substrate, and the member functioning as the light incident means and the microarray substrate have different configurations. Therefore, if the microarray substrate 70 is replaced, the microarray substrate 70 can be used for various types of microarray substrates, and is highly versatile. Further, the microarray reader 100 can perform imaging, and can detect a DNA microarray with high accuracy.

[0088] In the above description, a sample containing target DNA to which a fluorescent functional group is bound is used. For example, a sample containing a fluorescent substance that specifically binds to double-stranded DNA is used. It can also be used. In this case, as shown in FIG. 4, a sample containing the target DNA (single strand) 82 and the fluorescent material 83 on the surface 70a of the microarray substrate 70 on which the probe DNA (single strand) 80 is fixed. Distribute the solution. Double-stranded DNA 81 is formed by hybridizing the target DNA 82 of the specimen to the probe DNA 80 immobilized on the surface 70a of the microarray substrate 70.

Here, since the fluorescent substance 83 specifically binds to the double-stranded DNA, it adheres to the double-stranded DNA 81 generated by the hybridization on the surface 70a of the microarray substrate 70. At this time, since the evanescent field 60 is thinly generated on the upper surface side of the surface 70a on which the probe DNA 80 is immobilized on the microarray substrate 70, only the fluorescent substance 84 attached to the double-stranded DNA 81 is caused by the evanescent field 60. Emits fluorescence when excited. On the other hand, the free fluorescent material 83 is not excited by the evanescent field 60.

In this way, by exciting the fluorescent material 84 by illumination using the evanescent field 60, only the fluorescent material 84 near the surface 70a of the microarray substrate 70 can be selectively excited. Then, the change in fluorescence intensity over time is measured as a change in the amount of hybridization, and the final amount of hybridization can be predicted by fitting to a theoretical curve.

[Embodiment 2]

Another embodiment of the present invention will be described below with reference to FIG. 5 and FIG. In the present embodiment, components having the same functions as the components in the first embodiment are given the same reference numerals, and descriptions thereof are omitted. Real In the embodiment, differences from the first embodiment will be described.

In this embodiment, the case where single-stranded DNA is used as the probe substance and the target substance, that is, the case where a so-called DNA microarray is measured will be described as an example.

In the present embodiment, the microarray reader is configured with an inverted microscope as a base, and includes a microarray reader 200 that includes light incident means for making light incident on the microarray substrate 70 without passing through the objective lens. Will be described. That is, the microarray reader 200 according to the present embodiment has substantially the same configuration as the microarray reader 100 shown in the first embodiment, except that the illumination by the evanescent field is performed without passing through the microscope objective lens. The fluorescence detection is configured to be performed through a microscope optical system. In the microarray reading apparatus 200 according to the present embodiment, as an inverted microscope that can be used as a base, for example, TE-2000 made by Nico can be cited as in the first embodiment.

FIG. 5 is a diagram schematically showing a configuration of microarray reading apparatus 200 according to the present embodiment. FIG. 6 is a perspective view schematically showing a part of the configuration shown in FIG. 5 upside down. As shown in FIGS. 5 and 6, the microarray reader 200 includes a laser light source 11, a first lens 12, a reflecting surface 13, an objective lens 14, an excitation light cut filter 15, an imaging lens 16, and a photodetector. 17, a temperature adjusting unit 18, an arithmetic processing unit 19, a position changing unit 21, a light incident unit 30, and a light absorbing unit 32. Further, the microarray reader 200 uses the microarray substrate 70 as a reading target.

In the present embodiment, the objective lens 14 has only the function of the objective lens in the microscope optical system, and does not function as a light incident means as in the first embodiment.

The light incident part 30 has a reflection surface 31 for reflecting light emitted from the laser light source 11 and passing through the reflection surface 13. In the present embodiment, the reflecting surface 31 is light that passes from the laser light source 11 via the reflecting surface 13, that is, light that is parallel to the optical axis of the microscope optical system (for example, the objective lens 14), and the optical axis Is set to an angle at which the light that is offset by a certain distance is incident on the microarray substrate 70 at an incident angle that is greater than the angle at which the probe material on the surface of the microarray substrate 70 is fixedly reflected. . That is, the light incident part 30 having the reflecting surface 31 emits the laser light emitted by the laser light source 11 so as to generate an evanescent field on the surface of the microarray substrate 70 on which the probe DNA is fixed. Light incident means for entering the substrate 70. The probe material on the microarray substrate is fixed without passing the laser light from the laser light source 11 through the objective lens 14 (by passing through the outside of the objective lens 14). The incident light is incident on the microarray substrate 70 at an incident angle that is greater than the angle at which the light is totally reflected on the surface.

That is, the present embodiment is a feature that is greatly different from the first embodiment in that light is incident on the microarray substrate 70 without passing through the objective lens 14 to generate an evanescent field. As shown in Embodiment 1, when the incidence of light for generating an evanescent field on the surface of the microarray substrate 70 is performed by the objective lens 14 (via the objective lens 14), the viewing angle is reduced. There is a problem that many pots cannot be detected at once! /, And! /.

However, as shown in the present embodiment, incidence of light for generating an evanescent field on the surface of the microarray substrate 70 is performed from outside the objective lens 14 without passing through the objective lens 14. In this case, the viewing angle becomes larger and many DNA spots can be detected at one time. Specifically, as in the first embodiment, when light is incident on the microarray substrate 70 via the objective lens 14, the viewing angle is about 150 m × 150 m. As described above, when light is incident on the microarray substrate 70 without passing through the objective lens 14, the viewing angle is about 1 mm (1000 m) × 1 mm (1000 m), and the viewing angle is greatly widened.

[0100] In addition, the light incident portion 30 is preferably disposed between the microarray substrate 70 and the objective lens 14. It is more preferable that the light incident portion 30 is in contact with the objective lens 14. This is because the number of apertures of the objective lens 14 can be increased because the light incident portion 30 is in contact with the objective lens 14. This makes it possible to acquire weak fluorescence efficiently with the objective lens 14. In addition, the light emitted from the fluorescent substance existing in the vicinity of the microarray substrate 70 is a force that exists as a lot of light that travels through the microarray substrate 70 at a larger angle than the total reflection angle. Become. Ma An oil layer 20 is provided between the microarray substrate 70 and the light incident part 30. Such an oil layer 20 has a function similar to that of the oil layer 20 in the above-described first embodiment, and thus description thereof is omitted here.

[0101] The refractive index of the light incident section 30 is preferably substantially the same as the refractive index of the microarray substrate 70. By matching the refractive indices of the light incident part 30 and the microarray substrate 70, the light incident angle can be adjusted and controlled more accurately, and reflection at the interface can be suppressed. is there. Therefore, like the microarray substrate 70, the light incident portion 30 is preferably composed of a glass substrate, which is a material having high light transmittance, or a resin such as polycarbonate or PMMA, but when the temperature is changed. A glass substrate is more preferable because of a small change in refractive index.

In addition, the microarray reader 200 includes a light absorption unit 32. The light absorbing unit 32 functions as a beam trap for absorbing the light totally reflected on the surface of the microarray substrate 70. A conventionally known beam trap device can be suitably used as the light absorbing portion 32 that is powerful. For example, a beam diff user BD-40 manufactured by Sigma Kogyo can be used.

Next, the operation of the above-described microarray reader 200 will be described. First, as shown in FIGS. 5 and 6, the laser light source 11 emits laser light. The irradiated laser light is condensed by the first lens 12 and subsequently reflected by the reflecting surface 13. The reflecting surface 13 is a beam parallel to the optical axis of the microscope optical system (for example, the objective lens 14), which is a laser beam from the laser light source 11 that arrives via the first lens 12. The light is reflected from the reflecting surface 31 of the light incident part 30 as light offset by a certain distance.

The laser beam reflected by the reflecting surface 13 passes through the outside of the objective lens 14 (without passing through the objective lens 14) and enters the light incident portion 30. The light incident on the light incident part 30 is reflected by the reflecting surface 31, passes through the inside of the light incident part 30, and enters the microarray substrate 70. Here, the light incident on the light incident portion 30 has a large incident angle with respect to the microarray substrate 70 even though it is a parallel light beam at the exit of the light incident portion 30 due to the action of the light incident portion 30. Illuminates the DNA spot.

[0105] That is, the light incident part 30 converts the laser light into the probe DNA on the microarray substrate 70. The microarray substrate is fixed at an angle of incidence that is greater than the angle at which it is totally reflected on the surface.

It can be said that it functions as a light incident means for making the light incident on 70. Since the refractive index of the light incident part 30 and the refractive index of the microarray substrate 70 are substantially the same, the incident angle of the incident light with respect to the microarray substrate 70 hardly changes from the angle reflected by the reflecting surface 31.

At this time, an evanescent field is generated thinly on the upper surface side of the surface of the microarray substrate 70. The mechanism for detecting whether or not the probe DNA and the target DNA are hybridized by the evanescent field is the same as in the first embodiment, and thus the description thereof is omitted here.

The generated fluorescence is collected by the objective lens 14, imaged again through the excitation light cut filter 15, the imaging lens 16 t, and the mechanism of the microscope optical system, and detected by the photodetector 17. Also in this embodiment, the temperature control unit 18 is used to distribute the sample solution containing the target DNA in a state where the temperature of the sample solution is kept constant at 65 ° C. and the temperature is stabilized. Is in contact with. Therefore, the hybridization process can be evaluated in real time by observing the temporal change of the fluorescence image.

Therefore, according to the microarray reader 200, the time required for the hybridization process can be shortened, so that the detection time can be greatly reduced as compared with the conventional apparatus. Furthermore, since the viewing angle is large, many pots can be observed at one time, and the detection time can be further reduced and highly accurate detection can be performed.

[0109] In the present specification, the reading of the DNA microarray substrate has been described as an example, but the present invention is not limited to this. In other words, the present invention can be used as a measurement technique for detecting not only DNA but also molecules such as proteins, small molecules in vivo, and environmental hormones. Therefore, based on the technical level at the time of filing of the present application, those skilled in the art also fully include the scope in which the DNA microarray reading technology can be applied to the protein or other substance detection technology. Let me add that just in case.

[0110] Finally, the arithmetic processing unit 19 may be configured by hardware logic, or may be realized by software using a CPU as follows! /. That is, the arithmetic processing unit 19 includes a CPU (.central processing unit) that executes instructions of a control program that realizes each function, a ROM (read only memory) that stores the program, and a RAM (random access that expands the program). memory), a storage device (recording medium) such as a memory for storing the above programs and various data, etc. The object of the present invention is to control the arithmetic processing unit 19 which is software for realizing the functions described above. A recording medium in which the program code (executable program, intermediate code program, source program) of the program is recorded so as to be readable by a computer is supplied to the arithmetic processing unit 19, and the computer (or CPU or MPU) stores the recording medium in the recording medium. It can also be achieved by reading and executing the recorded program code.

[0112] The recording medium includes, for example, a tape system such as a magnetic tape and a cassette tape, a magnetic disk such as a floppy (registered trademark) Z hard disk, and an optical disk such as a CD-ROMZMOZ MD / DVD / CD-R. Disk systems, IC cards (including memory cards) Z optical cards and other card systems, or mask ROMZEPROMZEEPROMZ flash ROM and other semiconductor memory systems can be used.

[0113] The arithmetic processing unit 19 may be configured to be connectable to a communication network, and the program code may be supplied via the communication network. The communication network is not particularly limited. For example, the Internet, intranet, extranet, LAN, ISDN, VAN, CATV communication network, virtual private network, telephone line network, mobile communication network. Satellite communication networks can be used. In addition, the transmission medium constituting the communication network is not particularly limited. For example, IEEE1394, USB, power line carrier, cable TV line, telephone line, ADSL line, etc. (Registered trademark), 802.11 wireless, HDR, mobile phone network, satellite line, terrestrial digital network, etc. can also be used. The present invention can also be realized in the form of a computer data signal embedded in a carrier wave, in which the program code is embodied by electronic transmission.

[0114] Embodiments of the present invention will now be described in more detail with reference to examples. Of course, it goes without saying that the present invention is not limited to the following examples, and various modes are possible for details. Further, the present invention is limited to the above-described embodiment. Various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included in the technical scope of the present invention.

[0115] [Example]

Hereinafter, as a specific example of the present invention, the results of time series observation of the hybridization process of DNA molecules using the microarray reader 100 shown in the first embodiment will be shown. In this example, an hybridization experiment was performed using cDNA used for the detection of the gene relA, which is widely used in general DNA microarray experiments. relA is a gene (length 2.2 kbp) that is important for activation and inactivation of transcription factor NF κ Β, which moves from the cytoplasm to the nucleus and induces the expression of target genes, and is involved in immune responses and developmental differentiation. It is known that it is widely expressed in cells! In the hybridization experiment, this cDNA was spotted as a probe DNA on a cover glass substrate, and the same cDNA was flowed through a flow cell (hybridization 'cell) as a target DNA for detection.

[0116] In this example, the hybridization cell is placed on the sample stage of an inverted microscope (Nikon, TE-20000), and the illumination by the evanescent field is the objective lens (Nikon, TIRF mode). Things, NA = 1.45) Side force was also performed. The generated fluorescence was collected by an objective lens, imaged again through a microscope optical system, and detected by a cooled CCD camera (Hamamatsu Photonicus, ORCA-ER).

[0117] An experiment was carried out to detect target DNA by carrying out noise hybridization. The prepared DNA microarray was attached to a 98 ° C water bath and thermally denatured to make a single strand, which was then immersed in a NaBr aqueous solution for blocking treatment, dried and used in the experiment. The target DNA to be flowed into the hybridization cell is dispersed in TE buffer without staining, mixed with a fluorescent probe, adjusted in concentration, and then thermally denatured at 98 ° C for 5 minutes to form a single strand. Using. The fluorescent probe used is POPO-3 (Molecular Probes, Inc.), and it has been evaluated beforehand that double-stranded DNA can be selectively detected. While the hybridization cell was heated to 65 ° C and the temperature was stabilized, a solution containing the target DNA was allowed to flow, and the change in fluorescence image over time was observed. The results are shown in FIGS. 7 (a) to 7 (d). Fig. 7 (a) is t = 0, Fig. 7 (b) is t = l (min), Fig. 7 (c) is t = 5 (min), Fig. 7 (d) is t = 20 (min) FIG. Bright and fluorescent light is observed in the spot where the probe DNA is spotted, which indicates that hybridization has occurred in that spot.

[0119] In addition, as a target experiment (control), the target DNA solution prepared by mixing POPO-3 in the same manner was not heat denatured, and it was put into the hybridization cell in a state (double-stranded state). The experiment was conducted. Since it does not hybridize to the probe DNA in the double-stranded state, it may not be detected in the probe DNA spot.

[0120] The observation results are shown in Fig. 7 (e) to Fig. 7 (h). Fig. 7 (e) is t = 0, Fig. 7 (f) is t = 1 (min), Fig. 7 (g) is t = 5 (min), Fig. 7 (h) is t = 20 (min) FIG. Initially brightening power After that, the brightness has become almost constant.

[0121] Fig. 8 shows the results of plotting changes in the fluorescence intensity (average value in each spot) of each spot from the results of Figs. 7 (a) to 7 (h). In the graph, “Hybridization” indicates the result when the single-stranded DNA is hybridized, and “Control” indicates the result of the control experiment.

[0122] As shown in FIG. 8, as the target DNA flows in the flow cell, the intensity of fluorescence in the spot increases, but the intensity has reached a certain level. This corresponds to the peak intensity change as the target DNA amount decreases as predicted by Equation (1) above! /

[0123] In addition, the graph shows the force that the fluorescence intensity increases stepwise. This is because the DNA solution in the hybridization cell pulsates and the concentration of the DNA solution varies over time. thinking. Since the DNA solution flows by pushing out the buffer at the initial stage, it is speculated that the difference in the concentration of the DNA solution at this stage also caused the step. In the experiment in this example, the starting force was continuously observed until 50 minutes, but it was confirmed that the noise stabilization process was stable in about the first 10 minutes.

[0124] As shown in this example, by using a real-time observation type DNA microarray reader, it becomes possible to observe the hybridization process. It was found that the detection time can be greatly shortened compared to the array reader. The microarray reading apparatus according to the present invention is characterized in that the time required for the hybridization process can be shortened. At present, measurement can be performed with a hybridization time of about 15 minutes. Furthermore, by completing technical issues such as flow stability during hybridization, suppression of variation in solution concentration, and high efficiency of hybridization, the degree of completeness can be further improved. I think you can.

Industrial applicability

[0125] The microarray reader according to the present invention has an effect that the binding between the probe substance on the microarray substrate and the target substance contained in the sample can be detected with higher accuracy and efficiency. Furthermore, according to the microarray reader, DNA is not spotted on the prism. For this reason, it can be used for many commercially available microarray substrates.

[0126] Further, since the light incident means (for example, a high refractive index block) allows the light to be incident on the microarray substrate without passing through the objective lens, the viewing angle can be increased. Many spots can be read, and the detection time can be shortened.

[0127] By providing a temperature control means, the hybridization between the probe DNA and the target DNA can be detected in real time, so that the detection time can be shortened. Play.

[0128] As described above, according to the present invention, the detection time can be greatly shortened and higher accuracy can be achieved. Field-use microarray readers used in test laboratories can be provided, so they have industrial applicability in a wide range of fields such as the medical industry, food industry, pharmaceutical industry, and environment.

Claims

The scope of the claims

[1] Specific interaction between the probe substance and the target substance when a sample containing at least a fluorescent substance and a target substance is brought into contact with the microarray substrate on which the probe substance is immobilized. A microarray reader for detecting light irradiation means for irradiating light;

Light incident means for causing the light irradiated by the light irradiation means to enter the microarray substrate so as to generate an evanescent field on the surface of the microarray substrate on which the probe substance is fixed;

Photodetection means for detecting fluorescence emitted from a fluorescent material contained in a sample excited by the evanescent field, and

The microarray reading apparatus, wherein the light detection means includes an optical lens that functions as an objective lens, and the optical lens functions as the light incident means.

[2] Specific interaction between the probe substance and the target substance when a sample containing at least a fluorescent substance and a target substance is brought into contact with the microarray substrate on which the probe substance is immobilized. A microarray reader for detecting light irradiation means for irradiating light;

The light detection means includes an optical lens that functions as an objective lens, and the light incidence means causes light from the light irradiation means to enter the microarray substrate without passing through the optical lens. A microarray reader characterized by being a thing.

[3] The light incident means is for causing the light irradiated by the light irradiation means to be incident at an incident angle equal to or greater than an angle at which the probe material on the surface of the microarray substrate is totally reflected. The microarray according to claim 1 or 2, wherein Reader.

4. The microarray reading apparatus according to claim 2, wherein the light incident means is disposed between the microarray substrate and the optical lens.

[5] The light incident means has at least one reflection surface for reflecting the light irradiated by the light irradiation means,

The reflecting surface is provided so that light reflected by the reflecting surface is incident at an incident angle that is greater than an angle at which the reflected light is totally reflected on the surface of the microarray substrate on which the probe substance is fixed. The microarray reader according to claim 2, wherein

6. The microarray reader according to claim 2, wherein the refractive index of the light incident means is substantially the same as the refractive index of the microarray substrate.

[7] The light irradiation means and the light detection means are arranged on the back side of the surface of the microarray substrate on which the probe substance is fixed.

2. The microarray reader according to item 1.

8. The microarray reader according to any one of claims 1 to 7, wherein an oil layer is provided between the microarray substrate and the light incident means.

[9] The microarray reader according to any one of [1] to [8], further comprising temperature adjusting means for adjusting the temperature of the sample containing the target material on the microarray substrate. .

10. The microarray reader according to any one of claims 1 to 9, wherein the specific interaction between the probe substance and the target substance is detected in real time.

11. The microarray reader according to any one of claims 1 to 10, further comprising position changing means for changing a relative position between the microarray substrate and the light incident means.

[12] Furthermore, in order to estimate the amount of the target substance that interacts with the probe substance, using the detection data obtained by observing the process in which the target substance interacts with the probe substance in time series. The microarray reading device according to claim 1, further comprising an arithmetic processing unit that performs the arithmetic processing.

[13] The microarray reader according to any one of claims 1 to 12, wherein the probe substance and the target substance are single-stranded DNAs.

[14] The fluorescent substance is bonded to the target substance.

3. The microarray reader according to item 1 above.

[15] The fluorescent substance according to any one of claims 1 to 14, wherein the fluorescent substance specifically binds to a substance in a state where the probe substance and the target substance interact with each other. The microarray reader according to the item.