JP2004138420A - Biochip equipped with confocal optical system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive, compact biochip reading apparatus by providing biochips having a confocal optical system. <P>SOLUTION: The biochips comprise a substrate having a plurality of samples and a light-shielding member that is provided at a surface at a side opposite to a surface provided with the sample of the substrate. The substrate has a lens corresponding to each of the substrates. The light-shielding member has an opening corresponding to each of the samples, and the sample and the opening are in a confocal relationship via the lens. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biochip having a function of controlling a traveling direction of weak light in a predetermined direction, and more specifically, to a biochip provided with a confocal optical system. The present invention also relates to a biochip reader using the biochip and a light detection method.
[0002]
[Prior art]
In physical property evaluation tests and quantitative tests using biochips (including DNA chips, protein chips, DNA arrays and DNA microarrays) in which multiple samples are arranged and immobilized on a substrate such as glass or silicon, fluorescence or chemiluminescence is used. Detection is often performed. A typical device for detecting such weak light is a biochip reader using a confocal optical system (for example, see Patent Document 1).
[0003]
The confocal optical system is an optical system for obtaining a high-contrast image by placing a pinhole at a position where an image is formed (focused position), thereby eliminating light from other than the focused point at all.
[0004]
As shown in FIG. 6, a conventional biochip reader 22 using a confocal optical system includes a biochip 24 having a sample 23, an objective lens 25, a beam splitter 26, a mirror 27, an optical filter 28, and a light receiving lens 29. , A confocal pinhole 30, a light receiver 31, and the like. When excitation light 32 emitted from a light source (not shown) is reflected by the beam splitter 26 and irradiated on the sample 23, fluorescence 33 is generated from the excited sample 23. The fluorescent light 33 is captured by the objective lens 25 located above the sample 23, transmitted through the beam splitter 26, guided to the optical filter 28 by the mirror 27, collected by the light receiving lens 29, and condensed by the confocal pinhole 30. , And is detected by the light receiver 31. In the case of chemiluminescence detection, the excitation light 32 is not irradiated, but the process until the light emitted from the sample 23 is detected by the light receiver 31 is the same.
Such a confocal type biochip reader has a high S / N ratio of detection light, but is expensive because the optical system for reducing noise light is complicated and large.
[0005]
[Patent Document 1]
JP 2001-31690 "Biochip reader and electrophoresis apparatus"
[Problems to be solved by the invention]
In view of the above circumstances, an object of the present invention is to reduce the size and cost of a biochip reader.
[0006]
[Means for Solving the Problems]
The biochip according to the present invention is a biochip having a substrate provided with a plurality of samples, and a light blocking member provided on a surface of the substrate opposite to a surface provided with the samples, wherein the substrate Has a lens corresponding to each of the samples, the light shielding member has an opening corresponding to each of the samples, and the sample and the openings are in a confocal relationship via the lens. It is characterized by the following.
Since the biochip having such a configuration actually has a confocal optical system in the chip, the biochip reader does not require a complicated and large-sized optical system. That is, by the biochip, the light emitted to the substrate side out of the light generated from the excited sample on the substrate is condensed by the lens, and the opening provided on the light shielding member corresponding to each of the samples is formed. Can be passed. Therefore, if the irradiation of the excitation light and the detection of the light generated by the sample are performed on the opposite side of the substrate using the biochip, the biochip reader can be reduced in size and cost. It becomes.
[0007]
The substrate may be formed by stacking a plurality of substances having different refractive indexes.
By forming the substrate in this manner, a substrate having a lens function therein can be easily formed.
Further, it is preferable that, out of a plurality of substances forming the substrate, a substance in contact with the sample has a refractive index larger than that of the sample.
As described above, if a material having a higher refractive index than the sample is selected as the material of the substrate in contact with the sample, the fluorescent light generated from the sample and traveling toward the substrate is refracted in a direction normal to the light incident surface of the substrate. Therefore, the light-collecting efficiency is higher than the fluorescence generated on the side opposite to the substrate.
[0008]
On the surface of the substrate on which the sample is provided, a first microlens array is further formed with the sample interposed therebetween, and the first microlens array includes a first lens corresponding to each of the samples. And the focal point of the first lens may coincide with the position of the sample.
As described above, since the biochip includes the first microlens array, an objective lens for condensing light from the light source on the sample becomes unnecessary, and the apparatus can be downsized.
Furthermore, since excitation light can be efficiently condensed on each sample by such a configuration, a plurality of samples can be excited simultaneously and accurately.
[0009]
A second microlens array is disposed on the surface of the substrate opposite to the surface on which the sample is provided, with the light shielding member interposed therebetween, and the second microlens array corresponds to each of the openings. And a focal point of the second lens coincides with the position of the opening.
As described above, since the biochip includes the second microlens array, a light receiving lens that receives light passing through the opening of the light blocking member is not required, and the device can be downsized.
Furthermore, with such a configuration, the light generated by each sample can be efficiently collected, so that the light generated by a plurality of samples can be detected simultaneously and accurately.
[0010]
The sample may include genetic information of an organism.
Use of a biochip equipped with a sample containing genetic information of an organism to determine the sequence of a gene, confirm the presence or absence of a specific gene, analyze SNPs (single nucleotide polymorphisms), identify proteins or analyze functions, etc. Can be.
[0011]
The biochip reader of the present invention includes a light source, an objective lens for condensing excitation light from the light source on the sample, a biochip, and the opening of the light shielding member generated on the substrate side from the sample. A light-receiving lens for receiving the passed light, an optical filter for blocking or attenuating the excitation light, and a light-receiving device for receiving the light passed through the light-receiving lens and the optical filter, wherein the biochip is A biochip having a substrate provided with a plurality of samples, and a light blocking member provided on a surface of the substrate opposite to the surface provided with the samples, wherein the substrate is provided on each of the samples. A corresponding lens is provided, and the light shielding member has an opening corresponding to each of the samples, and the sample and the opening are in a confocal relationship via the lens.
Since the biochip reader having such a configuration has a confocal optical system in the biochip, it is smaller and less expensive than a conventional device.
[0012]
The light detection method according to the present invention is a light detection method using the biochip, a light source, and a light receiver, wherein light generated from the sample by irradiating the sample with excitation light from the light source. The light emitted to the substrate side is condensed by the lens, passed through an opening provided on the light shielding member so as to correspond to each of the samples, and detected by a light receiver. .
In this way, by using a biochip equipped with a confocal optical system, irradiation of excitation light and detection of light generated by the sample are performed on the opposite side across the substrate, as in a conventional photodetector. A complicated optical system becomes unnecessary, and the size of the apparatus can be reduced.
[0013]
In the above-described light detection method, after the irradiation of the excitation light from the light source is stopped, light detection may be performed by the light receiver.
As described above, by detecting the light generated by the sample while the irradiation of the excitation light is stopped, the excitation light is not detected as noise light, so that the SN ratio is dramatically improved.
[0014]
[Detailed description]
Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the drawings, taking a case of detecting fluorescence as an example. However, the dimensions, materials, shapes, relative arrangements, and the like of the components described in this embodiment are merely illustrative examples, and there is no intention to limit the scope of the present invention to them unless otherwise specified. In addition, each drawing is a schematic diagram, and is not drawn in consideration of a difference in refractive index of a component.
[0015]
FIG. 1 shows a biochip reader 1 according to one embodiment of the present invention. The biochip reader 1 includes a light source 2, an objective lens 4, a biochip 5, a light receiving lens 6, an optical filter 7, and a light receiver 8.
[0016]
First, the details of the biochip 5a used in the biochip reader 1 will be described with reference to FIG. In the biochip 5a, the substrate 11 is formed from substances 12 and 13 having different refractive indexes, and a sample 9 is provided on the surface of the substrate 11. Here, the boundary surface between the substances 12 and 13 forms a microlens array having lenses 120 corresponding to each of the samples 9. Desirably, the effective radius of each lens 120 is equal to the sample spacing. Further, a light blocking member 14 is formed on the surface of the substrate 11 opposite to the surface on which the sample 9 is provided, and the light blocking member 14 is provided with an opening 15 corresponding to each sample. Here, the sample 9 and the opening 15 have a confocal relationship via a microlens array formed of the substances 12 and 13.
[0017]
Next, the operation of the biochip reader 1 will be described. The excitation light 3 from the light source 2 is focused by the objective lens 4 and is irradiated on the sample 9 on the substrate 11. The sample 9 absorbs the excitation light 3 and generates fluorescence 10. The fluorescence 10 has a wavelength characteristic different from that of the excitation light 3. The fluorescence 10 generated from the sample 9 undergoes a refraction action by the microlens array formed by the substances 12 and 13 in the biochip 5a. At this time, since the sample 9 and the opening 15 have a confocal relationship, the fluorescent light 10 passes through the opening 15 of the light shielding member 14. Since the excitation light 3 similarly passes through the opening 15 by the refraction of the lens, an optical filter 7 having a wavelength selection characteristic is provided between the light receiving lens 6 and the light receiver 8 to transmit the fluorescent light 10 and excite the excitation light. Light 3 is not transmitted. The fluorescence 10 transmitted through the optical filter 7 is detected by the light receiver 8.
Here, the optical filter refers to a wavelength selection element having a function of transmitting or absorbing or reflecting light of a specific wavelength to block the light.
[0018]
Thus, unlike the conventional biochip, the biochip of the present invention has a configuration for detecting the fluorescence generated on the substrate side from the fluorescence generated from the sample. When detecting the fluorescence generated on the side opposite to the substrate using the conventional confocal biochip reader as shown in FIG. 6, the amount of detected light depends on the numerical aperture (NA) of the objective lens 25. And a little. On the other hand, when detecting fluorescence generated on the substrate side according to the present invention, if a material having a higher refractive index than that of the sample is selected as the material of the substrate, the fluorescence traveling toward the substrate side is normal to the light incident surface of the substrate. Since the light is refracted in the direction, the light can be collected more efficiently than the fluorescence generated on the side opposite to the substrate.
Here, since the refractive index of the sample is about 1.33 to 1.50, for example, when the refractive index of the sample is 1.4, a resin having a refractive index of about 1.6 is selected as the material of the substrate. For example, the refraction angle of the fluorescent light is about 60 degrees, and the light collection efficiency is good. Therefore, as a material used for the substrate, glass, resin, or the like that is transparent and has a large refractive index is suitable.
[0019]
The curvature of the lens formed inside the substrate and the thickness of the substrate are selected according to the refractive indices of the sample and the substrate so that the efficiency of condensing the fluorescence is optimized.
[0020]
The substrate 11 may be formed of a plurality of resins having different refractive indexes, or may be formed of a resin layer and an air layer. Since the refractive index of air is 1, by combining a resin layer with a large refractive index, a lens with a large NA can be formed, and more fluorescence can be detected.
[0021]
FIG. 3 shows another biochip 5b according to the present invention. As shown in FIG. 3, the substrate 11 may have a configuration in which two or more microlens arrays are stacked in the optical axis direction. Here, the refractive indices of the substances 16 and 18 may be the same or different.
By laminating two or more microlens arrays in this way, the synthetic NA of the lens can be increased, so that more fluorescence can be detected.
[0022]
FIG. 4 shows a biochip reader 19 according to still another embodiment of the present invention. The biochip reader 19 is characterized in that a plurality of samples included in a biochip are irradiated with excitation light at a time, and the light generated by each sample is simultaneously detected. FIG. 5 shows details of the biochip 5c used in the biochip reader 19.
[0023]
The biochip 5c includes a first microlens array 20 via a spacer 21 on the surface of the substrate 11 on which the sample 10 is provided, in addition to the configuration of the biochip 5b. Here, the first microlens array 20 has a first lens 200 corresponding to each of the samples, and the focal point of the first lens 200 coincides with the position of the sample.
[0024]
As described above, since the biochip 5c includes the first microlens array 20, an objective lens for condensing the excitation light 3 from the light source on the sample becomes unnecessary. That is, the excitation light 3 is efficiently condensed at the position of each sample 10 by the first microlens array 20, so that a plurality of samples 10 can be simultaneously and accurately excited. When an objective lens is used, the focal position needs to be adjusted to each sample. However, when the first micro lens array 20 is provided, the focal point of the first lens 200 matches the position of each sample. There is no need to adjust the focus position.
[0025]
Further, although not shown, a second microlens array may be provided on a surface of the substrate opposite to the surface on which the sample is provided, with a light shielding member interposed therebetween. The second micro lens array has a second lens corresponding to each of the openings of the light blocking member, and the focal point of the second lens is made to coincide with the position of the opening.
As described above, since the biochip 5c includes the second microlens array, the light generated by each sample is efficiently collected, so that the light generated by a plurality of samples can be simultaneously detected with high accuracy. A high-sensitivity CCD camera or the like is used as the light receiver.
[0026]
Therefore, if the biochip 5c is used, a plurality of samples can be excited at the same time, and the light generated by the plurality of samples can be detected at the same time, so that it is not necessary to scan the substrate.
However, when providing the first microlens array 20 and the second microlens array on the biochip 5b, highly accurate positioning is required. An alignment mark is used for alignment.
[0027]
The first lens 200 of the first microlens array 20 may be in contact with air, or the surface of the lens may be flattened using resins having different refractive indexes. Then, the lens surface can be protected from dust, and at the same time, the handling becomes easy.
[0028]
In the above embodiments, the example of the microlens array formed by laminating a plurality of materials having different refractive indexes as the lens included in the substrate has been described, but the GRIN lens in which the gradient index lenses (GRIN lenses) are arranged. Similar effects can be obtained as an array.
[0029]
The light detection method according to the present invention is a light detection method using the biochip described above, a light source, and a light receiver, and irradiates the sample with excitation light from the light source, and generates the light from the excited sample. The light emitted to the substrate side is collected by a lens inside the chip, passed through an opening provided on a light shielding member so as to correspond to each of the samples, and detected by a light receiver. I do.
In this way, by using a biochip equipped with a confocal optical system, irradiation of excitation light and detection of light generated by the sample are performed on the opposite side across the substrate, as in a conventional photodetector. A complicated optical system becomes unnecessary, and the size of the apparatus can be reduced.
[0030]
Further, in the above-described light detection method, if the irradiation of the excitation light from the light source is stopped and the light is detected by the light receiver, the excitation light is not detected as noise light, so that the SN ratio is dramatically improved. I do. Further, an optical filter for removing the excitation light is not required in the photodetector, and the device can be downsized.
[0031]
The case where fluorescence is detected has been described above as an example, but it is clear that the same effect can be obtained by the biochip according to the present invention even when chemiluminescence is detected.
[0032]
The photodetection method of the present invention includes determination of a gene sequence, confirmation of the presence or absence of a specific gene, measurement of the expression level of a specific gene, analysis of SNP (single nucleotide polymorphism), metabolism and absorption of a substance administered to an experimental mouse -It can be used for confirming the route or state of excretion, measuring intracellular ion concentration, identifying proteins or analyzing functions. Further, the present invention can be applied to a medical examination for determining the health condition of an individual, a test for personal security, and the like.
【The invention's effect】
In the biochip according to the present invention, for example, a substrate having a plurality of samples is formed by laminating substances having different refractive indices, so that the substrate has a lens function. Further, a light-shielding member having an opening corresponding to each of the samples is provided on the surface of the substrate opposite to the surface on which the samples are provided, so that a confocal optical system is provided inside the chip. By using the biochip of the present invention to perform irradiation of excitation light and detection of light generated by the sample on the opposite side of the substrate, a conventional complicated and large-sized optical system becomes unnecessary, and a biochip reader is used. It is possible to reduce the size and cost of the device.
[Brief description of the drawings]
FIG. 1 shows a biochip reader according to an embodiment of the present invention.
FIG. 2 shows a main part of the biochip reader according to the embodiment of the present invention.
FIG. 3 shows a biochip according to another embodiment of the present invention.
FIG. 4 shows a biochip reader according to another embodiment of the present invention.
FIG. 5 shows a main part of a biochip reader according to another embodiment of the present invention.
FIG. 6 shows a conventional confocal biochip reader.
[Explanation of symbols]
1, 19, 22 Biochip reader 2 Light source 3, 32 Excitation light 4, 25 Objective lens 5, 24 Biochip 6, 29 Light receiving lens 7, 28 Optical filter 8, 31 Light receiver 9, 23 Sample 10, 33 Fluorescence 11 Substrate 14 Light-blocking member 15 Opening 20 First micro-lens array 21 Spacer 26 Beam splitter 27 Mirror 30 Confocal pinhole 120 Lens 200 First lens

Claims (8)

A substrate provided with a plurality of samples, a biochip having a light-blocking member provided on a surface of the substrate opposite to the surface provided with the samples,
The substrate includes a lens corresponding to each of the samples,
The light blocking member has an opening corresponding to each of the samples,
A biochip, wherein the sample and the aperture are in a confocal relationship via the lens. The biochip according to claim 1, wherein the substrate is formed by stacking a plurality of substances having different refractive indexes. The biochip according to claim 2, wherein a refractive index of a substance in contact with the sample is larger than a refractive index of the sample among the plurality of substances forming the substrate. A first microlens array is arranged on the surface of the substrate on which the sample is provided, with the sample interposed therebetween,
The first micro lens array has a first lens corresponding to each of the samples,
The biochip of claim 1, wherein a focus of the first lens coincides with a position of the sample. The biochip according to claim 1, wherein the sample contains genetic information of an organism. The biochip according to claim 1, a light source, an objective lens for condensing excitation light from the light source on the sample, and the sample is generated on the substrate side from the sample and passed through the opening of the light shielding member. A biochip reader comprising: a light receiving lens for receiving light; an optical filter for blocking the excitation light; and a light receiver for receiving light passing through the light receiving lens and the optical filter. A light detection method using a biochip, a light source, and a light receiver according to claim 1, wherein the substrate is included in light generated from the sample by irradiating the sample with excitation light from the light source. A light detection method, wherein the light emitted to the side is condensed by the lens, passed through an opening provided on the light shielding member so as to correspond to each of the samples, and detected by a light receiver. The light detection method according to claim 7, wherein after the irradiation of the excitation light from the light source is stopped, light detection is performed by the light receiver.

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