JP2004257737A - Biochip reader - Google Patents

Abstract

A small and low-cost biochip reader is provided.
A semiconductor laser for irradiating a laser beam capable of exciting a fluorescent substance, a beam shaper for converting the laser beam emitted from the semiconductor laser from Gaussian light to a top hat light, and scattered fluorescent light are provided. A capillary plate 22 for collimating the light, an interference filter 24 for blocking the laser beam 11 of the light passing through the capillary plate 22 and transmitting the fluorescent light 21, an electronically cooled CCD 20 for receiving the fluorescent light 21 transmitted through the interference filter 24, .
[Selection diagram] Fig. 1

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biochip reader.
[0002]
[Prior art]
For the purpose of researching useful genes and diagnosing diseases, analysis of gene expression patterns and proteome analysis for elucidating the functions of proteins have been performed. For these analyses, a hybridization reaction is used. In the hybridization reaction, a DNA or protein having a known sequence (hereinafter, referred to as a probe) and a DNA or protein to be investigated (hereinafter, referred to as a sample) are complementarily bound (hybridized). It is. As a specific method, first, a biochip such as a DNA chip or a protein chip is formed. The biochip has a plurality of spots formed on the surface of a glass substrate on which probes having different arrangements are fixed. One sample is labeled with a fluorescent substance. Then, the biochip and the sample are placed in a reaction vessel and hybridization is performed. Thereafter, the biochip is irradiated with light to excite the fluorescent substance, and the intensity of the fluorescence emitted from each spot is measured to confirm the presence or absence of hybridization at each spot. Thereby, various analyzes relating to DNA, protein, and the like can be performed.
[0003]
This fluorescence measurement method includes the following two methods. FIG. 4 is an explanatory diagram of the first method. In the first method, a semiconductor laser 110 irradiates a laser beam 111 having an excitation wavelength of a fluorescent substance with a semiconductor laser 110, and the laser beam 111 is condensed by a condenser lens 114 and further reflected by a galvanomirror 116, and Is scanned. At the spot 102 where the hybridization has been performed on the biochip 101, the fluorescent substance is excited by the laser light to generate fluorescence 121. The scattered fluorescent light 121 is collected by the lens 126 and received by the photomultiplier 120 or the like. In the second method, an excitation light is created by combining a continuous light source such as xenon and an optical bandpass filter, and the excitation light is irradiated onto a biochip. Further, the fluorescent light scattered from each spot of the biochip is condensed by a lens, and is received two-dimensionally by a cooled CCD or the like.
[0004]
[Patent Document 1]
JP-A-11-094747 (FIGS. 1 and 2)
[0005]
[Problems to be solved by the invention]
In the first method described above, a condenser lens is used for condensing irradiated laser light and condensing scattered fluorescence. Since the condenser lens generally requires a long focal length (about several cm to 10 cm), there is a problem that the biochip reader becomes large. Further, the galvanomirror that scans the biochip with a laser has difficulty in setting the optical axis and timing, and has a problem that the biochip reader becomes complicated.
[0006]
In the above-described second method, a continuous light source such as xenon is used. However, a high-luminance continuous light source is required, and there is a problem that a biochip reader becomes large and expensive. In addition, a continuous light source such as xenon generates a large amount of heat and has a risk of damaging DNA or the like. Therefore, there is a problem that the size of the biochip reader increases. On the other hand, since a condensing lens is used for condensing the scattered fluorescence, there is still a problem that the biochip reader becomes large.
[0007]
As described above, there is a problem that the size of the biochip reader is increased or the cost is increased, regardless of which method is used. Therefore, the biochip reader could not be used in some clinical tests and food tests.
The present invention has been made in view of the above problems, and has as its object to provide a small and low-cost biochip reader.
[0008]
[Means for Solving the Problems]
In order to achieve the above object, a biochip reader according to the present invention irradiates a plurality of spots on a biochip with light, receives fluorescence generated thereby, and identifies the spot where a fluorescent substance is present. A chip reader, comprising: a semiconductor laser that irradiates laser light capable of exciting the fluorescent substance; and a beam shaper that converts the laser light irradiated from the semiconductor laser from Gaussian light to top hat light. Configuration.
[0009]
By using a beam shaper, a semiconductor laser can be used as a surface light source, and it is not necessary to use a condenser lens and a galvanomirror. Thus, the semiconductor laser can be arranged close to the biochip, and the size of the biochip reader can be reduced. In addition, since it is not necessary to use expensive and complicated components such as a galvanometer mirror and a continuous light source, a low-cost and highly reliable biochip reader can be provided.
[0010]
A biochip reader for irradiating a plurality of spots on a biochip with light, receiving fluorescence generated thereby, and identifying the spot where a fluorescent substance is present, wherein the capillary collimates the scattered fluorescence. A plate, among light passing through the capillary plate, an interference filter that blocks light that excites the fluorescent substance and transmits the fluorescence, and an image sensor that receives the fluorescence that has passed through the interference filter. Configuration.
[0011]
By using a capillary plate, it becomes possible to collimate the scattered fluorescence, and it is not necessary to use a condenser lens. This makes it possible to dispose the image sensor close to the biochip, and to reduce the size of the biochip reader.
[0012]
A biochip reader that irradiates a plurality of spots on the biochip with light, receives fluorescence generated thereby, and specifies the spot where a fluorescent substance is present, wherein the laser can excite the fluorescent substance. A semiconductor laser for irradiating light, a beam shaper for converting the laser light emitted from the semiconductor laser from Gaussian light to top hat light, a capillary plate for collimating the scattered fluorescence, and a light passing through the capillary plate Among the light, an interference filter that blocks the laser light and transmits the fluorescence, and an image sensor that receives the fluorescence transmitted through the interference filter are provided. Thereby, a small and low-cost biochip reader can be provided.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
Preferred embodiments of a biochip reader according to the present invention will be described in detail with reference to the accompanying drawings. Note that what is described below is merely an embodiment of the present invention, and the present invention is not limited thereto.
[0014]
FIG. 1 shows a side view of the biochip reader according to the present embodiment. The biochip reader 5 according to the present embodiment irradiates a plurality of spots on the biochip 1 with light, receives the fluorescent light 21 generated thereby, and specifies the spot where the fluorescent substance is present. A semiconductor laser 10 for irradiating a laser beam 11 capable of exciting a fluorescent substance, a beam shaper 12 for converting the laser beam 11 emitted from the semiconductor laser 10 from Gaussian light to a top hat light, and a scattered fluorescence 21 A capillary plate 22 for collimating the light, an interference filter 24 for blocking the laser light 11 and transmitting the fluorescent light 21 out of the light passing through the capillary plate 22, an electronically cooled CCD 20 for receiving the fluorescent light 21 transmitted through the interference filter 24, It has.
[0015]
As the semiconductor laser 10, a laser capable of irradiating a laser beam having a wavelength capable of exciting a fluorescent substance existing on the biochip 1 is used. For example, a semiconductor laser 10 that can emit laser light having a wavelength of 532 nm is used.
[0016]
A beam shaper 12 is provided downstream of the semiconductor laser 10. The beam shaper 12 converts Gaussian light such as laser light having an intensity distribution as shown in FIG. 2A into top hat light having an intensity distribution as shown in FIG. The beam shaper 12 includes a refraction method and a diffraction method. The refraction method uses an aspheric lens. That is, the light intensity is made uniform by using a concave lens that expands the light flux at the center of the Gaussian light with a high light density and using a convex lens that narrows the light flux at the periphery of the Gaussian light with a low light density (Japanese Patent Application Laid-Open No. 2002-2002). 139696). One diffraction method uses a diffraction type optical element. That is, by appropriately setting the crystal orientation and the spatial period in a diffractive optical element (holographic element), an incident wave is diffracted to a desired position and the light intensity is made uniform (see Japanese Patent Application Laid-Open No. 8-94839). ).
[0017]
Note that, even when a diffuser is used instead of the beam shaper, top hat light can be obtained. The diffuser diffuses incident light with a diffraction grating pattern etched on a transparent medium to obtain uniform light.
[0018]
On the other hand, a capillary plate 22 is provided downstream of the set position of the biochip 1. The capillary plate 22 is formed by providing a glass plate having a thickness of 0.4 to several tens mm with a large number of micro holes having a diameter of several μm. Each of the micro holes is aligned with the surface of the capillary plate 22 and is formed vertically from the surface of the capillary plate 22. In general, a capillary plate is used for rectification and filtering of a gas flow, and the capillary plate 22 is used as a collimator for extracting parallel light from scattered fluorescence by utilizing the fact that the hole diameter is extremely small with respect to the plate thickness. This eliminates the need to use a condenser lens, so that an image sensor such as an electronically cooled CCD 20 described later can be arranged close to the biochip 1, and the biochip reader 5 can be downsized. .
[0019]
An interference filter 24 is provided downstream of the capillary plate 22. In the biochip reader 5, it is necessary to image only the fluorescent light 21 which is much weaker than the laser light 11. Therefore, the interference filter 24 allows only the fluorescent light 21 of a specific wavelength to pass therethrough, and blocks the laser light 11 that hinders the fluorescent imaging.
[0020]
The interference filter 24 is an optical filter that removes light of a wavelength that is not transmitted by interference instead of absorption or scattering. The interference filters include a metal interference filter and an all-dielectric interference filter. The metal interference filter is obtained by alternately depositing a thin film of magnesium fluoride as a dielectric and a translucent metal thin film on a surface of a glass substrate. One all-dielectric interference filter is a multilayer filter in which a thin film having a high refractive index such as zinc sulfide or cesium oxide is deposited instead of a metal thin film. The all-dielectric interference filter can make the transmission wavelength band narrower than the metal filter. Therefore, when the transmitted light wavelength and the cutoff light wavelength are close to each other, an all-dielectric interference filter is effective. Here, the difference between the excitation light wavelength and the fluorescence wavelength is usually 20 to 30 nm, but may be about 6 nm. Therefore, in the present embodiment, the fluorescence can be prevented from being buried in the laser light even when the fluorescence and the excitation light as the laser light are simultaneously imaged by using the interference filter 24.
[0021]
An image sensor is provided downstream of the interference filter 24. In particular, an electronically cooled CCD 20 is employed as an image sensor for imaging weak fluorescence. In general, a CCD outputs a noise current called a dark current even when light is not input. When the amount of light is large, the dark current does not matter, but when imaging weak fluorescence, the dark current cannot be ignored. Since the dark current is mainly generated by accumulating electric charge by heat, the dark current can be reduced by cooling the CCD. As a cooling method, there is a cooling method using liquefied nitrogen or the like, but in this embodiment, an electronic cooling method is employed. The electronic cooling system is a system in which a thermoelectric conversion device such as a Peltier device is mounted on a CCD. The Peltier device absorbs heat at the contact surface by flowing a direct current through a contact surface of a dissimilar metal in a predetermined direction, and functions as a heat pump. In addition to the thermoelectric conversion device, a temperature sensor such as a thermistor is attached to the CCD to keep the CCD at a constant low temperature. By using such an electronically cooled CCD 20 to reduce dark current, it is possible to secure a high S / N ratio even after long-time exposure. The biochip reader according to the present embodiment configured as described above is used as follows.
[0022]
After performing the hybridization step, the biochip reader is used in the biochip reading step for checking the presence or absence of hybridization at each spot of the biochip. In the hybridization step, DNA or protein having a known sequence (hereinafter, referred to as a probe) and DNA or protein to be investigated (hereinafter, referred to as a sample) are complementarily bound (hybridized). Therefore, first, a biochip such as a DNA chip or a protein chip is formed. The biochip has a plurality of spots formed on the surface of a glass substrate on which probes having different arrangements are fixed. One sample is labeled with a fluorescent substance. Then, the biochip and the sample are placed in a reaction vessel and hybridization is performed. This causes probes and samples having complementary sequences to bind on specific spots on the biochip.
[0023]
In the biochip reading step, the biochip subjected to the hybridization step is mounted on the biochip reader 5 as shown in FIG. Next, the semiconductor laser 10 is started. The laser beam 11 emitted from the semiconductor laser 10 is a Gaussian beam whose light intensity has a Gaussian distribution as shown in FIG. That is, the intensity at the optical axis is the strongest, and the intensity decreases as the distance from the optical axis increases. If the laser light 11 is directly irradiated on the biochip 1, the intensity of the laser light irradiated on the peripheral part of the biochip 1 becomes weak, and the fluorescent substance existing in the spot 2 on the peripheral part cannot be excited. Then, the laser beam 11 emitted from the semiconductor laser 10 is made incident on the beam shaper 12. Inside the beam shaper 12, when the laser light passes through an aspheric lens or a diffractive optical element, the intensity of the laser light is made uniform. Thereby, the laser light is converted from the Gaussian light shown in FIG. 2A to the top hat light shown in FIG. That is, the intensity becomes constant within a predetermined distance from the optical axis, and the semiconductor laser 10 can be used as a surface light source. Then, the biochip 1 is irradiated with the laser light 13 output from the beam shaper 12. Thereby, each part of the biochip 1 is irradiated with the laser light of the same intensity. Therefore, the fluorescent substance present in each spot 2 of the biochip 1 can be excited reliably.
[0024]
The fluorescent substance excited by the laser light generates fluorescent light 21. Since the fluorescent light 21 is scattered from the fluorescent material, if the fluorescent light 21 is imaged as it is with a CCD or the like, it cannot be specified in which spot 2 the fluorescent material exists. Therefore, the capillary plate 22 is arranged downstream of the biochip 1. Thereby, as shown in FIG. 3, only the fluorescent light 21 a vertically emitted from the biochip 1 among the scattered fluorescent light 21 passes through the fine holes 22 a of the capillary plate 22. Then, by imaging the fluorescence 21 that has passed through the capillary plate 22, it is possible to specify which spot 2 on the biochip 1 contains the fluorescent substance.
[0025]
However, the laser light 11 transmitted through the biochip 1 also passes through the fine holes of the capillary plate 22 together with the fluorescent light 21. Since the intensity of the laser light 11 is much higher than the intensity of the fluorescent light 21, it is impossible to determine the reception of the fluorescent light in the result of imaging by a CCD or the like. Therefore, as shown in FIG. 1, the light transmitted through the capillary plate 22 is made incident on the interference filter 24. The interference filter 24 transmits only the fluorescent light 21 out of the light transmitted through the capillary plate 22 and blocks the laser light 11.
[0026]
Then, the fluorescence transmitted through the interference filter 24 is received by the electronically cooled CCD 20. The electronically cooled CCD 20 does not output dark current because the CCD is cooled by the thermoelectric conversion device. Therefore, only the received fluorescence can be output. This makes it possible to identify spots on the biochip 1 where the fluorescent substance is present, and to check the presence or absence of hybridization at each spot on the biochip 1.
With the configuration according to the present embodiment described in detail above, the size of the biochip reader can be reduced.
[0027]
That is, by using a beam shaper, a semiconductor laser can be used as a surface light source, and it is not necessary to use a condenser lens and a galvanomirror. Thereby, the semiconductor laser can be arranged close to the biochip, and the biochip reader can be downsized. In addition, since it is not necessary to use expensive and complicated components such as a galvanometer mirror and a continuous light source, a low-cost and highly reliable biochip reader can be provided.
[0028]
Also, by using a capillary plate, it becomes possible to collimate the scattered fluorescence, and it is not necessary to use a condenser lens. This makes it possible to arrange the electronically cooled CCD near the biochip, and to reduce the size of the biochip reader. As described above, if the excitation light source and the light receiver are arranged close to the biochip, the height of the biochip reader can be reduced to about 10 cm.
[0029]
As described above, a small-sized and low-cost biochip reader can be provided. Therefore, the range of use of the biochip reader can be expanded to fields such as clinical inspection and food inspection.
[0030]
【The invention's effect】
A biochip reader that irradiates a plurality of spots on a biochip with light, receives the fluorescence generated thereby, and specifies the spot where a fluorescent substance is present, and emits a laser beam capable of exciting the fluorescent substance. A semiconductor laser for irradiation, a beam shaper for converting the laser light emitted from the semiconductor laser from Gaussian light to top hat light, a capillary plate for collimating the scattered fluorescence, and a light beam passing through the capillary plate. Among them, an interference filter that blocks the laser beam and transmits the fluorescent light, and an image sensor that receives the fluorescent light transmitted through the interference filter, has a configuration that includes a small and low-cost biochip reader. Can be provided.
[Brief description of the drawings]
FIG. 1 is a side view of a biochip reader according to an embodiment.
FIG. 2A is an intensity distribution diagram of Gaussian light, and FIG. 2B is an intensity distribution diagram of top hat light.
FIG. 3 is an explanatory diagram of a collimating action in a capillary plate.
FIG. 4 is an explanatory view of a biochip reader according to the related art.
[Explanation of symbols]
1 biochip, 2 spot, 5 biochip reader, 10 semiconductor laser, 11 laser light, 12 beam shaper, 13 laser light, 20 ... Electro-cooled CCD, 21 ... Fluorescence, 22 ... Capillary plate, 24 ... Interference filter.

Claims (3)

A biochip reader that irradiates a plurality of spots on the biochip with light, receives fluorescence generated thereby, and specifies the spot where a fluorescent substance is present,
A semiconductor laser that emits a laser beam capable of exciting the fluorescent substance,
A beam shaper that converts the laser light emitted from the semiconductor laser from Gaussian light to top hat light,
A biochip reader, comprising: A biochip reader that irradiates a plurality of spots on the biochip with light, receives fluorescence generated thereby, and specifies the spot where a fluorescent substance is present,
A capillary plate for collimating the scattered fluorescence,
Among the light that has passed through the capillary plate, an interference filter that blocks light that excites the fluorescent substance and transmits the fluorescence,
An image sensor that receives the fluorescence transmitted through the interference filter;
A biochip reader, comprising: A biochip reader that irradiates a plurality of spots on the biochip with light, receives fluorescence generated thereby, and specifies the spot where a fluorescent substance is present,
A semiconductor laser that emits a laser beam capable of exciting the fluorescent substance,
A beam shaper that converts the laser light emitted from the semiconductor laser from Gaussian light to top hat light,
A capillary plate for collimating the scattered fluorescence,
Among the light that has passed through the capillary plate, an interference filter that blocks the laser light and transmits the fluorescence,
An image sensor that receives the fluorescence transmitted through the interference filter;
A biochip reader, comprising:

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