JP4452863B2 - Biochip and biochip reader - Google Patents

Description

  The present invention relates to a biochip such as a DNA chip or a protein chip and a biochip reader. In particular, the present invention relates to a biochip and a biochip reader with improved detection sensitivity.

  A biochip is obtained by pasting various types of DNA fragments (probes) and synthetic oligonucleotides on a substrate such as glass. The biochip includes a DNA chip, a protein chip, a DNA array, a DNA microarray, and the like.

  The reading of the biochip is performed by irradiating the sample with excitation light such as a laser and detecting light generated by fluorescence generated from the sample or detecting light generated by chemiluminescence generated from the sample.

  FIG. 10 is a diagram showing a configuration of a conventional biochip reader. Sample excitation light from the sample excitation light source 1 is reflected by the dichroic mirror 2. The dichroic mirror 2 is selected so that it does not transmit the wavelength of the sample excitation light but transmits the wavelength of the fluorescence generated from the sample. The sample excitation light reflected by the dichroic mirror 2 is condensed at the sample position of the biochip 4 by the objective lens 3. Part of the fluorescence generated from the sample passes through the objective lens 3, further passes through the dichroic mirror 2, and reaches the detector 5 to be detected. In the case of chemiluminescence detection, the sample excitation light is not irradiated, but the process until the light generated from the sample reaches the detector 5 is the same.

  In FIG. 10, the solid line indicates the optical path of the sample excitation light. The dotted line indicates the optical path of the fluorescence or chemiluminescence light generated from the sample 13.

  FIG. 11 is a diagram showing a configuration of a conventional biochip. The biochip 4 includes a substrate 11 and a sample 13 is disposed on one surface of the substrate 11. The sample excitation light is irradiated from the surface where the sample 13 is disposed. The substrate 11 can be formed of glass or a plus chip so as to transmit light by fluorescence or chemiluminescence. Here, when the sample 13 is irradiated with sample excitation light, fluorescence is generated from the sample 13. A part of the fluorescence returns to the objective lens 3 as described above and reaches the detector 5 and is detected. However, the other part of the fluorescence proceeds in a direction different from the direction of the objective lens and is not detected by the detector.

  Therefore, it has been difficult to improve detection sensitivity with conventional biochips.

  Regarding the configuration of a conventional biochip reader using a biochip, for example, Patent Document 1 (FIG. 1, etc.), Patent Document 2 (FIGS. 1, 24, etc.), and Patent Document 3 (FIGS. 2, 6, etc.) ) And the like.

JP 2001-194309 A JP 2001-31690 A JP 2004-138420 A

  In this way, the conventional biochip only detects the light emitted in the direction of the detector and cannot capture fluorescence or chemiluminescence that passes through the substrate medium as detection target light, thus improving detection sensitivity. Difficult to do.

  Therefore, there is a need for a biochip capable of improving detection sensitivity by increasing the detection target light.

  The biochip according to the present invention includes a sample on one side, and the sample is provided with a reflecting member at a position corresponding to the sample on the side opposite to the light detector by fluorescence or chemiluminescence generated by the sample. Includes substrate.

  A biochip reader according to the present invention includes a light source for sample excitation light, a dichroic mirror that reflects the sample excitation light and transmits fluorescence from the sample, an objective lens that collects the sample excitation light at the position of the sample, A biochip including a sample on one surface, the substrate being opposite to the light source of the sample excitation light with respect to the sample and having a reflecting member at a position corresponding to the sample, and a detector for detecting fluorescence from the sample With.

  Therefore, since the fluorescent or chemiluminescent light generated by the sample is reflected toward the detector by the reflecting member, the sensitivity of the detector can be improved.

  According to one embodiment of the present invention, the reflecting member is configured to reflect the fluorescent or chemiluminescent light generated by the sample to the position of the sample.

  Therefore, the fluorescence or chemiluminescence light generated by the sample is reflected by the reflecting member toward the position of the sample and then directed to the detector, so that the sensitivity of the detector can be improved.

  According to one embodiment of the present invention, m is an integer equal to or greater than 1, and the reflective diffraction grating is configured to maximize the diffraction efficiency of mth-order reflected diffracted light.

  Therefore, detection is possible because the ratio of the light reflected by the reflective diffraction grating to the detector is the maximum with respect to the light incident on the reflective diffraction grating, which is light generated by fluorescence or chemiluminescence generated by the sample. The sensitivity of the vessel can be further improved.

  According to one embodiment of the present invention, after the light generated by the fluorescence or chemiluminescence generated by the sample reflected by the reflecting member is reflected at the position of the sample, the objective lens whose focusing position is the position of the sample is provided. It is configured to pass through to the detector.

  The fluorescent or chemiluminescent light generated by the sample is reflected by the reflecting member toward the sample position, travels toward the objective lens having the sample position as the condensing position, and is made substantially parallel light by the objective lens. Therefore, since it goes to a detector as parallel light, it is easy to inject into the detection surface of a detector, and the sensitivity of a detector can further be improved.

  According to one embodiment of the present invention, the reflecting member is formed on the surface of the substrate opposite to the surface provided with the sample.

  Therefore, it is easy to manufacture the reflecting member by imprinting when the substrate is a glass material and by injection molding or cutting when the substrate is a plastic material.

  According to one embodiment of the present invention, the substrate is made of a glass material or a plastic material.

  Therefore, it is convenient for the substrate to transmit light by fluorescence or chemiluminescence.

  According to one embodiment of the present invention, the surface of the reflecting member is coated with a metal film.

  Therefore, a high reflectance can be obtained.

  According to one embodiment of the present invention, the surface of the reflecting member is coated with a dielectric multilayer film.

  A highly efficient reflective film can be obtained by alternately laminating a dielectric film having a high refractive index and a dielectric film having a low refractive index. By optimizing the film thickness of each layer, it is possible to improve the reflection characteristics of a desired wavelength.

  According to one embodiment of the present invention, the sample is placed in a well provided on one side of the substrate.

  Therefore, it is convenient because the position of the sample is determined by the position of the well.

  According to one embodiment of the present invention, the well has a cone or truncated cone shape that narrows toward the bottom surface.

  Therefore, the sample is conveniently arranged in a narrow area at the bottom of the well, and the position of the sample is specified in the narrow area.

  According to one embodiment of the present invention, when the sample excitation light is configured to be collected on the bottom of the well by the objective lens, the inclination of the side surface of the well cone or the truncated cone becomes the collection angle. Therefore, it is determined.

  According to one embodiment of the present invention, the reflective member is a reflective diffraction grating.

  Therefore, the reflecting member is compact and has high reflection efficiency. In particular, the thickness in the optical axis direction can be made extremely small.

  According to one embodiment of the present invention, the reflective member includes a Fresnel reflective surface.

  Therefore, the reflecting member is compact.

  According to one embodiment of the present invention, the reflective member includes a reflective spherical surface or a reflective aspheric surface.

  Therefore, the reflection member has high reflection efficiency.

  FIG. 1 is a diagram illustrating a configuration of a biochip reader according to an embodiment of the present invention. Sample excitation light from the sample excitation light source 1 is reflected by the dichroic mirror 2. The dichroic mirror 2 is selected so that it does not transmit the wavelength of the sample excitation light but transmits the wavelength of the fluorescence generated from the sample. The sample excitation light reflected by the dichroic mirror 2 is condensed at the sample position of the biochip 4 by the objective lens 3. Part of the fluorescence generated from the sample passes through the objective lens 3, further passes through the dichroic mirror 2, and reaches the detector 5 to be detected. In the case of chemiluminescence detection, the sample excitation light is not irradiated, but the process until the light generated from the sample reaches the detector 5 is the same.

  In FIG. 1, the solid line indicates the optical path of the sample excitation light. The dotted line indicates the optical path of the fluorescence or chemiluminescence light generated from the sample 13.

  FIG. 2 is a diagram illustrating a configuration of a biochip according to an embodiment of the present invention. The biochip 4 includes a substrate 11 and a well 12 is formed on one surface of the substrate 11. The sample excitation light is irradiated from the surface where the well 12 is formed. The substrate 11 can be formed of glass or a plus chip so as to transmit light by fluorescence or chemiluminescence. A sample is placed in the well 12. In the present embodiment, a reflective diffraction grating 14 is formed at a position corresponding to the well 12 on the surface of the substrate 11 opposite to the surface on which the well 12 is formed. That is, the reflective diffraction grating 14 is formed for each well 12. A reflective film 15 is coated on the grating surface of the reflective diffraction grating 14. Here, as shown in FIG. 2, the shape of the well 12 is preferably a conical shape or a truncated cone shape that narrows toward the bottom. A sample 13 is disposed at the bottom of the well 12. The shape of the well 12 will be further described later.

  FIG. 3 is an optical path diagram showing the concept of design of the reflective diffraction grating of the present invention. Collimated light (sample excitation light) incident from the light source side is condensed at the position of the sample by the objective lens 3. Part of the fluorescence generated from the sample 13 is directed toward the objective lens 3, converted into collimated light by the objective lens 3, and returns to the detector. The other part of the fluorescence generated from the sample 13 is directed toward the reflective diffraction grating 14. The optical path of chemiluminescent light from the sample 13 is the same as the optical path of fluorescence. The reflective diffraction grating 14 is formed concentrically. The reflective diffraction grating 14 is designed to reflect fluorescent or chemiluminescent light at the position of the sample 13, that is, at the bottom of the well 12. The fluorescent or chemiluminescent light transmitted through the sample 13 is directed in the direction of the objective lens 3, converted into collimated light by the objective lens 3, and returns to the detector. In this way, fluorescence or chemiluminescence light that has not been directed to the detector in the conventional biochip is directed to the detector, and detection sensitivity is improved.

  In FIG. 3, the solid line indicates the optical path of the sample excitation light. A dotted line indicates an optical path of light including fluorescence or chemiluminescence light generated from the sample 13 and reflected by the reflection diffraction grating 14 at the position of the sample 13. In FIG. 3, θ represents the light collection angle (full angle) of the objective lens.

  In general, the grating interval of the diffraction grating determines the diffraction angle. In the present embodiment, the lattice spacing is determined so that the light beam returns to the bottom of the well 12. The grating depth also determines how much energy is distributed to which diffraction order. In the present embodiment, since the first-order diffracted light is used, the grating depth is determined so that the energy of the first-order diffracted light is maximized. In the case of a blazed reflective diffraction grating, the grating depth is as follows so that the energy of the first-order diffracted light is maximized.

λ / (2n)

Here, is the wavelength of light, and n is the refractive index of the substrate. Further, the detailed shape of the grating is obtained by obtaining an optical path difference function from the optical path and changing it to the shape function.

  FIG. 4 shows the shape of the reflective diffraction grating 14 used in this embodiment. As described above, the reflective diffraction grating 14 is formed concentrically. The horizontal axis indicates the distance from the center of the concentric circle. The vertical axis represents the lattice depth. The grating depth is 200 nanometers. Here, glass is used for the substrate, and the refractive index is 1.516. The wavelength of light is optimized for two wavelengths of 570 nanometers and 665 nanometers.

  Although the first-order diffracted light is used in this embodiment, the reflective diffraction grating 14 can be similarly designed even when higher-order diffracted light is used.

  The reflective diffraction grating 14 is manufactured by imprinting when the substrate is a glass material, or by injection molding or cutting when the substrate is a plastic material.

The grating surface of the reflective diffraction grating 14 is coated with a metal or dielectric multilayer film. The coating may be performed on the entire surface of the substrate or only on the position of the reflective diffraction grating 14. In the case of a metal coat, one layer is coated with gold or aluminum. The physical film thickness of gold may be several hundred nanometers or more. In this embodiment, it is 300 nanometers. The reflection characteristics by the gold coat are shown in FIG. The horizontal axis represents wavelength and the vertical axis represents reflectance. In the case of a dielectric multilayer coating, a highly efficient reflective film can be obtained by alternately laminating a dielectric film having a high refractive index and a dielectric film having a low refractive index. In the case of this embodiment, the physical film thickness is 2.2 μm, and SiO _{2 is} used as the low refractive index material and Ti ₃ O ₅ is used as the high refractive index material to form a 21-layer structure. The film thickness of each layer is optimized so as to enhance the reflection characteristics at a desired wavelength. The reflection characteristics by the dielectric multilayer coating of this embodiment are shown in FIG. The horizontal axis represents wavelength and the vertical axis represents reflectance. The reflectance peaks in the vicinity of two wavelengths of 570 nanometers and 665 nanometers.

FIG. 7 shows an embodiment in which a Fresnel reflecting surface 16 is used instead of the reflective diffraction grating 14. The Fresnel reflection surface is obtained by dividing a surface represented by the following expression into annular zones (annular zones) having a radius of h, and arranging these zones on a flat substrate.

Here, c is a curvature, R is a radius of curvature, k is a conic constant, and Ai is an aspherical coefficient. In the present embodiment, k is zero. Other numerical values are as shown below. These numerical values are determined so that the light beam returns to the bottom of the well 12.

FIG. 8 shows an embodiment in which a reflective spherical surface 17 is used instead of the reflective diffraction grating 14. The specification of the reflective spherical surface 17 is shown in the following table.

  FIG. 9 shows the effective diameter 2a of the reflecting spherical surface and the sag amount b with respect to the effective diameter 2a. These numerical values are determined so that the light beam returns to the bottom of the well 12. Instead of the reflecting sphere, a reflecting aspherical surface such as a paraboloid, an ellipsoid, a hyperboloid, and a quartic surface can be used.

  The Fresnel reflection surface 16 and the reflection spherical surface 17 are also coated with a metal or dielectric multilayer film as in the case of using the reflection type diffraction grating 14.

  Here, the shape of the well 12 will be described. In the present invention, the shape of the reflecting member 14, 16, or 17 is designed so that the fluorescent or chemiluminescent light generated from the sample 13 disposed at the bottom of the well 12 is reflected toward the bottom of the well 12. Is done. Therefore, in the present invention, it is preferable that the sample 13 is concentrated in a narrow region at the bottom of the well 12. Therefore, the shape of the well 12 is, for example, that the cross section is triangular or trapezoidal, that is, conical or frustoconical, as shown in FIG. The one is more advantageous. Moreover, it is preferable to determine the inclination of the side surface of the cone or the truncated cone according to the light collection angle.

  Here, the converging angle of the objective lens 3 is 11 degrees half angle, 22 degrees full angle, the distance between the objective lens 3 and the objective lens side of the substrate 11 is 4.3 mm, the thickness of the substrate 11 is 1.0 mm, and the well 12 The radius on the surface of the substrate 11 is 0.3 mm, the depth of the well 12 is 0.4 mm, the radius of the bottom surface of the well 12 is 0.16 mm, and the diameter of the outer circumference of the reflecting member 14, 16 or 17 is 0.3 mm. is there.

It is a figure which shows the structure of the biochip reader by one Embodiment of this invention. It is a figure which shows the structure of the biochip by one Embodiment of this invention. It is an optical path diagram which shows the idea of the design of the reflection type diffraction grating of this invention. It is a figure which shows the shape of the reflection type diffraction grating used by this embodiment. It is a figure which shows the reflective characteristic by a gold coat. It is a figure which shows the reflective characteristic by the dielectric multilayer coating of this embodiment. It is an optical path figure which shows the idea of the design of the Fresnel reflective surface of this invention. It is an optical path figure which shows the view of the design of the reflective spherical surface of this invention. It is a figure which shows the shape of a reflective spherical surface. It is a figure which shows the structure of the conventional biochip reader. It is a figure which shows the structure of the conventional biochip.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Dichroic mirror 3 Objective lens 4 Biochip 5 Detector 11 Substrate 12 Well 13 Sample 14 Reflective diffraction grating 15 Reflective film 16 Fresnel reflective surface 17 Reflective spherical surface

Claims (16)

Includes a sample on one side, concerning said sample, a side opposite to the light detector by fluorescence or chemiluminescence sample occurs, look including a substrate a reflective member at positions corresponding to the sample, the reflection A biochip whose member is a reflective diffraction grating . m as an integer of 1 or more, the reflection type diffraction grating, biochip according to claim 1 that is configured to maximize the diffraction efficiency of the m-order reflected diffracted light. Includes a sample on one side, concerning said sample, a side opposite to the light detector by fluorescence or chemiluminescence sample occurs, look including a substrate a reflective member at positions corresponding to the sample, the reflection A biochip whose member includes a Fresnel reflective surface . The biochip according to any one of claims 1 to 3, wherein the reflecting member is configured to reflect light generated by the sample by fluorescence or chemiluminescence to a position of the sample. The reflected by the reflecting member, light from a fluorescent or chemiluminescent sample occurs, after being reflected in the position of the sample, as reaching the detector through the objective lens to the position of the sample with the focusing position The biochip according to any one of claims 1 to 4, wherein the biochip is configured as follows. The biochip according to any one of claims 1 to 5 , wherein the reflecting member is formed on a surface of the substrate opposite to the surface including the sample. The biochip according to any one of claims 1 to 6 , wherein the substrate is made of a glass material or a plastic material. The biochip according to any one of the 7 surface of the reflecting member claims 1 coated with a metal film. The biochip according to any one of claims 1 to 7 , wherein a surface of the reflecting member is coated with a dielectric multilayer film. The biochip according to any one of claims 1 to 9 , wherein the sample is arranged in a well provided on the one surface of the substrate. The biochip according to claim 10 , wherein the well has a cone shape or a truncated cone shape that becomes thinner toward the bottom surface. A sample excitation light source, a dichroic mirror that reflects the sample excitation light and transmits fluorescence from the sample, an objective lens that collects the sample excitation light at the position of the sample, and a sample on one surface, A biochip including a substrate provided with a reflecting member at a position corresponding to the sample on the opposite side of the sample excitation light source with respect to the sample, and a detector for detecting fluorescence from the sample , the reflecting member reflecting Biochip reader which is a type diffraction grating . 13. The biochip reader according to claim 12 , wherein m is an integer equal to or greater than 1, and the reflective diffraction grating is configured to maximize the diffraction efficiency of mth-order reflected diffracted light. A sample excitation light source, a dichroic mirror that reflects the sample excitation light and transmits fluorescence from the sample, an objective lens that collects the sample excitation light at the position of the sample, and a sample on one surface, A biochip including a substrate provided with a reflecting member at a position corresponding to the sample on the opposite side of the sample excitation light source with respect to the sample, and a detector for detecting fluorescence from the sample , the reflecting member being Fresnel A biochip reader including a reflective surface . The biochip reader according to any one of claims 12 to 14, wherein the reflecting member is configured to reflect fluorescence from the sample to a position of the sample. Fluorescence reflected by the reflecting member after being reflected in the position of the sample, biochip reading as claimed in any one of claims 12 to 15 configured to reaching the detector through the objective lens apparatus.

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