JP2002350349A - Fluorescence detector - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a high-sensitivity and high-accuracy fluorescence detection device that can be miniaturized. SOLUTION: A plurality of photodiodes 2-1 and 2-2 are provided.
An amplifier for amplifying electric signals from the photodiodes 2-1 and 2-2;
2-2 and a plurality of filters 8 and 9 having different optical transmission spectral characteristics from the semiconductor integrated circuit substrate on which the amplifier is formed.
And a fluorescence reaction tank 6 serving as a field for a fluorescence reaction, and constitute a fluorescence detection device. The fluorescent reaction tank 6 is formed on the photodiodes 2-1 and 2-2 via the filters 8 and 9. In the amplifier, based on a difference in a ratio between a fluorescence component signal and an excitation light signal according to a difference in light transmission spectral characteristics of each of the filters, a fluorescent signal is output by removing the influence of the excitation light.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fluorescence detection device for detecting a fluorescence reaction, for example, a fluorescence detection device useful for detecting a specific gene contained in a sample.

[0002]

2. Description of the Related Art In recent years, the progress of genome decoding research has been tremendous.
The entire nucleotide sequence of the human genome will be decoded in 2003. In addition, the genomes of other organisms are being decoded worldwide. With the progress of such genomic research, the importance of gene detection has been further increased from the viewpoint of elucidation of gene functions and medical diagnosis. Conventional gene detection methods include PCR (polymerase chain reactant).
ion) method, there is a gene amplification method.
Gene detection methods using NA chips have also become widely used.

[0003] DNA is obtained by immobilizing a large number of single-stranded DNAs on a glass chip or silicon chip of about 1 cm square. As a single-stranded DNA to be fixed, DN of a pathogenic gene is used.
A and so on. A genetic test using a DNA chip is performed, for example, as follows. First, a gene to be detected is extracted from a cell (for example, a blood cell). And PC
The gene to be detected is amplified by R. During this amplification,
The amplification product is labeled with a fluorescent substance. A DNA chip is put into a solution containing a nucleic acid chain labeled with the fluorescent dye, and a hybridization reaction is performed. afterwards,
The DNA chip is washed to remove non-hybridized nucleic acid chains.

Next, the excitation light is applied to the DNA chip,
Detect fluorescence. FIG. 4 shows an example of a fluorescence detection device used for this. In this apparatus, a light source 105 such as a laser is used.
Is reflected by the beam splitter 104 and enters the objective lens 106, where it is condensed and the nucleic acid probe (single-stranded DN
This corresponds to the fixing portion 107 of (A). When a double strand is formed by hybridization, fluorescence 110 is generated by the excitation light 109 because the fluorescent substance is present on the DNA chip 108. Usually, fluorescence 110 and excitation light 109
Has a wavelength difference of about several tens of nm. Part of fluorescence 11
1 and the reflected light of the excitation light 109 return to the objective lens 106,
The light enters the beam splitter 104. Most of the reflected light of the excitation light 109 is reflected by the beam splitter 104 and goes to the light source side, and a part 111 of the fluorescence passes through the beam splitter 104 and goes to the light receiver 101 side. Part 1 of the fluorescence transmitted through the beam splitter 104
11 passes through the filter 103 for limiting the wavelength, but the reflected light of the excitation light 109 is removed. Further, a part 111 of the fluorescent light passes through the light receiving lens 102 and enters the light receiving device 101 for measuring the fluorescence intensity, where the fluorescent light is detected.

[0005]

The conventional fluorescence detection device is a large-scale and complicated device, and has a problem that, since the optical path is long, a fluorescence loss occurs during this time, and the detection sensitivity is low.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a small and highly sensitive fluorescence detection device.

[0007]

In order to achieve the above object, the present invention provides a fluorescence detecting apparatus comprising: a first photodiode and a second photodiode; an amplifier for amplifying an electric signal from each of the photodiodes; A semiconductor integrated circuit board on which the photodiodes and the amplifiers are formed, a first optical filter and a second optical filter having different light transmission spectral characteristics from each other, and a fluorescent reaction tank serving as a fluorescent reaction field; The first optical filter is disposed on a first photodiode, the second optical filter is disposed on the second photodiode, and the fluorescent reaction tank is formed on both optical filters,
In the amplifier, based on the difference in the ratio between the fluorescence signal and the excitation light signal according to the difference in the optical transmission spectral characteristics of each of the optical filters, the configuration is such that the influence of the excitation light is removed and the fluorescence signal is output. is there.

As described above, in the fluorescence detection device of the present invention,
Since the fluorescence reaction tank is provided above the photodiode for detecting fluorescence, the optical path can be shortened, and as a result,
The fluorescence detection sensitivity is improved, and the entire apparatus can be downsized. Further, in this device, based on the difference in the ratio between the fluorescence signal and the excitation light signal according to the difference in the light transmission spectral characteristics of each optical filter, the fluorescence signal is output by removing the influence of the excitation light. , High-precision fluorescence detection is possible. Further, it is preferable that one detection unit is constituted by the first photodiode and the first optical filter, and the second photodiode and the second optical filter, and the plurality of detection units be provided. With such a configuration, different inspections can be performed for each of the detection units. It is preferable that a color filter having different colors is used as the optical filter.

[0009] In the apparatus of the present invention, single-stranded DNA may be fixed to the bottom surface of the fluorescence reaction tank. In this case, it is used as a DNA chip. Further, an antibody or an antigen may be fixed on the bottom surface of the fluorescent reaction tank. Further, a gene amplification reaction such as a PCR reaction may be performed in the fluorescent reaction tank, and the increased by-product may be detected by fluorescence.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Next, an example of the fluorescence detecting device of the present invention will be described with reference to the drawings.

FIG. 1 and FIG. 3 show an example of the configuration of the fluorescence detection device of the present invention. FIG. 1 is a sectional view showing a structure of a light detecting portion of the device, and FIG. 3 is a plan view showing a light detecting portion of the device. In both figures, the same parts are denoted by the same reference numerals.

As shown in FIG. 1, in this fluorescence detecting device, two n-type impurity layers 2-2 are provided on a p-type silicon substrate 1.
1 and 2-2 are formed, and a high concentration p-type impurity layer 3 is formed thereon.
Are formed to form two photodiodes (a first photodiode and a second photodiode). A green filter 8 is formed on the two photodiodes via an interlayer film and a passivation film 4.
A (first optical filter) and a blue filter 9 (second optical filter) are arranged, and a fluorescent reaction tank 6 is formed on these via a transparent flattening film 5. The color filter is a filter using a pigment. This fluorescence reaction tank 6 contains a fluorescence reaction solution 7. In FIG. 1, reference numeral 13 denotes excitation light. In this device,
The p-type silicon substrate 1 and the high-concentration p-type impurity layer 3 are grounded, and bring the photodiode into a reverse bias state before photoelectrically converting light. Even at that time, the high-concentration p-type impurity layer 3 is set to an impurity concentration that does not completely deplete.

The semiconductor integrated circuit substrate is, for example, a MOS (metal-based) for manufacturing an IC (integrated circuit) from the silicon substrate 1.
Although it can be manufactured by an oxide film-semiconductor process, the present invention is not limited to this, and any substrate material that can form a photodiode and a transistor may be used. For example, a polycrystalline silicon integrated circuit substrate formed on a glass substrate, an amorphous silicon integrated circuit substrate, a GaAs integrated circuit substrate, or the like may be used. The fluorescent reaction tank 6 can be formed of a transparent material, for example, quartz, glass, or PMMA (polymethyl methacrylate), but is not limited thereto. The fluorescent reaction tank 6 is formed of a material having a high light transmittance and minimal fluorescence. I just want it.

As shown in FIG. 3, in this device, each of the n-type impurity layers 2-1 and 2-1 of the two photodiodes is used.
The signal from −2 is input to the amplifier circuit 11, where the signal is calculated and output from the signal output 12. In addition, the n-type impurity layers 2-1 and 2-2 are connected to the reset transistor 10 in order to set the photodiode in a reverse bias state.

Next, the operation of this device will be described. That is, first, a pulse is input to the reset pulse terminal φr14, the reset transistor 10 is turned on, and the n-type impurity layers 2-1 and 2-2 are reset to the reset power supply Vr1.
Charge with 5. This operation puts the photodiode in a reverse bias state. Next, the reset transistor 10 is turned off to detect fluorescence. This detection,
Absorption peak wavelength 497 having the highest fluorescence efficiency as excitation light
nm light, and SYBR-GreenI as a fluorescent material.
The case where (intercalator) is used will be described as an example. FIG. 2A shows an absorption spectral characteristic 21 and a fluorescent spectral characteristic 22 of SYBR-Green I (fluorescent material).
Reference numeral 23 denotes a position at 497 nm of the excitation light wavelength. As shown in the figure, the fluorescence exhibits an emission intensity distribution having a peak on a longer wavelength side than the absorption peak. The excitation light and the fluorescent light enter the photodiode through the green filter 8 or the blue filter 9. FIG. 2B shows the transmission spectral characteristic 25 of the green filter and the transmission spectral characteristic 24 of the blue filter, respectively. As shown in FIG. 2A, the difference between the spectral characteristics of the excitation light and the fluorescence and the difference in the spectral characteristics of the filter as shown in FIG.
-1 and the n-type impurity layer 2 under the blue filter 9
1 and the signal is different. The green filter 8 has a light transmission peak close to the peak wavelength of the fluorescent light, and the fluorescent light is more efficiently photoelectrically converted by the photodiode including the n-type impurity layer 2-1 than the excitation light. On the other hand, in the blue filter 9, the light transmission peak is largely shifted from the peak wavelength of the fluorescence, and the excitation light is more efficiently photoelectrically converted by the photodiode including the n-type impurity layer 2-2 than the fluorescence. From the spectral characteristics of the excitation light and the fluorescence, the ratio of the light transmission efficiency of each of the green filter 8 and the blue filter 9 can be known in advance. By calculating from the ratio of the light transmission efficiency and the signals of the n-type impurity layers 2-1 and 2-2, the fluorescence intensity can be measured. In this case, since the green filter 8 transmits the fluorescence efficiently, the measurement can be performed with high sensitivity. The spectral characteristics of the filters are not limited to those shown in FIG. 2B, and it is sufficient that two or more filters having different transmission peak wavelengths and distribution shapes are provided. For example, for one of the filters, the peak wavelength of the transmitted light is set close to the peak wavelength of the fluorescent light so that the fluorescent light is transmitted efficiently and the transmission of the excitation light is reduced. At that time, the other filter is set to a distribution characteristic including more excitation light components. It is desirable that the difference between the peak wavelengths of the two filters be equal to or longer than the wavelengths of the excitation light and the fluorescence. The half width of the distribution of each filter is preferably small, and is preferably equal to or less than the wavelength difference between the excitation light and the fluorescence. The filters having different light transmission spectral characteristics are not limited to the pigment color filters, and may be, for example, interference filters, dye filters, colored glass, and the like. An example of the calculation is shown based on FIGS. 2 (a) and 2 (b). For example, the intensity of light passing through the filter having the spectral characteristic 25 when fluorescence is caused by excitation light of 497 nm or less is I1, the ratio between the excitation light component and the fluorescence component is 1: 2,
The intensity of light transmitted through the filter having the spectral characteristic 24 is I2
Where the ratio between the excitation light component and the fluorescence component is 2: 1. In this case, the fluorescence intensity is obtained by the following equation. Fluorescence intensity = 2/3 (I1−I2 / 2) In order to accurately obtain the fluorescence intensity in this calculation, it is desirable that the fluorescence component ratio in the spectral characteristic 24 be small.

The operation of detecting fluorescence of a gene using this apparatus is performed, for example, as follows. First, a single-stranded DNA complementary to the gene to be detected is immobilized in a fluorescent reaction tank. The fixing method is not particularly limited, and a general method can be applied. DNA (oligonucleotide) may be directly synthesized on the bottom of the fluorescent reaction tank,
It may be coated with a material to which NA easily binds, and the cloned DNA or PCR product may be immobilized thereon. Then, the sample solution is put into the fluorescence reaction tank. in this case,
When the target DNA itself is fluorescently labeled with Cy3, Cy5, or the like, the fluorescent reaction tank may be washed after the sample solution is charged. Even if the target DNA is not fluorescently labeled, a fluorescent intercalator such as SYBR-Green I may be placed in the sample solution or the fluorescent reaction tank. Then, excitation light is incident from the side of the fluorescent reaction tank. When the single-stranded DNA fixed to the bottom of the fluorescence reaction tank and the target DNA are hybridized to form a double-stranded DNA, the fluorescence is radially emitted by the fluorescent intercalator or the fluorescent label of the target DNA that has entered into the double-stranded DNA. appear. SYBR-GreenI
Is used, an SHG laser having a wavelength of 497 nm may be applied. A part of the generated fluorescence is detected by a photodiode, converted into an electric signal by photoelectric exchange, the electronic signal is input to an amplifier circuit, and thereafter, the signal is output after being processed as described above.

As described above, in this apparatus, if a plurality of the detection units are provided and a plurality of types of single-stranded DNAs are fixed in accordance with the detection units, a plurality of tests can be performed by one fluorescence detection.

In this example, a single-stranded DN is placed on the bottom of the fluorescent reaction tank.
Although an example in which A is immobilized has been described, an antibody or an antigen may be immobilized. In that case, a fluorescently labeled antigen or antibody sample solution is placed in a fluorescent reaction tank. Thereafter, the sample solution is removed, and the sample is irradiated with fluorescent excitation light.
Here, when an antigen-antibody complex is formed, fluorescence is generated, which can be detected by a photodiode. Also, an enzyme immunoassay (ELISA) can be applied. In this case, the first antibody is immobilized on the bottom surface of the fluorescent reaction tank, and a sample solution containing the antigen is supplied thereto. Then, an antigen-antibody complex is formed. Further, the enzyme-labeled second antibody is supplied to form a sandwich-structured first antibody-antigen-second antibody complex. Here, a substrate that is converted into a fluorescent substance by an enzymatic reaction is added to cause an enzymatic reaction. Then, irradiation with excitation light is performed, and the generated fluorescence of the fluorescent substance may be detected by the photodiode. In addition,
In the fluorescence detection by the antigen-antibody reaction, when a plurality of the detection units are formed, if a plurality of antibodies or antigens are immobilized accordingly, a plurality of antigens or antibodies can be detected simultaneously.

Further, in this apparatus, a gene amplification method such as a PCR method may be performed. In this case, a sample solution containing the target DNA and the target DNA
And a buffer solution containing a heat-stable DNA polymerase (such as Taq DNA polymerase), dNTPs, and a fluorescent intercalator. And
The target DNA is amplified by repeating a series of steps of thermal denaturation of the target DNA, annealing of the primer, and extension reaction by DNA polymerase. Since the fluorescent intercalator binds to the obtained amplification product, it may be irradiated with excitation light and the resulting fluorescence may be detected by a photodiode.

[0020]

As described above, the fluorescence detector of the present invention can be downsized and the optical path can be shortened, so that the detection sensitivity is high and the influence of the excitation light can be eliminated. Therefore, with the fluorescence detection device of the present invention, for example, gene detection by fluorescence and the like can be performed with high accuracy and high sensitivity.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view showing a structure of a light detection portion of an example of a fluorescence detection device of the present invention.

FIG. 2A is a graph showing the spectral characteristics of light absorption and fluorescence of a fluorescent material, and FIG. 2B is a graph showing the light transmission spectral characteristics of a filter.

FIG. 3 is a plan view showing the configuration of the device of the above example.

FIG. 4 is a configuration diagram of a conventional fluorescence detection device.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 p-type silicon substrate 2-1, 2-2 n-type impurity layer 3 high-concentration p-type impurity layer 4 interlayer insulating film and passivation film 5 planarization film 6 fluorescent reaction tank 7 fluorescent reaction solution 8 green filter 9 blue filter 10 reset Transistor 11 Amplification circuit 12 Signal output 13 Excitation light 14 Reset pulse terminal 15 Reset power supply 21 Absorption spectral characteristic 22 Fluorescence spectral characteristic 23 Absorption peak wavelength 24 Transmission spectral characteristic of blue filter 25 Transmission spectral characteristic of green filter 101 Optical receiver 102 Optical receiver lens 103 Filter for limiting wavelength 104 Beam splitter 105 Light source 106 Objective lens 107 Nucleic acid probe fixing portion 108 DNA chip 109 Excitation light 110 Fluorescence 111 Part of fluorescence transmitted through beam splitter

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 37/00 102 G01N 37/00 102

Claims (5)

[Claims] A first photodiode and a second photodiode, an amplifier for amplifying an electric signal from each of the photodiodes, a semiconductor integrated circuit substrate on which each of the photodiodes and the amplifier are formed, and a light transmission spectrometer. The first optical filter and the second optical filter having different characteristics from each other.
An optical filter; and a fluorescent reaction tank serving as a field for a fluorescent reaction, wherein the first optical filter is disposed on the first photodiode, and the second optical filter is disposed on the second photodiode. The fluorescent reaction tank is formed on the two optical filters, and in the amplifier, based on a difference in ratio between a fluorescence signal and an excitation light signal according to a difference in light transmission spectral characteristics of each of the optical filters,
A fluorescence detector that removes the influence of excitation light and outputs a fluorescence signal. 2. The apparatus according to claim 1, wherein one detection unit is constituted by the first photodiode and the first optical filter, and the second photodiode and the second optical filter, and has a plurality of the detection units. 3. The apparatus according to claim 1, wherein the first optical filter and the second optical filter are color filters having different colors. 4. A single-stranded DNA is provided on a bottom surface of the fluorescence reaction tank.
Device according to any of the preceding claims, wherein is fixed. 5. The apparatus according to claim 1, wherein an antibody or an antigen is fixed on a bottom surface of the fluorescence reaction tank.