JP4701781B2 - Biopolymer analyzer and biopolymer analysis method - Google Patents

Description

The present invention relates to a biopolymer analyzer and a biopolymer analysis method using a biopolymer analysis chip such as a DNA microarray.

  In recent years, we have been analyzing gene expression of various species. Gene expression analysis is to identify a gene expressed in a cell, and specifically, to identify mRNA transcribed from DNA encoding the gene.

  A DNA microarray and its reader have been developed for gene expression analysis. A DNA microarray is obtained by aligning and fixing cDNA having a known base sequence serving as a probe in a matrix on a solid support such as a slide glass (see, for example, Patent Document 1). Here, as a cDNA having a known base sequence, a cDNA having the same base sequence as that of a known mRNA in a specimen or a part thereof is used. Gene expression analysis using a DNA microarray and its reader is performed as follows.

  First, a DNA microarray is prepared in which a plurality of types of cDNAs with known sequences (hereinafter referred to as probe DNA) are aligned and fixed on a solid support such as a slide glass. Next, mRNA is extracted from the specimen, and cDNA labeled with a fluorescent substance (hereinafter referred to as sample DNA) is synthesized using reverse transcriptase. Next, when the sample DNA is labeled with a fluorescent substance and then added to the DNA microarray, the sample DNA is immobilized on the DNA microarray by hybridizing with the complementary probe DNA. When excited, the fluorescent substance that labels the sample DNA emits fluorescence from the position of the probe DNA to which the sample DNA is bound.

Next, the DNA microarray is set in a reader and analyzed by the reader. The reader moves the irradiation point of excitation light two-dimensionally with respect to the DNA microarray, and simultaneously scans the DNA microarray two-dimensionally with a condenser lens and a photomultiplier. The fluorescence emitted from the fluorescent material excited by the excitation light is collected by a condensing lens, and the fluorescence intensity is measured by a photomultiplier to measure the fluorescence intensity distribution in the surface of the DNA microarray. The fluorescence intensity distribution on the microarray is output as a two-dimensional image. In the output image, the portion having high fluorescence intensity indicates that sample DNA having a base sequence complementary to the base sequence of the probe DNA is included. Therefore, it is possible to identify which mRNA of the known sequence is expressed in the specimen depending on which part of the two-dimensional image has high fluorescence intensity.
JP 2000-1312237 A

  By the way, a biopolymer analysis chip has been developed in which molecules such as probe DNA are spotted on a light receiving surface of a solid-state imaging device in which a plurality of photoelectric conversion elements are arranged in a two-dimensional array. In such a biopolymer analysis chip, light from a labeling substance such as a fluorescent substance or a luminescent substance that labels a biopolymer such as sample DNA attached to a spot is measured by each photoelectric conversion element. Spots are scattered on the light-receiving surface of the solid-state imaging device, and light emitted from the labeling substance is incident on the light-receiving surface of the solid-state imaging device with almost no attenuation. Therefore, there is an advantage that even a small amount of light can be measured. is there.

  However, it was not possible to know where the spot was spotted before hybridization. For this reason, the presence / absence of fluorescence must be determined from data captured by all the photoelectric conversion elements, and there is a problem that the determination accuracy of the presence / absence of fluorescence itself is lowered when there is a lot of data including noise.

An object of the present invention is to provide an accurate biopolymer analyzer and a biopolymer analysis method .

In order to solve the above problems, the invention described in claim 1 is characterized in that a specific biopolymer is formed on a light receiving surface of an imaging device including a plurality of photoelectric conversion elements arranged in a two-dimensional array on a transparent substrate. A biopolymer analysis chip in which a solution of a probe to be bound is dropped to form a spot; a spot detection light source that irradiates the biopolymer analysis chip with spot detection light from the transparent substrate side; and the spot of the spot detection light Detecting means for detecting the presence or absence of the spot based on reflected light intensity on the surface , each spot is formed on a light receiving surface of a plurality of the photoelectric conversion elements, and the detecting means corresponds to each spot If a plurality of photoelectric conversion elements is determined that there is fluorescence of the photoelectric conversion element to have more than one average value of fluorescence data that fluorescent data of the spot And butterflies.

According to a fourth aspect of the present invention, a solution of a probe that binds to a specific biopolymer is dropped on a light-receiving surface of an imaging device including a plurality of photoelectric conversion elements arranged in a two-dimensional array on a transparent substrate. The biopolymer analysis chip having spots formed on the light receiving surfaces of the plurality of photoelectric conversion elements is irradiated with spot detection light from the transparent substrate side, and the reflected light intensity of the spot detection light on the surface of the spot is increased. When there are a plurality of photoelectric conversion elements determined to have fluorescence among the plurality of photoelectric conversion elements corresponding to each spot when detecting the presence / absence of the spot based on the average value of the fluorescence data Fluorescence data.

  It is preferable to include a spotter that is driven by the detection means and selectively drops a liquid that emits a fluorescent color and a liquid that does not emit a fluorescent color on the light receiving surface of the imaging device.

  The spotter preferably performs the dropping of the liquid that does not emit the fluorescent color after the dropping of the liquid that emits the fluorescent color.

  Here, as the liquid that does not emit fluorescent color, pure water or a sample close to pure water, for example, the same solvent as the solution of the probe can be used.

  According to the present invention, the accuracy of biopolymer analysis can be improved.

  The best mode for carrying out the present invention will be described below with reference to the drawings. However, although various technically preferable limitations for implementing the present invention are given to the embodiments described below, the scope of the invention is not limited to the following embodiments and illustrated examples.

[First Embodiment]
[1] Overall Configuration of Biopolymer Analysis Chip FIG. 1 is a schematic plan view of a biopolymer analysis chip 10 according to the first embodiment to which the present invention is applied, and FIG. 2 is a cross-sectional view II in FIG. FIG.

  The biopolymer analysis chip 10 is dotted on the transparent substrate 17, the solid-state imaging device 30 in which the photoelectric conversion elements are arranged in a two-dimensional array on the transparent substrate 17, and the light-receiving surface of the solid-state imaging device 30. Spots 60, 60,...

[2] Solid-State Imaging Device The solid-state imaging device 30 will be described in detail with reference to FIGS. Here, FIG. 3 is a plan view showing an electrode structure of a photoelectric conversion element that is a pixel of the solid-state imaging device 30, and FIG. 4 is an enlarged view showing the photoelectric conversion element of the solid-state imaging device 30 in FIG. .

  The solid-state imaging device 30 is provided on the transparent substrate 17. The transparent substrate 17 has a property of transmitting light (hereinafter referred to as “light transmitting property”) and has an insulating property, and is a glass substrate such as quartz glass or a plastic substrate such as polycarbonate or PMMA.

  In this solid-state imaging device 30, a double gate type field effect transistor (hereinafter referred to as a double gate transistor) 20 is used as a photoelectric conversion element, and a plurality of double gate transistors 20, 20,. The double gate transistors 20, 20,... Are collectively covered with a protective insulating film 32. The double gate transistors 20, 20,.

  Each of the double gate transistors 20, 20,... Includes a semiconductor film 23 that is a light receiving portion, a bottom gate electrode 21 formed under the semiconductor film 23 with the bottom gate insulating film 22 interposed therebetween, and a top gate insulating film 29. The top gate electrode 31 formed on the semiconductor film 23, the impurity semiconductor film 25 formed so as to overlap part of the semiconductor film 23, and the impurity semiconductor formed so as to overlap another part of the semiconductor film 23 A film 26, a source electrode 27 overlaid on the impurity semiconductor film 25, and a drain electrode 28 overlaid on the impurity semiconductor film 25 are converted into an electric signal having a level according to the amount of light received by the semiconductor film 23. is there.

  The bottom gate electrode 21 is formed on the transparent substrate 17 for each double gate transistor 20. Further, a plurality of bottom gate lines 41, 41,... Extending in the horizontal direction are formed on the transparent substrate 17, and the double gate transistors 20, 20,. Each bottom gate electrode 21 is formed integrally with a common bottom gate line 41. The bottom gate electrode 21 and the bottom gate line 41 have conductivity and light shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.

The bottom gate electrode 21 and the bottom gate lines 41, 41,... Of the double gate transistors 20, 20,. That is, the bottom gate insulating film 22 is a film formed in common to all the double gate transistors 20, 20,. The bottom gate insulating film 22 has insulating properties and light transmissive properties, and is made of, for example, silicon nitride (SiN) or silicon oxide (SiO ₂ ).

  A plurality of semiconductor films 23 are formed on the bottom gate insulating film 22 so as to be arranged in a matrix. The semiconductor film 23 is formed independently for each double gate transistor 20, and is disposed opposite to the bottom gate electrode 21 in each double gate transistor 20, and the bottom gate insulating film 22 is interposed between the bottom gate electrode 21. Is sandwiched. The semiconductor film 23 has a substantially rectangular shape in plan view, and is a layer formed of amorphous silicon or polysilicon that generates electron-hole pairs in an amount corresponding to the amount of received fluorescence.

  A channel protective film 24 is formed on the semiconductor film 23. The channel protective film 24 is patterned independently for each double gate transistor 20, and is formed on the central portion of the semiconductor film 23 in each double gate transistor 20. The channel protective film 24 has insulating properties and light transmissive properties, and is made of, for example, silicon nitride or silicon oxide. The channel protective film 24 protects the interface of the semiconductor film 23 from an etchant used for patterning. When light enters the semiconductor film 23, an amount of electron-hole pairs according to the amount of incident light is generated around the interface between the channel protective film 24 and the semiconductor film 23. In this case, holes are generated as carriers on the semiconductor film 23 side, and electrons are generated on the channel protective film 24 side.

An impurity semiconductor film 25 is formed so as to partially overlap the channel protective film 24 on one end portion of the semiconductor film 23, and an impurity semiconductor film 26 is partially channeled on the other end portion of the semiconductor film 23. It is formed so as to overlap the protective film 24. The impurity semiconductor films 25 and 26 are patterned independently for each double gate transistor 20. The impurity semiconductor films 25 and 26 are made of amorphous silicon (n ⁺ silicon) containing n-type impurity ions.

  A source electrode 27 is formed on the impurity semiconductor film 25, and a drain electrode 28 is formed on the impurity semiconductor film 26. The source electrode 27 and the drain electrode 28 are formed for each double gate transistor 20. A plurality of source lines 42, 42,... And drain lines 43, 43,... Extending in the vertical direction are formed on the bottom gate insulating film 22. The source electrodes 27 of the double gate transistors 20, 20,... In the same column arranged in the vertical direction are formed integrally with the common source line 42, and the double gate transistors 20 in the same column arranged in the vertical direction. , 20,... Are formed integrally with a common drain line 43. The source electrode 27, the drain electrode 28, the source line 42, and the drain line 43 have conductivity and light shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.

  .. Of the double gate transistors 20, 20,... And the source lines 42, 42... And the drain lines 43, 43,. The top gate insulating film 29 is a film formed in common to all the double gate transistors 20, 20,. The top gate insulating film 29 has insulating properties and light transmissive properties, and is made of, for example, silicon nitride or silicon oxide.

  A plurality of top gate electrodes 31 are formed for each double gate transistor 20 on the top gate insulating film 29. The top gate electrode 31 is disposed opposite to the semiconductor film 23 in each double gate transistor 20, and the top gate insulating film 29 and the channel protective film 24 are sandwiched between the top gate electrode 31 and the semiconductor film 23. Further, a plurality of top gate lines 44, 44,... Extending in the horizontal direction are formed on the top gate insulating film 29, and the double gate transistors 20, 20,. The top gate electrode 31 is formed integrally with a common top gate line 44. The top gate electrode 31 and the top gate line 44 are conductive and light transmissive, for example, indium oxide, zinc oxide, tin oxide, or a mixture containing at least one of them (for example, tin-doped indium oxide ( ITO) and zinc-doped indium oxide).

  The top gate electrode 31 and the top gate lines 44, 44,... Of the double gate transistors 20, 20,... Are collectively covered by the protective insulating film 32, and the protective insulating film 32 is common to all the double gate transistors 20, 20,. It is the film | membrane formed in this way. The protective insulating film 32 has insulating properties and light transmittance, and is made of silicon nitride or silicon oxide. An excitation light filter 33 is provided on the surface of the protective insulating film 32.

  The solid-state imaging device 30 configured as described above has the surface of the excitation light filter 33 as a light receiving surface, and is provided so as to convert the amount of light received by the semiconductor film 23 of each double gate transistor 20 into an electrical signal. Yes.

[3] Spot Next, the spot 60 will be described. As shown in FIGS. 1, 2, and 4, a plurality of types of spots 60, 60,... Are spaced apart from each other and arranged on the upper surface of the solid-state imaging device 30. One spot 60 is a group of many single-stranded probe DNAs, and many single-stranded probe DNAs included in one spot 60 have the same base sequence (nucleotide sequence). In addition, the base sequence of the single-stranded probe DNA is different for each spot 60. As the single-stranded probe DNA, a DNA having a base sequence identical to or complementary to the base sequence of a known mRNA in a specimen or a part thereof is used.

  One spot 60 overlaps with an arbitrary number of double gate transistors 20 and is formed so as to be separated from the other spots 60, 60,. Note that the number of double gate transistors 20 overlapping one spot 60 may be different.

[4] Analyzing apparatus Since the biopolymer analyzing chip 10 is used by setting the biopolymer analyzing chip 10 to the analyzing apparatus, the analyzing apparatus will be described first with reference to FIGS. FIG. 5 is a schematic side view in which the biopolymer analysis chip 10 is set in the analyzer 70, and FIG. 6 is a block diagram showing the configuration of the analyzer 70. In FIG. 5, the biopolymer analysis chip 10 is shown broken.

  The analysis apparatus 70 includes an analysis table 71 on which the biopolymer analysis chip 10 is set, a biopolymer analysis chip 10 that can be attached to and detached from the analysis table 71, and a light-receiving surface from the light-receiving surface of the solid-state imaging device 30. Excitation light irradiation device 72 for irradiating the excitation light toward the top, top gate driver 74, bottom gate driver 75 and drain driver 76 for driving solid-state imaging device 30, excitation light irradiation device 72, top gate driver 74, bottom gate driver And a computer 73 that controls the drain driver 76, an output device 77 that outputs (displays or prints) the signal output from the computer 73, and a spot detection light source 78 embedded in the analysis table 71.

  When the biopolymer analysis chip 10 is set on the analysis table 71, the top gate lines 44, 44,... Of the solid-state imaging device 30 are connected to the terminals of the top gate driver 74, respectively. Similarly, the bottom gate lines 41, 41,... Of the solid-state imaging device 30 are respectively connected to the terminals of the bottom gate driver 75, and the drain lines 43, 43,. Is connected to each of the terminals. When the biopolymer analysis chip 10 is set on the analysis table 71, the source lines 42, 42,... Of the solid-state imaging device 30 are connected to a constant voltage source, and in this example, the source lines 42, 42,. It has come to be.

  The excitation light irradiation device 72 faces the analysis table 71, and when the biopolymer analysis chip 10 is mounted on the analysis table 71, the excitation light emitted in a planar shape from the excitation light irradiation device 72 is biopolymer analysis. The chip 10 is irradiated. The excitation light irradiation device 72 may be provided so that the wavelength range of the emitted light can be varied.

  The output device 77 is a plotter, a printer, or a display. The spot detection light source 78 irradiates spot detection light 61 (visible light, for example, wavelength 550 nm) from below the biopolymer analysis chip 10.

  The top gate driver 74, the bottom gate driver 75, and the drain driver 76 cooperate to drive the solid-state imaging device 30.

  The computer 73 includes a CPU, RAM, ROM, etc. (not shown) and has a function of turning on the excitation light irradiation device 72 and the spot detection light source 78. In addition, the computer 73 outputs a control signal to the top gate driver 74, the bottom gate driver 75, and the drain driver 76, thereby driving the solid-state imaging device 30 to the top gate driver 74, the bottom gate driver 75, and the drain driver 76. Has the function to perform. The computer 73 has a function of causing the output device 77 to output an image according to the input two-dimensional image data.

  Further, the computer 73 has a function of acquiring the light intensity distribution along the light receiving surface of the solid-state imaging device 30 as two-dimensional image data by A / D converting the electric signal input from the drain driver 76. The computer 73 has a function of calculating two two-dimensional image data acquired from the solid-state imaging device 30 as will be described later.

  In the RAM of the computer 73, data output by each double gate transistor is recorded. The CPU of the computer 73 reads the recorded data from the RAM, performs a predetermined calculation described later, and outputs a calculation result.

  FIG. 7 is a spot determination table created in the RAM of the computer 73. In the spot determination table, cells of first to eighth rows × A to H columns are formed corresponding to the double gate transistors 20, 20,... Of the biopolymer analysis chip 10. As will be described later, a predetermined number is written in the spot determination table depending on the presence or absence of the spot 60.

  FIG. 8 is a spot block table created in the RAM of the computer 73. The spot block table is detected in the number sequence in which the spot block number is written, the position content column in which the position of the cell in which the spot block number is written in the spot determination table is written, and the cell in which the spot block number is written. It consists of an average value sequence in which the average value of fluorescence data is written.

[5] Spot Detection Principle The presence or absence of the spot 60 on the double gate transistor 20 is determined from the transparent substrate 17 side of the biopolymer analysis chip by the spot detection light source 78 of the analyzer 70 as shown in FIGS. The spot detection light 61 is irradiated, and the determination is made based on the amount of the reflected light 62 detected by each of the double gate transistors 20, 20,. Here, the principle of detecting the presence / absence of the spot 60 based on the amount of the reflected light 62 will be described.

The spot detection light 61 can be separated into a P-polarized component and an S-polarized component. When the refractive index of the protective insulating film 32 is n _prot and the refractive index of the excitation light filter 33 is n _FLT , the p-polarized reflectance r _12P at the interface between the protective insulating film 32 of the spot detection light 61 and the excitation light filter 33, r _12S satisfies the following expressions (1) and (2).

The θ ₁ is an incident angle when the spot detection light 61 is incident on the excitation light filter 33 from the protective insulating film 32, and the θ ₂ is a refraction angle emitted into the excitation light filter 33.

As shown on the left side of FIG. 4, when there is air in the excitation light filter 33 (no spot 60), assuming that the refractive index of air is n _AIR , the p-polarized reflectance at the interface between the excitation light filter 33 and air. r _24P and r _24S satisfy the following expressions (3) and (4).

The angle θ ₄ is the refraction angle of the spot detection light 61 that passes through the excitation light filter 33 and is emitted into the air.

The P-polarized energy reflectivity R _14P and the S-polarized energy reflectivity R _{14S obtained} by combining the two interfaces satisfy the following expressions (5) and (6), respectively.

Here, δ ₂ satisfies the following equation (7). D ₂ is the thickness of the excitation light filter 33.

FIG. 9 shows the P-polarized energy reflectance R _14P and S-polarized energy reflectance R _14S of the spot detection light 61 in the portion where the spot 60 of the biopolymer analysis chip 10 is not provided, based on the equations (5) and (6). It is the graph shown. Note that the calculation is performed with λ = 550 nm, n _prot = 1.87, n _FLT = 2.32, and n _AIR = 1.0.

On the other hand, as shown on the right side of FIG. 4, when the excitation light filter 33 has a spot 60, assuming that the refractive index of the solution constituting the spot is n _hyb , p-polarized light at the interface between the excitation light filter 33 and the spot 60. The reflectances r _23P and r _23S satisfy the following expressions (8) and (9).

The θ ₃ is a refraction angle that is emitted into the spot 60 of the spot detection light 61 that has passed through the excitation light filter 33.

The p-polarized reflectances r _34P and r _34S at the interface between the spot 60 and the air satisfy the following expressions (10) and (11).

The P-polarized energy reflectivity R _24P and the S-polarized energy reflectivity R _24S at the virtual interface obtained by synthesizing the interface between the excitation light filter 33 and the spot 60 and the interface between the spot 60 and the air are: Respectively, the following expressions (12) and (13) are satisfied.

Here, δ ₃ satisfies the following equation (14). D ₃ is the height of the spot 60.

The P-polarized reflectances r _234P and r _{234S at} the virtual boundary surface between these two interfaces satisfy the following expressions (15) and (16).

The P-polarized energy reflectivity R _P and the S-polarized energy reflectivity R _S at the virtual interface and the virtual interface obtained by synthesizing the interface between the spot 60 and the air are expressed by the following equations (17), ( 18) is satisfied.

Here, φ _3P is the P-polarization phase angle at the virtual boundary surface, and φ _3S is the S-polarization phase angle at the virtual boundary surface, which satisfies the following equations (19) and (20).

FIG. 10 is a graph showing the P-polarized energy reflectivity R _P and the S-polarized energy reflectivity R _S on the basis of the equations (17) and (18) in the portion where the spot 60 is provided on the biopolymer analysis chip 10. It is. Note that the calculation is performed with λ = 550 nm, n _prot = 1.87, n _FLT = 2.32, n _hyb = 1.52, and n _AIR = 1.0.

  When there is no spot 60, as shown in FIG. 9, at a low incident angle (20 degrees or less), the deflection average reflectance is 15% or more, whereas when there is a spot 60, as shown in FIG. At a low incident angle (20 degrees or less), the deflection average reflectance is about 5%. Therefore, the intensity of the reflected light 62 detected by each of the double gate transistors 20, 20,... Is different by 3 to 5 times depending on the presence or absence of the spot 60. It can be determined that there is a spot 60.

[6] Spot Detection Operation Hereinafter, an operation for detecting the spot 60 on the light receiving surface of the solid-state imaging device 30 of the biopolymer analysis chip 10 using the analyzer 70 will be described.

  In order to detect the spot, first, the biopolymer analysis chip 10 in which the spot 60 is formed on the light receiving surface of the solid-state imaging device 30 is set on the analysis table 71 of the analyzer 70.

  Next, the computer 73 turns on the spot detection light source 78 and irradiates the spot detection light 61 from the transparent substrate 17 side of the biopolymer analysis chip 10 as shown in FIG. ... Detect the amount of reflected light 62 and determine the presence or absence of the spot 60. The determination result of the presence / absence of the spot 60 is stored in the RAM of the computer 73.

  Here, the biopolymer analysis chip 10 is obtained by dividing the light-receiving surface of the solid-state imaging device 30 into several regions (spot blocks) in advance and forming one spot in each spot block. In other words, the spot positions are generally determined, and each spot block is set to an area in which one spot is entered in consideration of the spot position deviation. For example, as shown in FIG. 1, regions composed of A to H columns × 1st to 8th rows of 8 × 8 double gate transistors 20, 20,... Are represented by A1 to D4, E1 to H4, A5 to D8. , E5 to H8, and one spot 60 is formed in each region. In FIG. 1, (B, 1), (B, 2), (C, 1), (C, 2), (C, 5), (C, 6), (D, 5), (D , 6), (F, 2), (F, 3), (G, 2), (G, 3), (G, 6), (G, 7), (H, 6), (H, 7 A spot 60 is formed on each double-gate transistor 20 corresponding to).

  A spot block number is assigned to each divided spot block. For example, a spot block number of 1 is assigned to the area A1 to D4, 2 to the area E1 to H4, 3 to the area A5 to D8, and 4 to the area E5 to H8.

The determination result of the presence / absence of the spot 60 is written in the spot determination table. For example, as shown in FIG. 7, cells (B, 1), (B, 2), coordinates of coordinates corresponding to the double gate transistor 20 determined to have the spot 60 due to the difference in reflectance in the area of the spot block 1. A value of block number “1” is written in (C, 1) and (C, 2), and a value of “0” is written in other cells.
Similarly, cells (C, 5), (C, 6), (D, 5), and (D, 6) corresponding to the double gate transistor 20 determined to have the spot 60 in the area of the spot block 2 The value of the block number “2” is written, and the value of “0” is written in the other cells.
Similarly, the cells (F, 2), (F, 3), (G, 2), and (G, 3) corresponding to the double gate transistor 20 determined to have the spot 60 in the spot block 3 region include The value of the block number “3” is written, and the value of “0” is written in the other cells.
Similarly, cells (G, 6), (G, 7), (H, 6), (H, 7) corresponding to the double gate transistor 20 determined to have the spot 60 in the area of the spot block 4 The value of the block number “4” is written, and the value of “0” is written in the other cells.

  In addition, block numbers “1”, “2”, “3”, “4”,... Are written in advance in the cells corresponding to the respective spot blocks, and “0” is written in the cells determined to have no spot 60. A value may be written.

  The coordinates corresponding to the cell in which the block number of the spot determination table is written are written in the position content column corresponding to the block number of the spot block table and used for calculating the average value of the fluorescence data at each spot.

  For example, the coordinates of the cells (B, 1), (B, 2), (C, 1), (C, 2) in which the block number “1” is written are written in the position content column of the row of block number 1. In the position content column of the row of block number 2, the coordinates of the cells (C, 5), (C, 6), (D, 5), (D, 6) in which the block number “2” is written are shown. The coordinates of the cell (F, 2), (F, 3), (G, 2), (G, 3) in which the block number “3” is written are written in the position content column of the row of the block number 3. Is written in the position content column of the row of block number 4 in the cells (G, 6), (G, 7), (H, 6), (H, 7) in which the block number “4” is written. Coordinates are written.

  In FIG. 1, since each spot 60 is formed on each of the four double gate transistors 20, four coordinates are written in the position content column of each row, but the number of spots 60 is formed. It varies depending on the position and size.

[7] DNA Sample Processing Method A DNA sample processing method using the biopolymer analysis chip 10 will be described.
First, an operator collects cDNA from a specimen, optionally performs PCR amplification, binds a fluorescent substance to the obtained DNA, and labels the DNA with the fluorescent substance. As the fluorescent material, one that is excited by the excitation light emitted from the excitation light irradiation device of the analyzer and emits fluorescence by the excitation light is selected. For example, CyDye Cy2 (Amersham) Made). The obtained DNA is contained in a solution. Hereinafter, this DNA is referred to as sample DNA.

  Next, the worker applies a solution containing sample DNA (hereinafter referred to as sample DNA solution) to the light receiving surface of the solid-state imaging device 30. Here, in order to distribute the sample DNA on the light receiving surface of the solid-state imaging device 30, the sample DNA may be electrophoresed. It should be noted that the sample DNA solution may be dropped on the spots 60, 60,. At this time, the sample DNA solution is heated so that the sample DNA becomes a single strand.

  Thereafter, the biopolymer analysis chip 10 is cooled to a predetermined temperature so that the probe DNA and the sample DNA cause hybridization. Thus, if there is a probe DNA complementary to the sample DNA in the spots 60, 60,. On the other hand, if none of the spots 60, 60,... Is complementary to the sample DNA, the sample DNA will not bind to any of the spots 60, 60,.

  Thereafter, the sample DNA applied to the light receiving surface of the solid-state imaging device 30 that is not hybridized is washed away.

[8] Method for detecting DNA sample A method for detecting a DNA sample will be described. First, the solid-state imaging device 30 subjected to the above processing is set on the analysis table 71, the excitation light irradiation device 72 is opposed to the light receiving surface of the solid-state imaging device 30, and the top gate driver 74, the bottom gate driver 75, and the drain driver 76 are set. Connect to computer 73. Thereafter, the computer 73 is started, and the fluorescence data measurement operation by the analyzer 70 is started.

  Hereinafter, the measurement operation of fluorescence data by the analyzer 70 will be described. First, the computer 73 controls the excitation light irradiation device 72 to turn on the excitation light irradiation device 72, and emits excitation light from the excitation light irradiation device 72 toward the light receiving surface of the solid-state imaging device 30. By controlling the gate driver 74, the bottom gate driver 75, and the drain driver 76, the solid-state imaging device 30 is caused to perform an imaging operation.

  Since the sample DNA is labeled, fluorescence (mainly visible light wavelength region) is emitted from the spot 60 hybridized with the sample DNA among the spots 60, 60,..., And the fluorescence enters the corresponding double gate transistor 20. Thus, electron-hole pairs are generated.

  Then, the fluorescence data of each of the double gate transistors 20, 20,... Is acquired and stored in the RAM. At this time, the presence / absence of fluorescence is determined only from the fluorescence data of the double gate transistor 20 corresponding to the cell in which any one of the block numbers “1”, “2”, “3”, and “4” is written. judge. Thereby, since it is not necessary to determine the presence / absence of fluorescence from the fluorescence data of the block number without the spot 60, the number of fluorescence data for determining the presence / absence of fluorescence is limited, and data that becomes noise is reduced, so that the accuracy can be improved. .

  Next, the average value of the fluorescence data detected by the double gate transistor 20 with the spot 60 is calculated for each spot block.

That is, as shown in FIG. 8, in the spot block table, (B, 1), (B, 2), (C, 1), fluorescence data detected by each double gate transistor 20 corresponding to (C, 2) is added, and an average value V _t1 divided by the added number of cells is written.

Similarly, the cell of the average value column in the row of block number 2 corresponds to (C, 5), (C, 6), (D, 5), (D, 6) written in the position content column. The average value V _t2 of the fluorescence data detected by the double gate transistor 20 is written, and the cells of the average value column in the row of the block number 3 are written in the position content column (F, 2), (F, 3 ), (G, 2), the average value V _t3 of the fluorescence data detected by the double gate transistor 20 corresponding to (G, 3) is written, and the cells in the rank average value column in the row of the block number 4 are The average value V _t4 of the fluorescence data detected by the double gate transistor 20 corresponding to (G, 6), (G, 7), (H, 6), (H, 7) written in the position content sequence is written. It is.

The computer 73 uses the average fluorescence data V _t1 , V _t2 , V _t3 , V _t4 ,... Corresponding to the block numbers 1, 2, 3, 4 _,. As image data. The computer 73 inputs the acquired image data and outputs the image to the output device 77. Then, the processing of the computer 73 ends.

  The operator confirms the presence or absence of hybridization from the image data output by the output device 77, and if hybridization has occurred, specifies the base sequence of the sample DNA from the base sequence of the probe DNA. That is, since the base sequence of the sample DNA is a sequence complementary to the spot 60 that overlaps the pixel that emitted fluorescence by hybridization in the image, which part of the output image data emitted fluorescence. The gene expressed in the sample can be identified.

  As described above, in the present embodiment, since the spot position can be clearly determined in advance before emitting the excitation light and detecting the fluorescence after hybridization, the subsequent operations can be simplified. That is, for example, the operator may determine only the presence or absence of fluorescence in a spotted portion, or fluorescence only from the fluorescence data of the double gate transistor 20 corresponding to the cell in which any block number is written in the spot determination table. Therefore, the accuracy can be increased.

  Further, by obtaining the average value of the fluorescence data acquired by the plurality of double gate transistors 20, noise can be removed and the S / N ratio can be improved. Further, by setting the average value of the fluorescence data as the light intensity of one block, it is possible to cancel the influence of the variation in the size of the spot 60 on the luminance of each block in the image data.

[Modification 1]
The light receiving surface of the solid-state imaging device 30 may not be divided into a plurality of spot blocks. In this case, the determination result of the presence / absence of a spot is written in a spot determination table, for example, as shown in FIG. That is, the value “1” is written in the cell corresponding to the double gate transistor 20 determined to have the spot 60, and the value “0” is written in the other cells. Then, when acquiring the fluorescence data, it is only necessary to acquire only the fluorescence data of the double gate transistor 20 corresponding to the cell in which the value “1” is written in the spot determination table.

[Second Embodiment]
Next, a second embodiment of the present invention will be described. The solid-state imaging device 30 of the biopolymer analysis chip 10 used in the present embodiment is the same as that used in the first embodiment, and the description thereof is omitted.

  12 and 13 show an analysis support apparatus 80 used in the present embodiment. FIG. 12 is a schematic side view when the biopolymer analysis chip 10 is set in the analysis support apparatus 80, and FIG. 13 is a block diagram showing a circuit configuration of the analysis support apparatus 80. In FIG. 12, the biopolymer analysis chip 10 is shown broken.

  Similar to the analysis support apparatus 70 of the first embodiment, the analysis support apparatus 80 includes an analysis table 71, an excitation light irradiation device 72, a biopolymer analysis chip 10, a top gate driver 74, and a bottom gate driver 75. And a drain driver 76, a computer 73, an output device 77, and a spot detection light source 78. Further, the analysis support apparatus 80 includes a spotter 79 controlled by the computer 73.

  In the RAM of the computer 73 in this embodiment, a spot determination table and a spot block table are formed as in the first embodiment. As shown in FIG. 14, the spot block table of the present embodiment is provided with a number column, a position content column, and an average value column, as in the first embodiment. A block ID column is provided.

  In the block ID column, a predetermined ID name is written according to the type of spot solution dropped on each spot block. For example, “test site 1” is written in the block ID column in the row of block number 1 corresponding to spot block 1. Similarly, the block ID column in the row of block number 2 has “test site 2”, the block ID column in the row of block number 3 corresponding to spot block 3 has “positive control”, and the block number 4 in row. In the block ID column, “negative control” is written. The positive control means that the phosphor always emits fluorescence, and the negative control means that it does not always emit fluorescence even when irradiated with excitation light.

  The computer 73 drives the spotter 79 to drop a predetermined spot solution on each spot block according to the ID name written in the block ID column of the spot block table. For example, the spot block 1 corresponding to the ID of “test site 1” includes the test spot solution 1, the spot block 2 corresponding to the ID of “test site 2” includes the test spot solution 2, and the ID of “positive control”. A positive spot solution is dropped on the spot block 3 corresponding to, and a negative spot solution is dropped on the spot block 4 corresponding to the ID of “negative control”.

  Here, as test spot solutions 1 and 2, a solution of cDNA (probe DNA) of an arbitrary sequence, respectively, as a positive spot solution, a solution containing the same fluorescent substance as the label of sample DNA, or for example, label of sample DNA As a DNA solution of any sequence labeled with the same fluorescent substance as in the above, a solution that can fix this fluorescent substance at the spot spot, a negative spot solution does not contain the same fluorescent substance as the fluorescent substance attached to the sample DNA Use a liquid or a liquid that does not immobilize the fluorescent substance attached to the sample DNA, that is, a liquid that does not contain a substance that hybridizes with the sample DNA, such as pure water, or the same solvent (buffer solution) as the test spot solution that does not contain DNA. be able to.

  The spotter 79 includes a spot solution tank, a cleaning tank, a spot needle for spotting various spot solutions on the spot block, and a driving device for driving the spot needle.

  One spot solution tank is provided for each spot solution, and spot solutions to be spotted on each spot block are stored. A cleaning solution (pure water) for spot needles is stored in the cleaning tank.

The driving device drives the spot needle in the three axis directions of front and rear, left and right horizontal directions, and up and down directions on the spot solution tank, the washing tank, and the solid-state imaging device 30.
The spot needle is immersed in a spot solution tank to attach the spot solution to the tip and spot on each spot block.

The operation of forming the spots 60 in each spot block by the spotter 79 will be described.
First, the driving device driven by the computer 73 moves the spot needle horizontally on the spot solution tank in which the test spot solution 1 corresponding to the spot block 1 is stored, and then moves the spot needle up and down to move the tip of the spot needle. Immerse in the spot solution in the spot solution tank and attach the spot solution to the tip. Next, the driving device horizontally moves the spot needle with the spot solution attached to the tip onto the spot block 1 of the solid-state imaging device 30.

  Next, the driving device moves the spot needle up and down on the spot block 1 and drops the spot solution adhering to the tip of the spot needle onto the spot block 1 to form the test spot 1.

  Thereafter, the driving device moves the spot needle onto the cleaning tank, moves the spot needle up and down, immerses the tip of the spot needle in the cleaning solution in the cleaning tank, and cleans the tip of the spot needle.

  Thereafter, the driving device moves the cleaned spot needle onto the spot solution tank in which the test spot solution 2 corresponding to the spot block 2 is stored. Similarly, a spot 60 is formed by dropping the test spot solution 2 on the spot block 2. Thereafter, in the same manner, a positive spot solution is dropped on the spot block 3, and a negative spot solution is dropped on the spot block 4. Then, a negative spot detection operation is performed at the timing when the negative spot solution is dropped onto the spot block 4.

  That is, the computer 73 takes the timing signal of the driving device when the spot needle drops the negative spot solution on the spot block 4, and then the computer 73 turns on the spot detection light source 78 and changes the spot detection light 61 to the height of the living body. While irradiating from the transparent substrate 17 side of the molecular analysis chip 10, the amount of the reflected light 62 is detected by each double gate transistor 20, 20,.

  Since the negative spot solution is pure water, a buffer solution, or the like, it is very difficult to optically detect the spot position after the negative spot solution has substantially disappeared after drying. This is because the refractive index of the negative spot solution is higher than the refractive index of air (about 1.0), such as pure water (refractive index of about 1.4) or buffer solution (refractive index of about 1.4 or more) on the surface. This is because the position is optically detected based on the difference in refractive index from the portion without the negative spot solution and the positive spot solution, that is, the air portion. In the present embodiment, since the spot is detected immediately after spotting the negative spot solution, the spot of the negative spot solution can be reliably detected.

  The spot block to which the test spot solution, positive spot solution, and negative spot solution are dropped is arbitrary. Further, the dropping order is arbitrary, but it is preferable to drop the negative spot solution last. If the negative spot solution is not dropped last, it is necessary to detect the spot with another spot solution after the negative spot is detected, but the negative spot solution is dropped last. This is because detection of all other spots can be performed together with the spot of the negative spot solution.

  When the spot 60 is formed in the same manner as in FIG. 1, a spot determination table similar to that in FIG. 7 is created in the RAM of the computer 73 as in the first embodiment, and the spot block table created in the RAM is In the position content column, as shown in FIG. 14, coordinates corresponding to the cell in which the block number of the spot determination table is written are written.

Then, in the same manner as in the first embodiment, when a DNA sample is detected and fluorescence data is obtained, each double gate transistor 20 of the coordinates written in the corresponding position content column is displayed in the cells of the average value column. The fluorescence data detected in (1) is added, and average values V _t1 , V _t2 , V _t3 , V _t4 divided by the added number of cells are written.

In the present embodiment, the CPU of the computer 73 treats the average value of the fluorescence data written in the average value column of the row where “positive control” is written in the block ID column as the positive control data V _pc . That is, it is treated as V _pc = V _t3 . Further, the average value of the fluorescence data written in the average value column of the row in which “Negative control” is written in the block ID column is handled as negative control data V _nc . That is, V _nc = V _t4 is handled.

Then, the CPU of the computer 73 compares the average value V _nc of the fluorescence data in the negative control with the average values V _t1 and V _t2 of the fluorescence data in the other test sites 1 and 2, thereby comparing each test site. The presence or absence of hybridization is detected.

Since the negative spot solution does not contain DNA, the fluorescently labeled sample DNA does not bind to the negative spot, so theoretically no fluorescence is detected from the negative spot, but impurities such as dust are present on the double gate transistor 20. In some cases, fluorescence may be detected as noise. That is, V _nc is the noise value itself.

When V _nc <V _t1 , it is considered that the sample DNA fluorescently labeled to test spot 1 is hybridized. When V _nc ≧ V _t1 , the sample fluorescently labeled to test spot 1 It is thought that DNA did not hybridize. The same applies to _Vt2 .

Further, the CPU of the computer 73 compares the average value V _pc of the fluorescence data in the positive control with the average values V _t1 and V _t2 of the fluorescence data at the other test sites 1 and 2, thereby comparing the magnitude of the experiment system itself. Consider success or failure.

Fluorescently labeled DNA is attached to the positive spot. For this reason, regardless of the composition of the sample DNA solution, fluorescence should always be detected from the positive spot. If fluorescence is not detected from the positive spot, there is some problem in the experimental system, for example, when the excitation light is not sufficiently irradiated or the excitation light filter 33 or the double gate transistor 20 is damaged. It is determined that the experimental system itself does not hold. On the other hand, when the fluorescence intensity values V _t1 , V _t2 ,... _Are smaller than V _pc , it is considered that there is no problem in the experimental system, and the reliability of the fluorescence data can be increased.

  The present invention is not limited to the above embodiment, and various improvements and design changes may be made without departing from the spirit of the present invention.

  For example, in the above embodiment, a single-stranded DNA of a known base sequence is used for the spot 60, but other known biopolymers, low molecules, and the like may be used. For example, a peptide or protein having a known amino acid sequence, an antibody that binds to the protein, a low molecular ligand, a known cell, or the like may be used.

1 is a schematic plan view of a biopolymer analysis chip 10 in an embodiment of the present invention. It is arrow sectional drawing along the cut surface II of FIG. 3 is a plan view showing an electrode structure of a photoelectric conversion element that is a pixel of the solid-state imaging device 30. FIG. It is an enlarged view which shows the photoelectric conversion element of the solid-state imaging device 30 in FIG. 4 is a schematic side view in which the biopolymer analysis chip 10 is set in the analysis device 70. FIG. 3 is a block diagram illustrating a configuration of an analysis device 70. FIG. It is a spot determination table created in the RAM of the computer 73. It is a spot block table created in the RAM of the computer 73. It is the graph which showed P polarization energy reflectance _R14P and S polarization energy reflectance _{R14S based} on Formula (5) and (6). It is the graph which showed P polarization energy reflectance _RP and S polarization energy reflectance _{RS based} on Formula (17) and (18). It is a spot determination table created in the RAM of the computer 73. FIG. 4 is a schematic side view in which the biopolymer analysis chip 10 is set in the analyzer 80. 2 is a block diagram showing a configuration of an analysis device 80. FIG. It is a spot block table created in the RAM of the computer 73.

Explanation of symbols

1 Biopolymer analysis chip 3 Solid-state imaging device (imaging device)
17 Transparent substrate 20 Double gate transistor (photoelectric conversion element)
60 Spot 73 Computer 78 Spot detection light source

Claims (4)

A biopolymer analysis chip in which spots are formed by dropping a solution of a probe that binds to a specific biopolymer onto a light-receiving surface of an image sensor including a plurality of photoelectric conversion elements arranged in a two-dimensional array on a transparent substrate ,
A spot detection light source for irradiating the biopolymer analysis chip with spot detection light from the transparent substrate side;
Detecting means for detecting the presence or absence of the spot based on the reflected light intensity at the surface of the spot of the spot detection light;
Equipped with a,
Each spot is formed on a light receiving surface of a plurality of the photoelectric conversion elements,
In the case where there are a plurality of photoelectric conversion elements determined to have fluorescence among the plurality of photoelectric conversion elements corresponding to each spot, the detection means uses the average value of the fluorescence data as the fluorescence data of the spot. A characteristic biopolymer analyzer. The living body according to claim 1 , further comprising a spotter that is driven by the detection unit and selectively drops a liquid that emits a fluorescent color and a liquid that does not emit a fluorescent color on a light receiving surface of the imaging device. Polymer analyzer. The spotter over the biopolymer analyzer according to claim 2, wherein the dropping of the liquid which does not fluoresce color, and performing after the dropping of the liquid which emits the fluorescent color. A solution of a probe that binds to a specific biopolymer is dropped on a light-receiving surface of an image sensor including a plurality of photoelectric conversion elements arranged in a two-dimensional array on a transparent substrate, and light reception by the plurality of photoelectric conversion elements is performed. Irradiate the biopolymer analysis chip having spots formed on the surface with spot detection light from the transparent substrate side,
When detecting the presence or absence of the spot based on the reflected light intensity on the surface of the spot of the spot detection light, a photoelectric conversion element determined to have fluorescence among a plurality of photoelectric conversion elements corresponding to each spot A biopolymer analysis method characterized in that, when there are a plurality of spots, the average value of the fluorescence data is used as the fluorescence data of the spot.

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