JP2006226887A - Biopolymer analysis chip, analysis support device, and biopolymer analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biopolymer analysis chip, an analysis support device and a biopolymer analyzing method of high sensitivity. <P>SOLUTION: This biopolymer analysis chip 1 is provided with a nonmagnetic substrate 2 recessed with a plurality of wells 3 on a surface thereof, and a magnetic substance fine particle 60 stored in each of the wells 3, and having a probe 61 coupled with a specified biopolymer 62. A buffer solution 64 containing the biopolymer 62 is filled in the each well 3 for storing the magnetic substance fine particle 60 in an inside thereof, the inside of the well 3 is stirred by fluctuating a magnetic field in the periphery of the nonmagnetic substrate 2 to move the magnetic substance fine particle 60, so as to enhance coupling probability of the specified biopolymer 62 onto the probe 61. The well 3 is washed with the buffer solution 64 containing no biopolymer 62, under the condition where the magnetic substance fine particle 60 is attracted by a magnet from an under side of the nonmagnetic substrate 2, so as to remove the biopolymer 62 not coupled with the probe 61. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a biopolymer analysis chip, an analysis support apparatus, and a biopolymer analysis method.

  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 in which a plurality of types of cDNA having a known base sequence (hereinafter referred to as probe DNA) are aligned and fixed on a solid support such as a slide glass is prepared. 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, mRNA expressed in the specimen can be identified based on which part of the two-dimensional image has high fluorescence intensity.

In order to form probe DNA on a fixed carrier in a conventional DNA microarray, there is a method in which nucleic acids are polymerized one by one on the fixed carrier by photolithography to synthesize an oligonucleotide that becomes probe DNA. There is also a method in which a solution containing probe DNA is spotted on a fixed carrier with a collection pin whose interval can be changed (see, for example, Patent Document 2).
JP 2000-1312237 A JP 2001-99847 A

  However, in the above-described DNA microarray, since various types of probe DNA are formed on a limited plane such as a slide glass, the amount of probe DNA per spot is limited. For this reason, the amount of sample DNA hybridized with the probe DNA is also limited, and the luminance of the fluorescence is also limited. Therefore, a high-sensitivity photosensor and a high-power excitation light source are required, and the apparatus is expensive. There was a problem.

  Further, in the above-described DNA microarray, since the probe DNA is planarly arranged on the fixed carrier, the contact with the buffer solution containing the sample DNA is limited, and the hybridization efficiency is poor.

  The present invention has been made to solve the above-described problems, and an object thereof is to provide a biopolymer analysis chip, an analysis support apparatus, and a biopolymer analysis method with good sensitivity.

  In order to solve the above problems, the invention according to claim 1 is a non-magnetic substrate having a plurality of wells provided on the surface thereof, and is accommodated in each well and binds to a specific biopolymer. A biopolymer analysis chip comprising a magnetic fine particle having a probe.

  According to the first aspect of the present invention, a buffer containing a biopolymer is placed in a well containing therein magnetic fine particles having a probe that binds to a specific biopolymer, and a magnetic field around the nonmagnetic substrate is placed. By moving the magnetic fine particles, the inside of the well is stirred to increase the binding probability of a specific biopolymer to the probe. Then, in a state where the magnetic fine particles are attracted from below the non-magnetic substrate with a magnet, the well can be washed with a buffer that does not contain the biopolymer to remove the biopolymer that does not bind to the probe.

The invention described in claim 6
A magnetic fine particle having a probe that binds to a specific biopolymer is accommodated in each well provided on the surface of the non-magnetic substrate,
A biopolymer sample labeled with a labeling substance is injected into each well,
Washing off each well of the non-magnetic substrate with a magnetic field formed on the non-magnetic substrate,
In this biopolymer analysis method, the labeling substance remaining in each well is detected.

  Here, a chemiluminescent substance, a fluorescent substance, or the like may be used as a labeling substance, and light emitted from these labeling substances may be detected. According to the sixth aspect of the present invention, a biopolymer sample is placed in a well containing therein magnetic fine particles having a probe that binds to a specific biopolymer, and is magnetized by a magnet from below the nonmagnetic substrate. The wells can be washed in a state in which the body fine particles are attracted to remove biopolymers that do not bind to the probe. Then, by detecting the labeling substance remaining in the well, it can be determined whether or not the specific biopolymer bound to the probe is contained in the biopolymer sample.

  According to the present invention, a buffer containing a biopolymer is placed in a well containing therein magnetic fine particles having a probe that binds to a specific biopolymer, and the magnet is moved to move the magnetic field around the non-magnetic substrate. By moving the magnetic fine particles, the inside of the well is stirred to increase the binding probability of a specific biopolymer to the probe.

  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 plan view showing a biopolymer analysis chip 1 in an embodiment to which the present invention is applied. The biopolymer analysis chip 1 includes a non-magnetic substrate 2 on which a plurality of wells 3 are formed, and magnetic fine particles 60 (shown in FIG. 2 and the like) accommodated in each well 3.

  The non-magnetic substrate 2 is made of a non-magnetic material such as a slide glass having a property of transmitting light (hereinafter referred to as “light transmissive”), and has a glass substrate such as quartz glass or a plastic substrate such as polycarbonate or PMMA. ing. On the upper surface of the non-magnetic substrate 2, a plurality of wells 3 are recessed in a substantially hemispherical shape having a diameter of about several hundred μm to 1 mm. These wells 3 are arranged in a matrix along the surface of the nonmagnetic substrate 2 at intervals of several hundred μm to 1 mm. In the figure, an 8 × 8 matrix is shown, but the arrangement of the wells 3 is not limited to this.

FIG. 2 is a vertical sectional view showing one of the wells 3. Each well 3 is filled with a buffer solution 64, and the buffer solution 64 contains magnetic fine particles 60 having optical transparency. There are one or more magnetic fine particles 60 in each well 3, and they float in the buffer solution 64. The light-transmitting magnetic fine particles 60 are realized by a composite material or the like doped with an element exhibiting ferromagnetism on a light-transmitting base material, specifically, manganese-doped zinc oxide or cobalt-doped oxide. There are composite materials using titanium and neodymium magnets (Nd ₂ Fe ₁₄ B). The buffer solution 64 contains no magnetic substance or contains only a very weak magnetic substance.

  A plurality of DNA molecules having a known base sequence serving as a probe (hereinafter referred to as probe DNA 61) are attached to the surface of the magnetic fine particle 60 in a radial pattern. Here, as the probe DNA 61, a DNA having a base sequence identical to or complementary to a base sequence of mRNA already known in the specimen or a part thereof is used. The base sequences of the probe DNAs 61 attached to the magnetic fine particles 60 in one well 3 are all the same, and probe DNAs 61 having different base sequences are used for each well 3. The probe DNA 61 is fixed to the magnetic fine particles 60 by attaching a solution containing the probe DNA 61 to the surface of the magnetic fine particles 60 and drying the solution.

[2] Analysis Support Device Next, an analysis support device 50 that supports imaging by the biopolymer analysis chip 1 will be described with reference to FIG. FIG. 3 is a schematic diagram showing the analysis support apparatus 50. The analysis support device 50 includes a biopolymer analysis chip 1, a support base 5 on which the biopolymer analysis chip 1 is detachably set horizontally, a magnetic field forming base 6 provided below the support base 5, and an excitation. A laser oscillation device 7 that oscillates light, a detection device 8 that detects fluorescence, and an optical system are provided.

The support 5 is provided with a temperature adjusting device that appropriately adjusts the temperature range in which the probe DNA 61 hybridizes with DNA having a complementary base sequence and the temperature range in which the probe DNA 61 cannot hybridize.
The magnetic field forming table 6 is provided with a permanent magnet or an electromagnet that forms a magnetic field by flowing a current at a position corresponding to the well 3 of the nonmagnetic substrate 2. The magnetic field forming base 6 forms a magnetic field on the non-magnetic substrate 2 and is provided so that the magnetic pole can be moved in the horizontal direction with respect to the non-magnetic substrate 2. The magnetic pole may be movable in the horizontal direction relative to the nonmagnetic substrate 2 by making only the support base 5 movable in the horizontal direction without moving the nonmagnetic substrate 2, and supporting. The magnetic pole may be movable in the horizontal direction relative to the non-magnetic substrate 2 by allowing only the magnetic field forming table 6 to move in the horizontal direction with respect to the support table 5 without moving the table 5. The nonmagnetic substrate 2 and the support 5 may be moved together so that the magnetic pole can be moved in the horizontal direction relative to the nonmagnetic substrate 2. The magnetic field forming table 6 preferably forms a magnetic field evenly at a position corresponding to each well 3, and it is preferable that magnets be arranged at intervals of each well 3. When each magnet of the magnetic field forming table 6 moves in the horizontal direction with respect to the non-magnetic substrate 2, the magnetic pole of each magnet moves in the horizontal direction with respect to the well 3, and the magnetic fine particles 60 are moved horizontally in the well 3. Can be moved. In addition, the magnetic fine particles 60 can be moved in the vertical direction in the well 3 by changing the magnetic force of the magnetic field forming table 6. When the magnetic field of the magnetic field forming table 6 is formed by a permanent magnet, the magnetic force can be adjusted by moving the magnetic pole of the permanent magnet closer to or away from the non-magnetic substrate 2, and in the case of an electromagnet, the magnetic force is adjusted by controlling the flowing current. be able to. In this way, the magnetic fine particles 60 in the well 3 can be moved in an arbitrary direction.

The optical system includes a dichroic mirror 9a, a mirror 9b, and other lenses (not shown).
The dichroic mirror 9a reflects excitation light 51 having an excitation light wavelength of a fluorescent material 63 described later to irradiate the well 3, and transmits the fluorescence 52 emitted from the fluorescent material 63 in the well 3. The mirror 9 b reflects the fluorescence 52 that has passed through the dichroic mirror 9 a so as to enter the detection device 8.
The optical paths of the excitation light 51 and the fluorescence 52 in the optical system lens are confocal, and the optical system can be two-dimensionally scanned with respect to the biopolymer analysis chip.

  Next, an analysis method using the analysis support apparatus 50 will be described.

[3] Preparation of sample An operator collects mRNA from a specimen, prepares cDNA using reverse transcriptase, performs PCR amplification as necessary, and attaches or binds the fluorescent substance 63. The fluorescent material 63 is selected from materials that are excited by the excitation light 51 of the analysis support device 50 and emit fluorescence (light having a wavelength that passes through the dichroic mirror 9a) 52 by the excitation light 51. Examples of the material 63 include CyDye Cy2 (manufactured by Amersham). The obtained cDNA is contained in a buffer. Hereinafter, the cDNA labeled with the fluorescent material 63 is referred to as sample DNA 62.

[4] Analysis Method An operator applies the buffer solution 64 containing the sample DNA 62 to the surface of the biopolymer analysis chip 1 set on the support 5 where the well 3 is provided. At this time, since the magnetic fine particles 60 are fixed to the bottom of the well 3 by the magnetic force of the magnet of the magnetic field forming table 6, they do not flow out of the well 3 by the application of the buffer solution 64.

  Next, the biopolymer analysis chip 1 is heated to denature the sample DNA 62 into a single strand. Thereafter, in order to cause hybridization of the sample DNA 62, the biopolymer analysis chip 1 is gradually cooled to a predetermined temperature. At this time, the magnetic poles of the magnetic field forming table 6 are moved in the horizontal direction and the vertical direction with respect to the well 3, and the magnetic fine particles 60 are moved to arbitrary positions in the well 3 to stir the inside of the well 3 and complement each other. The probability that a typical sample DNA 62 and probe DNA 61 will hybridize can be increased.

  The biopolymer analysis chip 1 is cooled to a temperature range in which the complementary sample DNA 62 and probe DNA 61 cause hybridization. During this time, the magnetic pole of the magnetic field forming table 6 may be moved horizontally and vertically with respect to the well 3. Thereafter, the surface of the biopolymer analysis chip 1 provided with the well 3 is washed with a buffer that does not contain the sample DNA 62, and the sample DNA 62 that has not hybridized with the probe DNA 61 is washed away. Until it is washed away, the temperature in the well 3 is controlled within a temperature range that causes hybridization. Also at this time, since the magnetic fine particles 60 are attracted to the bottom of the well 3 by the magnet of the magnetic field forming table 6, they do not flow out of the well 3 by the buffer. Therefore, the sample DNA 62 labeled with the fluorescent material 63 is maintained in a state of being bound to the sample DNA 62 in the well 3 where the magnetic fine particles 60 to which the sample DNA 62 causing hybridization is fixed are washed away. There is no.

  Thereafter, the irradiation point of the excitation light 51 is moved two-dimensionally with respect to the DNA microarray by the laser oscillation device 7 and the optical system. At this time, fluorescence 52 is emitted from the excited fluorescent material 63 in the well 3 where hybridization has occurred, and the detection device 8 detects the fluorescence 52 by controlling the optical system. In this way, the fluorescence intensity for each well 3 can be acquired as two-dimensional image data. Further, position information data of the well 3 that emits fluorescence having an intensity exceeding the threshold value may be acquired. Since the magnetic fine particles 60 are transparent and transmit the excitation light 51 and the fluorescence 52, the fluorescent material 63 of the sample DNA 62 hybridized to the probe DNA 61 below the magnetic fine particles 60 can also be excited. Can be detected. In the well 3, even if a plurality of magnetic fine particles 60 overlap the optical path of the fluorescence 52 of the detection device 8, the fluorescence 52 can pass through the magnetic fine particles 60 and reach the detection device 8. A portion having a high fluorescence intensity in the obtained image indicates that the sample DNA 62 complementary to the probe DNA 61 is hybridized. Therefore, the base sequence of the sample DNA 62 is specified from the base sequence of the probe DNA 61 of the well 3 having a high fluorescence intensity, and it can be understood which gene is expressed in the sample.

  As described above, according to the present embodiment, the buffer in the well 3 is agitated by moving the magnetic fine particles 60 provided with the probe DNA 61 on the surface in the well 3 containing the buffer containing the sample DNA 62. In addition, the probability that the sample DNA 62 and the probe DNA 61 in the buffer are hybridized can be increased. Further, unlike the probe DNA 61 that is planarly fixed to the spot as in the prior art, the probe DNA 61 that is fixed to the magnetic fine particles 60 is arranged in an arbitrary three-dimensional direction in the buffer, so that the sample DNA 62 and the probe DNA 61 The probability of hybridizing with can be increased.

  Conventionally, the amount of the probe DNA 61 that can be immobilized on the spot is limited by the area of the spot, that is, two-dimensionally, but in this embodiment, the probe DNA 61 is immobilized on the surface of the magnetic fine particle 60. Therefore, it is possible to perform hybridization in the front, rear, left, right, top and bottom of one magnetic fine particle 60, and it is also possible for a plurality of magnetic fine particles 60 to be positioned one above the other in the vertical direction. It is also possible to fix the probe DNA 61 in an area larger than the area of the well 3 in FIG. Therefore, the amount of sample DNA 62 hybridized with the probe DNA 61 can be increased, the fluorescence intensity can be increased, the S / N ratio can be improved, and a high-sensitivity photosensor and a high-output excitation light source are not required. Thus, the apparatus can be made inexpensive.

  As shown in FIG. 4, a reflective coating 4 that reflects excitation light and fluorescence may be formed on the inner peripheral surface of the well 3. In this case, since the excitation light 51 irradiated to the well 3 is reflected by the reflective coating 4 and the excitation light is irradiated to the fluorescent material 63 from the lateral direction or the lower direction, the excitation light rate can be improved. . Further, since the fluorescence 52 emitted from the fluorescent material 63 in the lateral direction or the downward direction is reflected upward by the reflective coating 4, the detected fluorescence intensity can be increased.

Further, instead of two-dimensionally scanning the surface of the biopolymer analysis chip 1 with the optical system and the detection device 8, the line sensor is moved along the surface of the biopolymer analysis chip 1, thereby The fluorescence intensity may be acquired as two-dimensional image data. Or you may acquire the fluorescence intensity for every well 3 as two-dimensional image data by imaging with the electronic image sensor which consists of a solid-state image sensor and a lens.
In addition, the probe DNA 61 and the sample DNA 62 in the well 3 can be easily removed by removing the non-magnetic substrate 2 from the support base 5 and washing the well 3 after weakening the magnetic force of the magnetic field forming stage 6 after the analysis. The nonmagnetic substrate 2 can be reused.

[Second Embodiment]
Next, a second embodiment of the present invention will be described.
[5] Overall Configuration of Biopolymer Analysis Chip FIG. 5 is a schematic plan view of the biopolymer analysis chip 1 in the embodiment to which the present invention is applied, and FIG. 6 shows the biopolymer analysis chip 1 of FIG. It is sectional drawing which looked at the cut surface VI cut | disconnected in the thickness direction in the arrow direction. In the biopolymer analysis chip 1 of the present embodiment, an image sensor is integrally formed on a nonmagnetic substrate 2 similar to that of the first embodiment.

  The biopolymer analysis chip 1 has a solid-state imaging device 10 formed by arranging photoelectric conversion elements as pixels in a two-dimensional array on a transparent substrate 17 on the transparent substrate 17. A nonmagnetic substrate 2 similar to that of the first embodiment is in close contact. An excitation light reflecting coating 4 a that reflects light in the wavelength region of excitation light and transmits light in the wavelength region of fluorescence may be formed on the inner peripheral surface of the well 3. In this case, since the excitation light 51 irradiated to the well 3 from the excitation light irradiation device 72 described later is reflected by the excitation light reflection coating 4a, the excitation light is irradiated to the fluorescent material 63 from the lateral direction or the downward direction. The excitation light rate can be improved.

[6] Solid-State Imaging Device The solid-state imaging device 10 will be described in detail with reference to FIGS. Here, FIG. 7 is a plan view showing an electrode structure of a photoelectric conversion element that is a pixel of the solid-state imaging device 10, and FIG. 8 is a cross-sectional view of the photoelectric conversion element of the solid-state imaging device 10 in FIG. It is sectional drawing which looked at the cut surface VIII which looked in the arrow direction.
The solid-state imaging device 10 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 the solid-state imaging device 10, 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,. .. Are arranged in an array, particularly in a matrix, and these 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 30 formed on the semiconductor film 23 with the sandwich, the impurity semiconductor film 25 formed so as to overlap a 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 on one end portion of the semiconductor film 23 so as to partially overlap the channel protective film 24, 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 30 are formed for each double gate transistor 20 on the top gate insulating film 29. The top gate electrode 30 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 30 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,. Are formed integrally with a common top gate line 44. The top gate electrode 30 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 30 and the top gate lines 44, 44,... Of the double gate transistors 20, 20,... Are collectively covered with a protective insulating film 31, and the protective insulating film 31 is common to all the double gate transistors 20, 20,. It is the film | membrane formed in this way. The protective insulating film 31 has insulating properties and light transmissive properties, and is made of silicon nitride or silicon oxide.

  The solid-state imaging device 10 configured as described above has the surface of the protective insulating film 31 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. Further, the nonmagnetic substrate 2 similar to that of the first embodiment is adhered to the upper portion of the protective insulating film 31 so that one well 3 is disposed on the upper portions of the double gate transistors 20, 20,. Is done. It should be noted that several adjacent double gate transistors 20, 20,... Per well 3 may overlap, but in this case, the number of double gate transistors 20 overlapped in any well 3 is the same.

[7] Analysis Support Device Next, an analysis support device that supports imaging of the biopolymer analysis chip 1 by the solid-state imaging device 10 will be described with reference to FIGS. 9 and 10. FIG. 9 is a block diagram showing a circuit configuration of the analysis support apparatus 70, and FIG. 10 is a side view when the biopolymer analysis chip 1 is set in the analysis support apparatus 70. In FIG. 10, the biopolymer analysis chip 1 is shown broken.

  The analysis support apparatus 70 emits excitation light from above the light receiving surface of the solid-state imaging device 10 toward the light receiving surface, the biopolymer analyzing chip 1, the support base 71 on which the biopolymer analyzing chip 1 is detachably set. Excitation light irradiation device 72 for irradiation, top gate driver 74, bottom gate driver 75 and drain driver 76 for driving solid-state imaging device 10, excitation light irradiation device 72, top gate driver 74, bottom gate driver 75 and drain driver 76 , A controller 73 that controls the output, an output device 77 that performs output (display or print) according to a signal output from the controller 73, and a magnetic field forming table 78 similar to the magnetic field generating table 6 of the first embodiment.

The support base 71 is provided with a temperature adjusting device for appropriately adjusting the temperature range in which the probe DNA 61 is hybridized with DNA having a complementary base sequence and the temperature range in which the probe DNA 61 cannot be hybridized.
When the biopolymer analysis chip 1 is set on the support base 71, the top gate lines 44, 44,... Of the solid-state imaging device 10 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 10 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 1 is set on the support base 71, the source lines 42, 42,... Of the solid-state imaging device 10 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 support base 71, and when the biopolymer analysis chip 1 is mounted on the support base 71, the excitation light emitted in a planar shape from the excitation light irradiation device 72 is biopolymer analysis. The chip 1 is irradiated. Excitation light emitted by the excitation light irradiation device 72 is light in the ultraviolet wavelength region. The excitation light irradiation device 72 may be provided so that the wavelength range of the emitted excitation light can be varied.

The output device 77 is a plotter, a printer, or a display.
The magnetic field forming table 78 is provided below the support table 71 and has the same configuration and function as the magnetic field forming table 6 of the first embodiment.

  The top gate driver 74, the bottom gate driver 75, and the drain driver 76 cooperate to drive the solid-state imaging device 10. The top gate driver 74 is a shift register. That is, as shown in FIG. 11, the top gate driver 74 sequentially outputs reset pulses to the top gate lines 44, 44,. The level of the reset pulse is a high level of +5 [V]. On the other hand, the top gate driver 74 applies a low level potential of −20 [V] to each top gate line 44 when no reset pulse is output.

  The bottom gate driver 75 is a shift register. That is, as shown in FIG. 11, read pulses are sequentially output to the bottom gate lines 41, 41,. The level of the read pulse is a high level of +10 [V], and the level when the read pulse is not output is a low level of ± 0 [V].

  The top gate driver 74 outputs a read pulse to the bottom gate line 41 of the same row after the carrier accumulation period after the top gate driver 74 outputs the reset pulse to the top gate line 44 of any row. 74 and bottom gate driver 75 shift the output signal. That is, in each row, the timing at which the read pulse is output is delayed from the timing at which the reset pulse is output. The period from the start of reset pulse input to the top gate line 44 of any row to the end of read pulse input to the bottom gate line 41 of the same row is the selection period of that row. is there. The level of the reset pulse is a high level of +5 [V], and the level when the reset pulse is not output is a low level of −20 [V].

  As shown in FIG. 11, the drain driver 76 precharges all the drain lines 43, 43,... Between the reset pulse output and the read pulse output in the selection period of each row. A pulse is output. The level of the precharge pulse is a high level of +10 [V], and the level when the precharge pulse is not output is a low level of ± 0 [V]. Further, the drain driver 76 amplifies the voltages of the drain lines 43, 43,... After outputting the precharge pulse, and outputs the amplified voltages to the controller 73.

  The controller 73 has a function of turning on the excitation light irradiation device 72. Further, the controller 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 10 to the top gate driver 74, the bottom gate driver 75, and the drain driver 76. Has the function to perform. The controller 73 has a function of acquiring the light intensity distribution along the light receiving surface of the solid-state imaging device 10 as two-dimensional image data by A / D converting the electric signal input from the drain driver 76. The controller 73 has a function of causing the output device 77 to output an image according to the input two-dimensional image data.

  Next, operations of the biopolymer analysis chip 1 and the analysis support apparatus 70 and a DNA analysis method (identification method) will be described. The sample adjustment is the same as in the first embodiment.

[8] Analysis Method An operator applies a buffer containing the sample DNA 62 to the surface of the non-magnetic substrate 2 of the biopolymer analysis chip 1 set on the support 5. At this time, since the magnetic fine particles 60 are fixed to the bottom of the well 3 by the magnetic force of the magnet of the magnetic field forming table 78, the magnetic fine particles 60 do not flow out of the well 3 by the application of the buffer solution 64.

  Next, the biopolymer analysis chip 1 is heated to denature the sample DNA 62 into a single strand. Thereafter, in order to cause hybridization of the sample DNA 62, the biopolymer analysis chip 1 is gradually cooled to a predetermined temperature. At this time, the magnetic pole of the magnetic field forming stand 78 is moved in the horizontal direction with respect to the well 3, and the magnetic fine particles 60 are moved in the well 3 to stir the well 3 so that the sample DNA 62 and the probe DNA 61 are hybridized. Increase the probability of doing.

  When the biopolymer analysis chip 1 is cooled to a predetermined temperature, the surface of the non-magnetic substrate 2 of the biopolymer analysis chip 1 is washed with a buffer not containing the sample DNA 62, and the sample DNA 62 that has not hybridized with the probe DNA 61 is allowed to flow. leave. Also at this time, the magnetic fine particles 60 are fixed to the bottom of the well 3 by the magnetic force of the magnet of the magnetic field forming table 78, and therefore do not flow out of the well 3 by the application of the buffer solution 64.

  Next, the excitation light irradiation device 72 is opposed to the surface of the nonmagnetic substrate 2, and the top gate driver 74, the bottom gate driver 75, and the drain driver 76 are connected to the controller 73.

  Thereafter, when the controller 73 is activated, the controller 73 controls the excitation light irradiation device 72 to turn on the excitation light irradiation device 72, and excitation light is emitted from the excitation light irradiation device 72 toward the light receiving surface of the solid-state imaging device 10. .

  Since the sample DNA 62 is labeled, the well 3 hybridized with the sample DNA 62 out of the wells 3, 3, 3... Emits fluorescence (mainly in the visible light wavelength region), and from the well 3 not bound to the sample DNA 62. Does not fluoresce. Therefore, high-intensity fluorescence is incident on the double gate transistor 20 corresponding to the well 3 bonded to the sample DNA 62, and almost no fluorescence is incident on the double gate transistor 20 corresponding to the well 3 not bonded to the sample DNA 62. Since the non-magnetic substrate 2 is fixed immediately above the light receiving surface of the solid-state imaging device 10, the fluorescence emitted from the well 3 combined with the sample DNA 62 is not attenuated so much, and the double gate transistor 20 corresponding to the well 3. To generate electron-hole pairs. Therefore, even if the sensitivity of the double gate transistors 20, 20,... Is low, the strength can be sufficiently detected. Even if a plurality of magnetic fine particles 60 overlap the optical path of the fluorescence 52 of the double gate transistor 20 in the well 3, the fluorescence 52 can pass through the magnetic fine particles 60 and reach the double gate transistor 20.

  Thereafter, the controller 73 controls the top gate driver 74, the bottom gate driver 75, and the drain driver 76 in a state where the excitation light irradiation device 72 is lit, thereby causing the solid-state imaging device 10 to perform an imaging operation. As a result, the solid-state imaging device 10 detects the light intensity or the light amount by each of the double gate transistors 20, 20,..., And acquires the light intensity distribution along the light receiving surface as two-dimensional image data. The controller 73 inputs the image data acquired by the solid-state imaging device 10 and outputs the image to the output device 77. Then, the controller process ends.

  The operator confirms the presence or absence of hybridization from the image data output by the output device 77. If hybridization has occurred, the base sequence of the sample DNA 62 is specified from the base sequence of the probe DNA 6161, and which gene is in the sample. You can see if it is expressed.

Here, the operation of the solid-state imaging device 10 by the top gate driver 74, the bottom gate driver 75, and the drain driver 76 will be described.
The top gate driver 74 sequentially outputs reset pulses from the top gate line 44 of the first row to the top gate line 44 of the last row, and the bottom gate driver 75 sequentially reads the read pulses to the bottom gate lines 41, 41, 41,. Is output. At that time, the drain driver 76 outputs a precharge pulse to all the drain lines 43, 43,... Between the reset period in which the reset pulse is output in each row and the period in which the read pulse is output in each row. .

  The operation of each double gate transistor 20 in the i-th row will be described in detail. When the top gate driver 74 outputs a reset pulse to the i-th top gate line 44, the i-th top gate line 44 goes to a high level. While the i-th top gate line 44 is at a high level (this period is referred to as a reset period), in each double-gate transistor 20 in the i-th row, the semiconductor film 23 and the channel protective film 24 are included in the semiconductor film 23. The carriers (here, holes) accumulated near the interface with the surface are repelled and discharged by the voltage of the top gate electrode 30.

  Next, the top gate driver 74 finishes outputting the reset pulse to the i-th top gate line 44. The amount of light depends on the amount of light during the period from when the reset pulse of the top gate line 44 in the i-th row ends to when the read pulse is output to the bottom gate line 41 in the i-th row (this period is referred to as a carrier accumulation period). An amount of electron-hole pairs is generated in the semiconductor film 23, and the holes are accumulated in the semiconductor film 23 or in the vicinity of the interface between the semiconductor film 23 and the channel protective film 24 by the electric field of the top gate electrode 30. The

  Next, during the carrier accumulation period, the drain driver 76 outputs a precharge pulse to all the drain lines 43, 43,. While the precharge pulse is output (referred to as a precharge period), in each double gate transistor 20 in the i-th row, the potential applied to the top gate electrode 30 is −20 [V], and the bottom Since the potential applied to the gate electrode 21 is ± 0 [V], even if only the charge of holes accumulated in the semiconductor film 23 or in the vicinity of the interface between the semiconductor film 23 and the channel protective film 24 is gate-source. Since the interpotential is low, no channel is formed in the semiconductor film 23, and no current flows between the drain electrode 28 and the source electrode 27. Since no current flows between the drain electrode 28 and the source electrode 27 during the precharge period, the drain electrode 28 of each double gate transistor 20 in the i-th row is output by the precharge pulse output to the drain lines 43, 43,. Is charged.

  Next, the drain driver 76 finishes outputting the precharge pulse, and the bottom gate driver 75 outputs a read pulse to the i-th bottom gate line 41. While the bottom gate driver 75 outputs a read pulse to the i-th bottom gate line 41 (this period is referred to as a read period), +10 is applied to the bottom gate electrode 21 of each i-th double gate transistor 20. Since the potential of [V] is applied, each double gate transistor 20 in the i-th row is turned on.

  In the read period, the carriers accumulated in the carrier accumulation period work so as to alleviate the negative electric field of the top gate electrode 30, so that an n channel is formed in the semiconductor film 23 by the positive electric field of the bottom gate electrode 21, and the drain electrode Current flows from 28 to the source electrode 27. Therefore, in the read period, the voltages of the drain lines 43, 43,... Tend to gradually decrease with time due to the drain-source current.

  Here, as the amount of light incident on the semiconductor film 23 in the carrier accumulation period increases, the number of accumulated carriers also increases. As the number of accumulated carriers increases, the current flowing from the drain electrode 28 to the source electrode 27 in the read period is increased. The level also increases. Therefore, the voltage change tendency of the drain lines 43, 43,... During the read period is deeply related to the amount of light incident on the semiconductor film 23 during the carrier accumulation period. The drain lines 43 and 43 after the elapse of a predetermined time from the start of the read period via the drain driver 76 during the period from the i-th read period to the next (i + 1) -th precharge period. ,... Are detected and A / D converted. Thereby, it converts into the intensity | strength of light. Note that the time required to reach a predetermined threshold voltage may be detected via the drain driver 76 between the i-th read period and the next (i + 1) -th precharge period. Even in this case, the light intensity is converted. In FIG. 10, the rising timing of the reset pulse of the (i + 1) th row of the top gate driver 74 is after the read pulse of the i-th row of the bottom gate driver 75 falls. The rising timing of the reset pulse of the (i + 1) -th row of the gate driver 74 is from immediately after the falling edge of the reset pulse of the i-th row of the top gate driver 74 to the falling edge of the read pulse of the i-th row of the bottom gate driver 75. It may be. However, the output of the precharge pulse output to the drain lines 43, 43,... For the double gate transistor 20 in the (i + 1) -th row is after the falling edge of the read pulse in the i-th row of the bottom gate driver 75. Is set to

  The above-described series of image reading operations is set as one cycle, and the same processing procedure is repeated for each double gate transistor 20 in all rows, whereby the light intensity distribution on the biopolymer analysis chip 1 is acquired as an image. . An image representing the light intensity distribution is input to the controller.

  As described above, according to the present embodiment, since the nonmagnetic substrate 2 is in close contact with the light receiving surface of the solid-state imaging device 10, only imaging with the solid-state imaging device 10 is performed without scanning excitation light. A two-dimensional image can be obtained. Furthermore, since the clear image can be obtained with the solid-state imaging device 10 without providing a lens in the analysis support apparatus 70, the analysis support apparatus 70 can be downsized. Furthermore, since the light emitted from the well 3 is incident on the light receiving surface of the solid-state imaging device 10 without being attenuated, the sensitivity of the solid-state imaging device 10 may not be high.

  Note that an antireflection film may be formed on the light receiving surface of the solid-state imaging device 10 to improve the fluorescence transmittance. Thereby, even if the fluorescence sensitivity of the solid-state imaging device 10 is low, a clear image can be obtained by the solid-state imaging device 10.

  In addition, the probe DNA 61 and the sample DNA 62 in the well 3 can be easily removed by removing the non-magnetic substrate 2 from the support base 5 and washing the well 3 after weakening the magnetic force of the magnetic field forming stage 78 after the analysis. The nonmagnetic substrate 2 can be reused.

  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.

[Modification 1]
In the second embodiment, the excitation light irradiation device 72 is installed above the biopolymer analysis chip 1 and irradiates the excitation light toward the well 3 of the nonmagnetic substrate 2. On the other hand, as shown in FIG. 12, the excitation light irradiation device 72 may be installed below the biopolymer analysis chip 1 and the solid-state imaging device 10. In this case, the biopolymer analysis chip 1 is set with the back surface of the solid-state imaging device 10 facing the excitation light irradiation device 72, and the excitation light is irradiated from below the solid-state imaging device 10 by the excitation light irradiation device 72. Irradiated toward. Since the solid-state imaging device 10 is light-transmitting except for the bottom gate electrode 21, the bottom gate line 41, the source electrode 27, the source line 42, the drain electrode 28, and the drain line 43, the excitation light is transmitted to the double gate transistor 20, The light is emitted upward from the light receiving surface of the solid-state imaging device 10 between 20,. Further, in this case, since the bottom gate electrode 21 is shielded from light, the excitation light is not directly incident on the semiconductor film 23 from the excitation light irradiation device 72, and therefore the excitation light reflection coating 4a may not be provided.

[Modification 2]
In the second embodiment, the solid-state imaging device 10 using the double gate transistors 20, 20,... As the pixels is used as the photoelectric conversion element. However, a solid-state imaging device using another type of photoelectric conversion element as the pixel is used. You may use for a biopolymer analysis chip. For example, a solid-state imaging device such as a CCD image sensor or a CMOS image sensor using a photodiode as a pixel may be used. In a CCD image sensor, photodiodes are arranged in a matrix on a substrate, and around each photodiode, a vertical CCD and a horizontal CCD for transferring an electrical signal photoelectrically converted by the photodiode. Is formed. In a CMOS image sensor, photodiodes are arranged in a matrix on a substrate, and a CMOS circuit for amplifying an electric signal photoelectrically converted by the photodiodes is provided around each photodiode. Yes. Whether it is a CCD image sensor or a CMOS image sensor, an antireflection film similar to the antireflection film 35 is formed on the light receiving surface, and a plurality of types of spots are spotted on the antireflection film.

[Modification 3]
In the above embodiment, the controller 73 outputs an image according to the image data input from the solid-state imaging device 10 to the output device 77, and the operator specifies expression information from the output image data. Expression information may be specified. That is, the controller specifies which part of the image data has high fluorescence intensity by the feature extraction process, specifies the spot 60 corresponding to the part having high fluorescence intensity, and a base sequence complementary to the specified spot 60 Is output from the output device.

[Modification 4]
In the first and second embodiments, the excitation light is ultraviolet light, and the fluorescence emitted from the sample DNA 62 by the excitation light is visible light. However, the wavelength range of such light is not limited. However, the excitation light is light in a wavelength region that excites the fluorescent material 63 bonded to the sample DNA 62, and the main wavelength region of the fluorescence emitted from the fluorescent material 63 by the excitation light is sufficiently different from the main wavelength region of the excitation light. is required.

[Modification 5]
In the first and second embodiments, the fluorescence intensity emitted from the fluorescent material 63 is measured. However, instead of the fluorescent material 63, a chemiluminescent material may be used as a labeling material to measure the emission intensity. In this case, an excitation light irradiation device is not necessary. However, it is necessary for the detection apparatus 8 or the solid-state imaging device 10 to show sensitivity to light emitted from the chemiluminescent substance.

[Modification 6]
In the first and second embodiments, DNA is used for both the probe and the sample, but the probe only needs to specifically bind to the biopolymer as the sample. For example, as a combination of the probe and the sample, Alternatively, a combination of an antigen and an antibody or a combination of an enzyme and a substrate may be used.

[Modification 7]
In the second embodiment, since the refractive index of the well 3 (buffer solution 64) and the refractive index of the nonmagnetic substrate 2 are not necessarily equal, the well 3 is moved from the well 3 to the nonmagnetic substrate 2 at the interface. Although there is a risk that the light incident on the interface will be reflected at this interface, an antireflection film that transmits light in the same wavelength range as the fluorescence incident from the well 3 to this interface may be provided at this interface. .

  A plurality of the above modifications may be combined.

1 is a schematic plan view of a biopolymer analysis chip 1 in an embodiment of the present invention. 3 is a cross-sectional view showing one well 3 of the biopolymer analysis chip 1. FIG. 2 is a schematic diagram illustrating an overall configuration of an analysis support apparatus 50. FIG. It is sectional drawing which shows the other form of one well 3 of the biopolymer analysis chip | tip 1. FIG. 1 is a schematic plan view of a biopolymer analysis chip 1 in an embodiment of the present invention. FIG. 6 is a cross-sectional view taken along a cutting plane VI in FIG. 5. 2 is a plan view of one pixel of the solid-state imaging device 10. FIG. FIG. 8 is a cross-sectional view taken along a cutting plane VIII in FIG. 7. 3 is a block diagram showing a circuit configuration of an analysis support apparatus 70. FIG. 3 is a schematic side view of an analysis support apparatus 70. FIG. 6 is a timing chart showing the transition of the level of an electrical signal output to the solid-state imaging device 10 by a driver. It is a schematic side view of 70 A of analysis assistance apparatuses of another example.

Explanation of symbols

1 Biopolymer Analysis Chip 2 Nonmagnetic Substrate 3 Well 4 Reflective Coating 4a Excitation Light Reflective Coating 6 Magnetic Field Forming Table (Magnet)
10 Solid-state imaging device (photoelectric conversion element)
60 Magnetic particles 61 Probe DNA (probe)
62 Sample DNA (Biopolymer)
63 Fluorescent substance (labeling substance)
64 Buffer solution

Claims (6)

A non-magnetic substrate having a plurality of wells on the surface;
A biopolymer analysis chip comprising: magnetic fine particles that are housed in each well and have a probe that binds to a specific biopolymer.   The biopolymer analysis chip according to claim 1, wherein a reflection coating that reflects light is formed on an inner peripheral surface of the well. A plurality of photoelectric conversion elements arranged in a two-dimensional array are provided below the non-magnetic substrate,
The biopolymer analysis chip according to claim 1 or 2, wherein the non-magnetic substrate has optical transparency.   An excitation light reflecting coating that reflects excitation light that excites the fluorescent substance attached to the biopolymer and transmits fluorescence emitted from the fluorescent substance is formed on the inner peripheral surface of the well. The biopolymer analysis chip according to claim 3. The biopolymer analysis chip according to any one of claims 1 to 4,
Magnetic field forming means capable of moving the magnetic fine particles in the well by forming a magnetic field;
An analysis support apparatus comprising: A magnetic fine particle having a probe that binds to a specific biopolymer is accommodated in each well provided on the surface of the non-magnetic substrate,
A biopolymer sample labeled with a labeling substance is injected into each well,
Washing off each well of the non-magnetic substrate with a magnetic field formed on the non-magnetic substrate,
A biopolymer analysis method comprising detecting a labeling substance remaining in each well.

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