JP4179169B2 - Analysis equipment - Google Patents

Description

  The present invention relates to a process for generating a reaction product from a specimen and an analyzer used for performing a process for analyzing the reaction product.

  In recent years, genetic information on living organisms has been used in a wide range of fields such as the medical field and the agricultural field. However, elucidation of the DNA structure is indispensable when using genes. DNA has two polynucleotide strands that are twisted in a spiral shape, and each polynucleotide strand is one-dimensionally composed of four types of bases (adenine: A, guanine: G, cytosine: C, thymine: T). Based on the complementarity of adenine and thymine and guanine and cytosine, the bases of one polynucleotide chain are bonded to the bases of the other polynucleotide chain.

In order to analyze the DNA structure of the sample, the DNA is extracted from the sample, the step of amplifying the DNA, the step of attaching a fluorescent label to the DNA, etc., and the obtained DNA is analyzed using a thermal lens microscope or the like. (For example, refer to Patent Document 1).
JP 2000-287682 A

  However, there is a problem that it takes a long time through complicated steps to obtain DNA from a specimen. Even if a thermal lens microscope is used to analyze the obtained DNA, a highly sensitive and expensive thermal lens microscope is required to detect the DNA, and the cost of DNA analysis increases.

  Therefore, the present invention has been made in order to solve the above-described problems, and is an analysis apparatus that can easily generate a reaction product such as DNA from a sample and analyze the reaction product in a short time. The purpose is to provide.

In order to solve the above problems, the analyzer of the present invention provides:
A reactor provided on a substrate and having a reaction furnace for generating DNA formed therein;
A solid-state imaging device provided on the substrate, and spots having a probe DNA fragment having a known base sequence scattered in a matrix on the light-receiving surface of the solid-state imaging device, and generated in the reactor An inspection device for specifically inspecting the DNA sequence;
A groove communicating the inspection device with the previous reactor;
A temperature adjusting means for heating the inspection apparatus provided on the light receiving surface of the solid-state imaging device so as to be orthogonal to the groove along an edge closer to the reactor among the outer edges of the solid-state imaging device. , equipped with a,
The spot is characterized Rukoto which have a lower said probe DNA fragments of duplex binding temperature increasing distance from said temperature adjusting means.

As described above, since the reactor and the inspection apparatus are integrated, the reaction product generated by the reaction process in the reactor can be directly sent to the inspection apparatus without being separated. For this reason, even if it is a trace amount reaction material, it becomes possible to detect the presence efficiently with an inspection apparatus.
In particular, when the DNA base sequence is specified, if the specimen injection opening and the reagent injection opening leading to the reaction furnace are formed in the reactor, the operator injects the specimen into the specimen injection opening. When the operator injects the reagent into the reagent injection opening, the specimen and the reagent react in the reaction furnace, and the reaction product flows out from the outlet of the reaction furnace. If the inspection apparatus is an optical solid-state imaging device, the reaction product spreads on the light-receiving surface of the solid-state imaging device because the outlet of the reaction furnace is guided to the light-receiving surface of the solid-state imaging device. If imaging is performed with the solid-state imaging device, the reaction product that has spread on the light-receiving surface of the solid-state imaging device can be analyzed. Thus, even without an expensive apparatus such as a thermal lens microscope, the reaction product can be analyzed with a solid-state imaging device that is less expensive than such an apparatus.

  Thus, when the reactor, the specimen injection opening, and the reagent injection opening are formed in the reactor, the operator injects the specimen into the specimen injection opening and the reagent into the reagent injection opening. A reaction product can be easily produced simply by injection. Therefore, no complicated chemical operation technique is required, and anyone can produce a reaction product. In addition, since the reactor and the inspection device are integrated, the process from the generation of the reaction product to the analysis of the reaction product can be performed in a short time.

  When temperature adjusting means for heating or cooling the inspection apparatus or the reactor is provided, it is possible to easily control the temperature of hybridization during DNA base sequence inspection. As the temperature adjusting means, there is a thin film electric heating resistor, and the entire apparatus can be made thin.

  In particular, since it is provided between the reactor and the solid-state imaging device, a temperature gradient is generated in each region of the solid-state device.

  The inspection apparatus includes: a solid-state imaging device; and a protrusion provided on a light-receiving surface of the solid-state imaging device in parallel with a pixel row of the solid-state imaging device, and between the plurality of protrusions The formed groove is preferably communicated with the reaction furnace.

  As described above, since a plurality of protrusions become walls, it becomes possible to move the reaction product along the groove formed between the protrusions, and the reaction product can be efficiently applied to the pixels of the solid-state imaging device. Can be transported.

  It is preferable that a voltage applying means for applying a voltage to the grooves between the protrusions is provided.

  As the voltage applying means, an electrode provided in the vicinity of the groove is particularly preferable. As described above, since an electric field along the groove can be generated, if the reaction product is filled with an electrophoresis medium during hybridization in the case of DNA base sequence inspection, the reaction product is moved along the groove. Electrophoresis.

  It is preferable that there is at least one column of pixels of the solid-state imaging device per groove formed between the plurality of protrusions.

  The inspection apparatus includes a solid-state imaging device and a plurality of spots made of a probe DNA fragment having a known base sequence and scattered on a light receiving surface of the solid-state imaging device, and the solid-state imaging device for each spot It is preferable that at least one of the pixels overlap.

  Since spots of known base sequences are scattered on the light-receiving surface of the solid-state imaging device, if the spot where the sample DNA fragment as a reaction product is hybridized is detected by the solid-state imaging device, the detected spot is complementary. A simple base sequence is the base sequence of the sample DNA fragment. Therefore, the base sequence of the sample DNA fragment as the reaction product can be identified.

  According to the present invention, even without an expensive apparatus such as a thermal lens microscope, the reaction product can be analyzed with a solid-state imaging device that is less expensive than such an apparatus. Further, a complicated chemical operation technique is not required, and the process from the generation of the reaction product to the analysis of the reaction product can be easily performed in a short time. In particular, if the reaction product is transferred to an inspection device from a flask, test tube, or petri dish as in the past, the residue may remain and the detection accuracy may be reduced in the case of a small amount of reaction product. Since the product and inspection can be performed in a series of processes, they can be processed efficiently.

  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]
FIG. 1 is a plan view of a DNA analyzer 1 according to an embodiment to which the analyzer of the present invention is applied.

  As shown in FIG. 1, a DNA analyzer 1 includes a reactor 50 for generating DNA from a specimen, and a DNA array chip 2 as an inspection device for specific inspection of the sequence of DNA generated in the reactor 50. Are integrally provided on the same substrate 51.

  2 is a cross-sectional view taken along the line II-II in FIG. As shown in FIG. 2, a reactor 50 includes a substrate 51 made of a transparent member such as glass and a substrate 52 formed in a plate shape from at least one material of ceramic, silicon, aluminum, and glass. These surfaces are superposed and bonded together via a polyimide layer 58 in which a groove 59 serving as a microchannel 53 is formed. The groove 59 of the polyimide layer 58 is formed in a twisted shape on the joint surface with the substrate 52 by an etching method. By bonding the substrate 52 to the polyimide layer 58 so as to cover the groove 59, the groove 59 is formed. The space formed by 59 becomes a micro flow path 53 as a reaction furnace.

  In the substrate 52, four openings 54, 55, 56, 57 (shown in FIG. 1) penetrating from the front surface to the back surface of the substrate 52 are formed. These openings 54, 55, 56 and 57 communicate with the microchannel 53. The path of the micro flow path 53 will be described. As shown in FIG. 1, the path from the opening 54 to the merge part 53a merges with the path from the opening 55 to the merge part 53a at the merge part 53a. The path from the merging portion 53a to the merging portion 53b merges with the path from the opening 56 to the merging portion 53b at the merging portion 53b. The path from the merging portion 53b to the merging portion 53c is joined at the merging portion 53c. The micro flow channel 53 is continuous from the merging portion 53c to the branching portion 53d and branches into six paths at the branching portion 53d. Each of the six paths branched at the branch part 53d is open on the side surface of the reactor 50, and the ends of the six paths branched at the branch part 53d are outlets.

  Although details will be described later, when the DNA analyzer 1 is used, a specimen containing DNA such as blood is injected from the opening 54 and poured into the microchannel 53, and a DNA separation and extraction reagent is injected from the opening 55. In the microchannel 53, the DNA fragments in the specimen are each separated into a helical molecular structure, and then an enzyme reagent called a polymerase is injected from the opening 56 and mixed with the DNA separated in the microchannel 53. Then, a fluorescent substance reagent that becomes a fluorescent label is injected from the opening 57 and physically or chemically combined with the amplified DNA fragment in the microchannel 53.

  The DNA array chip 2 will be described with reference to FIGS. 3 is a cross-sectional view taken along the line III-III in FIG. 1, and FIG. 4 is a plan view of the DNA array chip 2. FIG. 3 and 4, the DNA array chip 2 includes a solid-state imaging device 3, spots 60, 60,... Scattered in a matrix on the light-receiving surface of the solid-state imaging device 3, and the light reception of the solid-state imaging device 3. A plurality of protrusions 62, 62, ... provided on the surface.

  The solid-state imaging device 3 includes a substantially flat transparent substrate 51 shared with the reactor 50, and a plurality of double-gate field effect transistors (6 × 6 matrix arranged on one surface of the transparent substrate 51). .., And a protective insulating layer 31 that collectively covers the double gate transistors 20, 20,.

  The transparent substrate 51 has a property of transmitting light (hereinafter referred to as light transmissive property) and has an insulating property, and is a glass substrate such as quartz glass or a plastic substrate such as polycarbonate or PMMA. A reactor 50 is provided, and the solid-state imaging device 3 is provided in a downstream region.

  The double gate transistor 20 will be described with reference to FIGS. Here, FIG. 5 is a plan view showing one pixel of the solid-state imaging device 3, and FIG. 6 is a cross-sectional view taken along the line VI-VI in FIG.

  The double gate transistors 20, 20,... Are photoelectric conversion elements serving as pixels. Each of the double gate transistors 20, 20,... Has a bottom gate electrode 21 formed on the transparent substrate 51, a bottom gate insulating film 22 formed on the bottom gate electrode 21, and a bottom gate insulating film 22 as a bottom gate electrode. Intrinsic semiconductor film 23 sandwiched between 21 and facing bottom gate electrode 21, channel protective film 24 formed on the central portion of semiconductor film 23, and formed on both ends of semiconductor film 23 so as to be spaced apart from each other Impurity semiconductor films 25 and 26, source electrode 27 formed on impurity semiconductor film 25, drain electrode 28 formed on impurity semiconductor film 26, and top gate formed on source electrode 27 and drain electrode 28 The insulating film 29, the top gate insulating film 29, and the channel protective film 24 are sandwiched between the semiconductor film 23 and the semiconductor film 23. A top gate electrode 30 which comprises a.

  The bottom gate electrode 21 is formed on the transparent substrate 51 for each double gate transistor 20. Further, six bottom gate lines 41, 41,... Extending in the horizontal direction are formed on the transparent substrate 51, and the six double gate transistors 20, 20, 20 in the same row arranged in the horizontal direction are formed. ... 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 insulating film 22 is formed in common to all the double gate transistors 20, 20,... And collects the bottom gate electrode 21 and the bottom gate lines 41, 41,. Covered. 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 ).

  On the bottom gate insulating film 22, a semiconductor film 23 is formed for each double gate transistor 20. The semiconductor film 23 has a substantially rectangular shape in plan view, and is not sufficiently excited even when receiving ultraviolet light (wavelength range of less than 400 nm) and receives visible light (400 nm or more) having a longer wavelength than ultraviolet light. Then, it is a layer formed of amorphous silicon or polysilicon that is sufficiently excited to generate an amount of electron-hole pairs corresponding to the amount of light. A channel protective film 24 is formed on the semiconductor film 23. 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 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 patterned for each double gate transistor 20 is formed on the impurity semiconductor film 25. A drain electrode 28 patterned for each double gate transistor 20 is formed on the impurity semiconductor film 26. .. And drain lines 43, 43,... Extending in the vertical direction are formed on the bottom gate insulating film 22. Each of the six double gate transistors 20, 20,... In the same column arranged in the vertical direction is integrally formed with a common source line 42, and the six source electrodes 27 in the same column arranged in the vertical direction. The drain electrodes 28 of the double gate transistors 20, 20,... Are integrally formed 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.

  The top gate insulating film 29 is formed in common to all the double gate transistors 20, 20,..., And the channel protective film 24, the source electrode 27, the drain electrode 28, and the source line of the double gate transistors 20, 20,. .. And the drain lines 43, 43,... Are collectively covered. 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 top gate electrode 30 patterned for each double gate transistor 20 is formed on the top gate insulating film 29. Further, six top gate lines 44, 44,... Extending in the lateral direction are formed on the top gate insulating film 29, and the six double gate transistors 20, 20 in the same row arranged in the lateral direction are formed. 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 protective insulating layer 31 collectively covers the top gate electrode 30 and the top gate lines 44, 44,... Of the double gate transistors 20, 20,. The protective insulating layer 31 has insulating properties and light transmittance, and is made of silicon nitride or silicon oxide.

  The solid-state imaging device 3 configured as described above uses the surface of the protective insulating layer 31 as a light receiving surface, and each double gate transistor 20 is a photoelectric conversion element that converts the amount of light received by the semiconductor film 23 into an electrical signal. is there.

  On the light receiving surface of the solid-state imaging device 3, that is, on the protective insulating layer 31, the excitation light irradiated from the upper side toward the double gate transistor 20 to excite the phosphor is shielded, and the phosphor is emitted by the excitation light. An excitation light shielding film 32 that transmits light in the long wavelength region is formed on the entire surface.

  On the excitation light shielding film 32, an overcoat layer 33 having optical transparency is formed on the entire surface. The overcoat layer 33 protects the excitation light shielding film 32 and fixes the spots 60, 60,... To the light receiving surface of the solid-state imaging device 3.

  Next, the spot 60 will be described. 1, 3, 4, etc., a plurality of types of spots 60, 60,... Are separated from each other, and are formed in a matrix of 6 rows and 6 columns on the overcoat layer 33 for easy understanding. It is arranged. As shown in FIG. 6, one spot 60 is a group in which a large number of single-stranded probe DNA fragments 61 are gathered, and a large number of single-stranded probe DNA fragments 61 contained in one spot 60 have the same base sequence (nucleotide sequence). ). Further, the single-stranded probe DNA fragment 61 has a different base sequence for each spot 60. Each spot 60 has a known base sequence.

  The spots 60, 60,... Are arranged in correspondence with the double gate transistors 20, 20,. That is, when the solid-state imaging device 3 is viewed in plan as mainly shown in FIG. 4, one spot 60 overlaps one double gate transistor 20, and in particular, the semiconductor film 23 of the double gate transistor 20 has one spot 60. It overlaps with.

As a method for fixing the spots 60, 60,... To the light receiving surface of the solid-state imaging device 3, a solid-state imaging device in which a probe DNA fragment prepared in advance is surface-treated with a polycation (poly-L-lysine, polyethyleneimine, etc.). 3 is applied to the surface of the solid-state imaging device 3 by spotting on the light receiving surface 3 using a dispensing device and utilizing the charge of DNA.
As another fixing method, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group or the like is also used. In this case, since an amino group, an aldehyde group, or the like is introduced to the surface of the solid-state imaging device 3 through a covalent bond, the amino group or aldehyde group is stably present on the surface of the solid-state imaging device 3 as compared with the case of using a polycation.
As another immobilization method, there is a method in which an oligonucleotide having a reactive group introduced therein is synthesized, the oligonucleotide is spotted on the surface of the surface-treated solid-state imaging device 3 and covalently bonded.

  On the light-receiving surface of the solid-state imaging device 3, ridges 62, 62,... Formed integrally with the polyimide layer 58 of the reactor 50 are provided. The ridges 62, 62, ... extend in the vertical direction between the rows of spots 60, 60, ... arranged in the vertical direction. ... are formed between the protrusions 62, 62, ..., and six spots 60, 60, ... are arranged in the vertical direction for each groove 63. The groove 63 communicates integrally with the groove 59 of the polyimide layer 58, and the outlets at the ends of the six branches in the branch portion 53d of the reactor 50 are connected to the grooves 63, 63,. ing. The longitudinal direction of the groove 63 is parallel to the drain line 43.

  Next, a DNA reader using the DNA analyzer 1 configured as described above will be described with reference to FIGS.

  As shown in FIGS. 7 and 8, the DNA reader 70 includes a display device 71, an arithmetic processing device 72 that performs overall control, and a light irradiation unit that irradiates the surface of the solid-state imaging device 3 with excitation light in a planar shape. 73 and a driving device (comprising a top gate driver 74, a bottom gate driver 75, a drain driver 76, and a driving circuit 77) for driving the solid-state imaging device 3 to acquire an image.

  The light irradiation unit 73 includes, for example, a light source 78 that emits excitation light such as ultraviolet light that does not include a wavelength region that sufficiently excites the semiconductor film 23 and sufficiently excites a phosphor that will be described later, and ultraviolet light emitted from the light source 78. Includes a light guide 79 that is emitted from the emission surface 79a to the outside. The DNA analyzer 1 is detachable from the DNA reader 70, and the light receiving surface of the solid-state imaging device 3 of the DNA analyzer 1 mounted on the DNA reader 70 is close to the emission surface 79a of the phosphor excitation light. And come to face each other. When the light receiving surface of the solid-state imaging device 3 faces the emission surface 79a of the light guide 79, the light emitted from the emission surface 79a is evenly applied to the surface of the solid-state imaging device 3.

  When the DNA analyzer 1 is attached to the DNA reader 70, the top gate lines 44, 44,... Of the solid-state imaging device 3 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 3 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 DNA analyzer 1 is mounted on the DNA reader 70, the source lines 42, 42,... Of the solid-state imaging device 3 are connected to a constant voltage source, and in this example, the source lines 42, 42,. It has become so.

  The top gate driver 74 is a shift register. In other words, the top gate driver 74 receives the control signal Tcnt from the drive circuit 77, and in this order from the top gate line 44 of the first row to the top gate line 44 of the sixth row (if the sixth row is reached, as necessary. (Returns to the first line.) A reset pulse (shown in FIG. 9) is output. 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, read pulses (shown in FIG. 9) are output in the order from the bottom gate line 41 of the first row to the bottom gate line 41 of the sixth row (when the sixth row is reached, return to the first row as necessary). It has become. 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].

  After the top gate driver 74 outputs a reset pulse to the i-th top gate line 44 (i is an integer from 1 to 6), the bottom gate driver 75 passes through the carrier accumulation period and the bottom gate driver 75 then enters the i-th bottom gate line. The top gate driver 74 and the bottom gate driver 75 shift the output signal so that a read pulse is output to 41. 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. Also, after the reset pulse is input to the top gate line 44 of the i-th row (i is any one of 1 to 6), the read pulse is input to the i-th bottom gate line 41. The period until ending is the selection period of the i-th row. 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].

  The drain driver 76 applies precharge pulses (shown in FIG. 9) to all the drain lines 43, 43,... Between the reset pulse output and the read pulse output in the selection period of each row. 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]. 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 drive circuit 77.

  The drive circuit 77 is driven by the arithmetic processing unit 72 to output control signals Bcnt, Tcnt, Dcnt to the bottom gate driver 75, the top gate driver 74, and the drain driver 76, respectively, thereby causing the bottom gate driver 75, the top gate, The driver 74 and the drain driver 76 are configured to appropriately output pulses. Further, the drive circuit 77 detects the voltage of the drain lines 43, 43,... After a predetermined time has elapsed after the read pulse is output, or the drain circuit 43, 43,. By detecting the time until the voltage reaches a predetermined threshold voltage, an image is acquired and the image is output to the arithmetic processing device 72. The arithmetic processing device 72 is configured to display an image input from the drive circuit 77 on the display device 71.

  As described above, since the spots 60, 60,... Are arranged on the surface of the solid-state imaging device 3, a clear image is obtained with the solid-state imaging device 3 without providing the DNA reader 70 with an optical system such as a lens / microscope. Can be imaged. Therefore, the DNA reader 70 can be reduced in size.

A DNA analysis method (identification method) using the DNA analyzer 1 and the DNA reader 70 will be described.
First, an operator injects a specimen into the opening 54, injects a DNA separation / extraction reagent into the opening 55, injects an enzyme reagent called a polymerase into the opening 56, and injects a fluorescent substance reagent into the opening 57. . Note that the fluorescent material injected into the opening 57 emits visible light when excited by ultraviolet rays.

Then, a sample (for example, blood, saliva, etc.) that flows in the microchannel 53 and reaches the confluence section 53a is mixed with the DNA separation / extraction reagent and heated by a heater (temperature adjusting means) provided in the reactor 50. When heated, the DNA of the specimen is extracted in a state where the helical structure is released. Note that the sample is heated together with the reactor 50 when the heater provided in the reactor 50 generates heat.
Thereafter, when the extracted single-stranded DNA reaches the merging portion 53b, the DNA is mixed with an enzyme reagent, whereby the DNA is amplified by a PCR reaction (polymerase chain reaction). Thereafter, when the amplified DNA reaches the merging portion 53c, the DNA is mixed with a fluorescent substance, and thereby the DNA is fluorescently labeled. Thereafter, the fluorescently labeled DNA is diverted at the branching portion 53d, and flows out into the grooves 63, 63,... From the ends of the six paths branched at the branching portion 53d. Here, the DNA is denatured into single-stranded DNA fragments before flowing into the grooves 63, 63,. Hereinafter, a single-stranded DNA fragment denatured from a specimen is referred to as a sample DNA fragment.

  As described above, since the microchannel 53 is minute, a sample DNA fragment labeled with at least the amount of the specimen, the DNA separation / extraction reagent, the enzyme reagent, and the fluorescent substance reagent can be obtained. In addition, a fluorescently labeled sample DNA fragment can be easily generated simply by injecting a specimen, a DNA separation / extraction reagent, an enzyme reagent, and a fluorescent substance reagent into the openings 54, 55, 56, and 57, respectively.

  The sample DNA fragment that has flowed into the grooves 63, 63,... Flows to the opposite end of the grooves 63, 63,. Here, like the reactor 50, the DNA array chip 2 is heated to a temperature at which the sample DNA fragments are unwound. When sample DNA fragments are distributed throughout all spots 60, the DNA array chip 2 is cooled to the extent that complementary bases form hydrogen bonds. As a cooling means (temperature adjusting means) for cooling the DNA array chip 2, an electrically cooled cooler or a heat radiating plate may be provided, but heat from the heater of the reactor 50 may be insulated. The heat insulating structure may be formed by providing a groove in the substrate 51 at a position between the reactor 50 and the DNA array chip 2. When cooled in this way, the sample DNA fragment binds to the probe DNA fragment 61 of the spot 60 having complementarity among the spots 60, 60,... By hybridization, and the probe fragment 61 of the spot 60 having no complementarity. And do not combine. Next, the operator flows pure water through the groove 63 to wash away sample DNA fragments that have not been hybridized. At this time, the sample DNA fragment hybridized with the probe DNA fragment 61 remains immobilized on the spot 60 without flowing along with the fluorescent substance.

  Next, the operator attaches the DNA analyzer 1 to the DNA reader 70, makes the light receiving surface of the solid-state imaging device 3 face the emitting surface 79 a of the light guide 79, and sets the spots 60, 60,. Soft contact with 79a. When the operator sets the DNA analyzer 1 to the DNA reader 70, the top gate lines 44, 44,... Are connected to the terminals of the top gate driver 74, and the bottom gate lines 41, 41,. The drain lines 43, 43,... Are connected to the terminals of the drain driver 76, respectively.

  Next, the light source 78 of the light irradiation unit 73 is turned on, and excitation light by ultraviolet rays is emitted from the emission surface 79 a toward the light receiving surface of the solid-state imaging device 3. Thus, of the spots 60, 60,..., The spot 60 hybridized with the sample DNA fragment emits fluorescence (mainly visible light) from the phosphor attached to the sample DNA fragment and does not bind to the sample DNA fragment. 60 does not emit fluorescence. Therefore, the excitation light from the light source 78 is not incident on the double gate transistor 20 corresponding to the spot 60 combined with the sample DNA fragment by the excitation light shielding film 32, but the fluorescence from the phosphor is incident, and the sample DNA fragment and Exciting light and fluorescence are hardly incident on the double gate transistor 20 corresponding to the uncoupled spot 60. Since the probe DNA fragment 61 of the spots 60, 60,... Is fixed on the surface of the solid-state imaging device 3, the fluorescence emitted from the spot 60 combined with the sample DNA fragment does not attenuate so much and corresponds to the spot 60. It enters the double gate transistor 20 and generates electron-hole pairs. On the other hand, sufficient electron-hole pairs are not generated in the double gate transistor 20 in which excitation light and fluorescence are not incident. Therefore, even if the sensitivity of the double gate transistors 20, 20,... Is low, it can be detected with sufficient sensitivity.

  The drive device (top gate driver 74, bottom gate driver 75, drain driver 76, and drive circuit 77) of the DNA reader 70 drives the solid-state image pickup device 3, so that the solid-state image pickup device 3 becomes the double gate transistor 20, 20,..., Respectively, detects the fluorescence intensity or the amount of fluorescence light, and acquires the light intensity distribution along the light receiving surface as two-dimensional image data.

The image acquisition operation of the DNA reader 70 is as follows.
That is, the drive circuit 77 outputs a control signal Tcnt to the top gate driver 74, and the top gate driver 74 sequentially outputs reset pulses from the top gate line 44 in the first row to the top gate line 44 in the sixth row. The drive circuit 77 outputs a control signal Bcnt to the bottom gate driver 75, and the bottom gate driver 75 sequentially outputs read pulses from the bottom gate line 41 in the first row to the bottom gate line 41 in the sixth row. The drive circuit 77 outputs a control signal Dcnt to the drain driver 76, and the incident light between the reset period in which the drain driver 76 outputs a reset pulse in each row and the read period in which a read pulse is output in each row. Are output to all the drain lines 43, 43,... During the carrier accumulation period for accumulating carriers generated by.

  The operation of each double gate transistor 20 in the i-th row will be described in detail. As shown in FIG. 9, 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), each double-gate transistor 20 in the i-th row is in the vicinity of the interface between the semiconductor film 23 and the channel protective film 24. The accumulated carriers (here, holes) 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. Fluorescence from a phosphor contained in a pair of the probe DNA fragment 61 and the sample DNA fragment of the closest spot 60 is incident on the semiconductor film 23 of the double gate transistor 20. During the period from the end of the reset pulse until the read pulse is output to the i-th bottom gate line 41 (this period is referred to as a carrier accumulation period), the amount of fluorescence in the semiconductor film 23 is followed. An amount of electron-hole pairs is generated, and the holes are accumulated near the interface between the semiconductor film 23 and the channel protective film 24 by the negative electric field of the top gate electrode 30.

  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, for example, −20 [V] and a negative potential. Since the potential applied to the bottom gate electrode 21 is ± 0 [V], holes accumulated near the interface between the semiconductor film 23 and the channel protective film 24 due to the negative electric field of the top gate electrode 30 Since the gate-source potential is low with only this charge, 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 negative electric field of the top gate electrode 30 is reduced according to the amount of carriers accumulated in the carrier accumulation period. Therefore, if the amount of carriers is sufficient, the positive voltage applied to the bottom gate electrode 21 is increased. A channel is formed in the semiconductor film 23 by the voltage, and a current flows from the drain electrode 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 fluorescent light incident on the semiconductor film 23 in the carrier accumulation period increases, the amount of accumulated carriers also increases. As the amount of accumulated carriers increases, the source from the drain electrode 28 in the lead period increases. The level of current flowing through the electrode 27 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. That is, the greater the amount of incident light, the greater the drop in precharged voltage. Then, the drive circuit 77 passes through the drain driver 76 between the i-th read period and the next (i + 1) -th precharge period, and after a predetermined time has elapsed since the start of the read period. The voltage of the drain lines 43, 43,... Is detected and A / D converted. By analyzing the A / D converted signal, it is converted into light intensity. Note that the drive circuit 77 detects the time until the predetermined threshold voltage is reached via the drain driver 76 between the read period of the i-th row and the precharge period of the next (i + 1) -th row. Also good. Even in this case, the light intensity is converted. In FIG. 9, 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 needs to make the carrier accumulation time of each row constant and the precharge period and the read of each row do not overlap, so the i-th row of the top gate driver 74 It may be from immediately after the fall of the reset pulse to the fall of the i-th read pulse of the bottom gate driver 75. 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

  By repeating the same processing procedure for each double-gate transistor 20 in all rows with the above-described series of image reading operations as one cycle, the light intensity distribution on the DNA analyzer 1 is acquired as an image. The image representing the light intensity distribution is output from the drive circuit 77 to the arithmetic processing device 72. The arithmetic processing device 72 causes the display device 71 to display an image representing the light intensity distribution. The base sequence of the sample DNA fragment is specified according to which part of the displayed image has a high light intensity.

  Note that the sensitivity of the double gate transistor 20 of the solid-state imaging device 3 can be adjusted by adjusting the length of the carrier accumulation period. For example, if the carrier accumulation period is lengthened, even when the intensity of light emitted from the hybridized spot 60 is weak, the time of the generated electron-hole pair becomes longer, so the amount of holes accumulated accordingly. Therefore, the light of the hybridized spot 60 can be detected.

  As described above, according to the present embodiment, since the outlet of the microchannel 53 is guided to the light receiving surface of the solid-state imaging device 3, the sample DNA fragment spreads on the light-receiving surface of the solid-state imaging device 3, and the solid-state imaging device By measuring the fluorescence distribution intensity along the light receiving surface 3 with the solid-state imaging device 3, the base sequence of the sample DNA fragment can be identified. Thus, even without an expensive apparatus such as a thermal lens microscope, the reaction product can be analyzed with the solid-state imaging device 3 that is less expensive than such an apparatus.

  Furthermore, since the microchannel 53 and the openings 54, 55, 56, 57 are formed in the reactor 50, the operator injects the specimen into the opening 54 and also supplies the reagent to the openings 55, 56, 57. By simply injecting, a fluorescently labeled sample DNA fragment can be easily generated. Therefore, no complicated chemical operation technique is required for the operator, and anyone can generate a sample DNA fragment. Further, since the reactor 50 and the solid-state imaging device 3 are integrated, it is possible to perform from the generation of the fluorescently labeled sample DNA fragment to the identification of the sample DNA fragment in a short time.

  Moreover, since the sample DNA fragment produced | generated in the microchannel 53 flows out into the groove | channels 63,63, ... from the exit of the microchannel 53 as it is, the fluorescence intensity distribution along the groove | channel 63 is solid solid imaging device 3 for every groove | channel 63. , And light detection of the spots 60, 60,... Can be performed at a time.

[Second Embodiment]
FIG. 10 is a plan view showing the DNA analyzer 101 according to the second embodiment. As shown in FIG. 10, in the DNA analyzer 101, the same reference numerals are given to the same parts as any part of the DNA analyzer 1 of the first embodiment, and the description of the same parts is as follows. Omitted. Also in the following description, the DNA analyzer 101 will be described using the same reference numerals.

  In the DNA analyzer 101, a strip-shaped thin film heater (temperature adjusting means) 102 is formed on the light receiving surface of the solid-state imaging device 3. The longitudinal direction of the thin film heater 102 is orthogonal to the longitudinal direction of the grooves 63, 63,. The thin film heater 102 is partially exposed at the bottom of the grooves 63, 63,... And partially covered by the protrusion 62. The thin film heater 102 is disposed along the edge near the reactor 50 among the outer edges of the solid-state imaging device 3. The thin film heater 102 is an electrothermal film that generates heat by electric energy. Specifically, an electric resistance heating element and a semiconductor heating element are formed into a thin film.

  When the DNA analyzer 101 is attached to the DNA reader 70 shown in FIGS. 7 and 8, the thin film heater 102 contacts a terminal provided in the DNA reader 70, and power is supplied to the thin film heater 102 through the terminal. It has come to be. Thereby, the thin film heater 102 generates heat.

  Focusing on one groove 63, the thin film heater 102 generates heat, so that the outlet side of the microchannel 53 becomes high temperature, and the temperature decreases as the distance from the outlet of the microchannel 53 increases. Thereby, the temperature dependence of the sample DNA fragment can be measured.

  In addition, a spot 60 having a high double-strand binding temperature (temperature at which single-stranded DNA fragments hybridize) is disposed closer to the thin film heater 102, and the double strands as the spots 60, 60,. If the binding temperature is low, it is possible to prevent the sample DNA fragment from being mishybridized with the non-complementary spot 60. Furthermore, the thin film heater 102 can perform good hybridization as long as it is heated to such an extent that the helical structure of the sample DNA fragment passing immediately above is unwound.

[Third Embodiment]
FIG. 11 is a plan view showing a DNA analyzer 201 in the third embodiment. As shown in FIG. 11, in the DNA analyzer 201, the same reference numerals are given to the same parts as any part of the DNA analyzer 1 of the first embodiment, and the description of the same parts is as follows. Omitted. Also in the following description, the DNA analyzer 201 will be described using the same reference numerals.

  In the DNA analyzer 201, a strip-shaped electrode 202 is formed on the light receiving surface of the solid-state imaging device 3. The longitudinal direction of the electrode 202 is orthogonal to the longitudinal direction of the grooves 63, 63,. The electrode 202 is partially exposed at the bottom of the grooves 63, 63,... And is partially covered by the protrusion 62. The electrodes 202 are arranged along a pair of edges that are separated from the reactor 50 among the outer edges of the solid-state imaging device 3.

  When the DNA analyzer 201 is attached to the DNA reader 70 shown in FIGS. 7 and 8, the electrode 202 contacts a DC voltage source provided in the DNA reader 70, and a DC voltage is applied to the electrode 202 by the DC voltage source. It is to be applied.

  When this DNA analyzer 201 is used, an electrophoretic medium (a colloidal solution (sol) in which particles are dispersed in a dispersion medium, an electrolytic solution containing a gel, other electrolytic solutions, etc.) is provided in the grooves 63, 63,. Meet. Focusing on one groove 63, the sample DNA fragment that has flowed into the groove 63 from the outlet of the microchannel 53 is applied to the electrode 202 by electrophoresis through the electrophoresis medium filled in the groove 63. To do. In this case, it is not necessary to provide the spots 60, 60,... On the light receiving surface of the solid-state imaging device 3, but by detecting the fluorescence distribution along the light receiving surface by the solid-state imaging device 3, the degree of electrophoresis of the sample DNA fragment can be determined. Can be measured.

The present invention is not limited to the above embodiments, and various improvements and design changes may be made without departing from the spirit of the present invention.
For example, in each of the above embodiments, the pixels (double gate transistors 20) of the solid-state imaging device 3 are arranged in 6 rows and 6 columns, but are not limited to 6 rows and 6 columns. The grooves 63 are also in six rows, but are not limited to six rows. It is sufficient that at least one vertical column of pixels is included in one groove 63 and the outlet of the groove 59 of the microchannel 53 communicates with each groove 63. Further, although one spot 60 is scattered for each pixel, one spot 60 may be scattered for a plurality of adjacent pixels.

  In each of the above embodiments, the solid-state imaging device 3 using the double gate transistors 20, 20,... Is described as an example of the photoelectric conversion element, but the present invention is applied to a solid-state imaging device using a photodiode as the photoelectric conversion element. May be applied. Solid-state imaging devices using photodiodes include CCD image sensors and CMOS image sensors.

  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. Similarly to the solid-state imaging device 3, the protective insulating layer is formed on one surface so as to cover a plurality of photodiodes, and the conductor layer is formed on the protective insulating layer. A plurality of types of spots are arranged on the conductor layer via the overcoat film, but one spot overlaps one photodiode in plan view.

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. Similarly to the solid-state imaging device 3, the protective insulating layer is formed on one surface so as to cover a plurality of photodiodes, and the conductor layer is formed on the protective insulating layer. A plurality of types of spots are arranged on the conductor layer via the overcoat film, but one spot overlaps one photodiode in plan view.
In addition, you may combine suitably the structure of said each embodiment.

It is a top view of the DNA analyzer 1 in the 1st Embodiment of this invention. 2 is a cross-sectional view of a reactor 50. FIG. 2 is a cross-sectional view of a DNA array chip 2. FIG. 2 is a plan view of a DNA array chip 2. FIG. It is a top view of one pixel. It is sectional drawing of one pixel. 2 is a block diagram showing a schematic configuration of a DNA reading device 70. FIG. FIG. 3 is a cross-sectional view showing a form when the DNA analyzer 1 is set in a DNA reader 70. 6 is a timing chart showing the transition of the level of an electric signal output to the solid-state imaging device 3 by a driver. It is a top view of the DNA analyzer 101 in the 2nd Embodiment of this invention. It is a top view of the DNA analyzer 201 in the 3rd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 101, 201 ... DNA analyzer 3 ... Solid-state imaging device (test | inspection apparatus)
20 ... Double gate transistor (pixel)
50 ... Reactor 53 ... Micro flow path (reactor)
54 ... Opening (specimen injection opening)
55 ... Opening (reagent injection opening)
56 ... Opening (reagent injection opening)
57… Opening (reagent injection opening)
62 ... ridge 63 ... groove 102 ... thin film heater (temperature adjusting means)
202 ... Electrode (voltage application means)

Claims (3)

A reactor provided on a substrate and having a reaction furnace for generating DNA formed therein;
A solid-state imaging device provided on the substrate, and spots having a probe DNA fragment having a known base sequence scattered in a matrix on the light-receiving surface of the solid-state imaging device, and generated in the reactor An inspection device for specifically inspecting the DNA sequence;
A groove communicating the inspection device with the previous reactor;
A temperature adjusting means for heating the inspection apparatus provided on the light receiving surface of the solid-state imaging device so as to be orthogonal to the groove along an edge closer to the reactor among the outer edges of the solid-state imaging device. , equipped with a,
The spot analysis apparatus according to claim Rukoto which have a lower said probe DNA fragments of duplex binding temperature increasing distance from said temperature adjusting means.   The analyzer according to claim 1, wherein there is at least one column of pixels of the solid-state imaging device per groove. The inspection apparatus has a protrusion provided on the light receiving surface of the solid-state imaging device in parallel with a pixel row of the solid-state imaging device,
The analysis apparatus according to claim 1 or 2, wherein a groove formed between the plurality of protrusions communicates with the reaction furnace.

Images