WO2000001848A1 - Polynucleotide assay apparatus and polynucleotide assay method - Google Patents

Abstract

A polynucleotide assay method with the use of a polynucleotide detection cell provided with a first electrode (111) containing DNA probes (13, 14, 15, 16) fixed to respective different compartments (3, 4, 5, 6) and second electrodes (113-1, 113-2) opposite to the first electrode. The method comprises capturing a target polynucleotide by complementary strand binding between each DNA probe fixed to each compartment and the target polynucleotide, conducting an elongation reaction by using a base (dNTP) labeled with electrochemiluminescence to elongate the DNA probe bonded by complementary strand binding, and detecting electrochemiluminescence created by applying a voltage across the first electrode and the second electrodes to detect whether or not an elongated chain produced by the elongation reaction is present. The DNA detection cell having a simple constitution and an assay apparatus using the same serve to quickly detect a hybrid of a target DNA fragment with a DNA probe and to assay a large amount of probes in a short time.

Description

 Description Polynucleotide testing device and polynucleotide testing method

 The present invention relates to a polynucleotide detection cell for detecting DNA, mRNA, and the like for testing, a detection apparatus using the cell, and a detection method. Background art

 Using a DNA detection cell on which 16000 probes are immobilized, a hybrid of the probe and the fluorescently labeled target DNA is formed. Using a confocal microscope, the entire area of the DNA detection cell is scanned with laser light in less than 15 minutes. A technique for detecting a hybrid by exciting a fluorescent label and detecting the resulting fluorescence is known (Nature Biotec hnology 14, 1675-1680 (1996)).

 The sample DNA is modified with a biotin group, and the sample DNA is captured by beads using a biotin-avidin bond. The complementary DNA strand is bound to the sample probe by electrochemiluminescence-labeled DNA probe with a known base sequence. A probe method using an electrochemiluminescent-labeled DNA probe, which detects the presence or absence of complementary strand binding by detecting the electrochemical emission of DNA, has been reported (Clinical Chemistry 37, No. 9, 1626). — 1632 (1991).

Electrochemical luminescence (ECL) -labeled nucleotides and oligos labeled with electrochemical luminescence (ECL) label are known (Japanese Patent Application Laid-Open No. Hei 9-1505464). Various complexes used in the electrochemiluminescence reaction are widely known (C 1 inical Chemistry 37, No. 9, 1534-1539 (1991)), J. Electrochem em. Soc., Vol. 132, No. 4, 842-849 (1995), JP-A-7-173185, JP-A-7-309836). Disclosure of the invention

 In the above conventional technology, laser light scanning is performed on a large number of sites, up to 160,000, on which the probes of the DNA detection cell are fixed, so that the above conventional technology is routinely used as a diagnostic technology in the clinical field. There was a problem that the throughput for detecting hybrids was not sufficient to apply the method.

 An object of the present invention is to provide a polynucleotide detection cell for performing high-speed detection of a hybrid between a target polynucleotide and a probe, a polynucleotide detection device using the cell, and a test method.

 In the polynucleotide detection cell of the present invention, a substrate under the DNA detection cell in which different DNA probes are immobilized in a plurality of compartments is formed, and a transparent DNA in which a predetermined counter electrode is formed. A space is formed between the substrate on the detection cell and the reagent solution involved in the siege reaction and the electrochemiluminescence reaction.

 In the test device using the polynucleotide detection cell of the present invention, the extension of the hybrid DNA probe between the target DNA fragment (target polynucleotide) and the DNA probe fixed in each section is performed, and the detection of the extended strand is performed. This is performed using an electrochemiluminescence reaction.

 In the inspection device of the present invention, the progress and stop of the electrochemiluminescence reaction are controlled at high speed by high-speed control of the voltage applied between the working electrode and the counter electrode. Presence can be detected at high speed. In other words, a large number of probe tests can be performed in a short time with a simple device configuration.

In the test device of the present invention, first, a DNA fragment group obtained from a DNA sample and a DNA probe immobilized in each compartment are subjected to a complementary strand reaction to capture a DNA fragment in each compartment. Next, an extension reaction is performed using Taq DNA polymerase and adenine, thymine, guacin, and cytosine to which the electrochemiluminescent label is bound, and the DNA probe complementary to the DNA fragment captured in each compartment is extended. Then, d NTP (N = A, T, G, C) to which the electrochemiluminescent label is bound is added to the extended strand. Embed. A reducing agent is introduced into the DNA detection cell, and a voltage is applied between the working electrode and the counter electrode to measure the electrochemiluminescence generated on and near the working electrode. Detection of the position of the compartment where the electrochemiluminescence occurs and the intensity of the electrochemiluminescence is determined by combining a light transmission means such as an optical fiber with a solid-state photodetector, a microchannel plate for performing optical amplification, and a TV camera. This is done separately for each working electrode section.

 Further, in the inspection apparatus of the present invention, a polynucleotide detection cell for applying a voltage to a local portion between a selected section of the working electrode and the counter electrode is integrated and configured, and an electrochemical cell generated at each local portion is formed. By measuring the luminescence, the presence or absence of the target DNA fragment that complementarily binds to the DNA probe immobilized in the selected compartment can be quickly detected.

 In the test apparatus of the present invention, a DNA fragment group obtained by amplifying a target DNA fragment by PCR using a primer to which an electrochemiluminescent label is bound, and an oligomer to which the electrochemiluminescent label is bound are ligated by a ligation reaction. A DNA fragment group obtained by binding to each DNA fragment can be used. In this case, using an adenine, thymine, guasine, or cytosine not bound to an electrochemiluminescent label and Taq DNA polymerase, an extension reaction of a DNA probe complementary to the DNA fragment is performed.

 In the first configuration of the test method of the present invention, a polynucleotide comprising a first electrode in which different DNA probes are fixed to different sections for each type, and a second electrode opposed to the first electrode A step of capturing the target polynucleotide by complementarily binding the DNA probe immobilized in the detection cell compartment to the target polynucleotide, and extending the complementary strand-bound DNA probe using an electrochemiluminescent-labeled base. Performing a reaction to extend the complementary DNA probe, applying a voltage between the first electrode and the second electrode, and detecting the presence or absence of electrochemiluminescence generated by the application of the voltage. And detecting the presence or absence of an extended chain generated by the elongation reaction.

In the second configuration of the test method of the present invention, different DNA probes are different for each type. A DNA probe immobilized in a compartment of a polynucleotide detection cell comprising a first electrode fixed in a compartment, and a second electrode facing the first electrode, and an oligonucleotide labeled with electrochemiluminescence. Capturing the target polynucleotide by binding the bound target polynucleotide to the complementary strand, and applying a voltage between the first electrode and the second electrode to generate an electrochemical signal generated by applying the voltage. And a step of detecting light emission.

 In the third configuration of the inspection method of the present invention, a poly-electrode comprising a first electrode in which different DNA probes are fixed to different sections for each type, and a second electrode opposed to the first electrode. A step of binding the DNA probe immobilized in the compartment of the nucleotide detection cell and the target polynucleotide labeled with electrochemiluminescence to capture the target polynucleotide; The method is characterized in that a voltage is applied in between and a step of detecting the electrochemiluminescence generated by the application of the voltage.

 In the configuration of the present invention, electrochemiluminescence is used, so that the optical system and the photodetector are simpler in configuration than the conventional configuration in which a fluorescent label is used to excite the fluorescent label with excitation light. It can be close to the detection cell, and the efficiency of electrochemiluminescence can be maximized.

 According to the DNA detection cell and the inspection apparatus of the present invention, even if a very large number of types of DNA probes are used, the time required for the inspection is short, and the inspection can be sped up. Since no mechanical or optical moving elements are required for the electrochemiluminescence measurement system, handling and adjustment can be simplified. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing a configuration of a DNA detection cell according to a first embodiment of the present invention. FIG. 2 shows a hybrid formed by complementary strand binding between a DNA probe fixed to a compartment of a DNA detection cell and a partial base sequence of a target DNA fragment in the first embodiment of the present invention. FIG.

FIG. 3 shows the ruthenium used in the extension reaction in the first embodiment of the present invention. FIG. 3 is a view showing dATP to which a complex is bound.

FIG. 4 is a diagram showing a dCTP to which a ruthenium complex used in an elongation reaction is bound in the first embodiment of the present invention.

FIG. 5 is a diagram showing a dGTP to which a ruthenium complex used in an extension reaction is bound in a first embodiment of the present invention.

FIG. 6 is a diagram showing a dTTP to which a ruthenium complex used in an elongation reaction is bound in the first embodiment of the present invention.

FIG. 7 is a diagram for explaining an extension reaction of a DNA probe bound to a target DNA fragment in a complementary manner in the first example of the present invention.

FIG. 8 is a diagram showing an example of an electrochemiluminescence detection system in the first embodiment of the present invention.

FIG. 9 is a diagram showing an example of a display screen showing a detection result in the first embodiment of the present invention.

FIG. 10 is a diagram showing an example of the configuration of an inspection apparatus for measuring electrochemiluminescence near a working electrode of a DNA detection cell using a TV camera in the second embodiment of the present invention.

FIG. 11 is a diagram showing a configuration of a DNA detection cell in which a working electrode and a counter electrode are formed on the same plane in a third embodiment of the present invention.

FIG. 12 is a view showing a configuration of a DNA detection cell in which a working electrode and a plurality of independent force counter electrodes are formed on the same plane in a fourth embodiment of the present invention. FIG. 13 is a view for explaining an optical system for collecting and detecting electrochemical light emission from a plurality of sections in the fourth embodiment of the present invention.

FIG. 14 shows the size of the section observed on the imaging surface of the TV camera and the image pickup device when the electrochemical camera emits the electrochemiluminescence from the section of the DNA detection cell in the fifth embodiment of the present invention. FIG. 4 is a diagram for explaining the relationship between the sizes of.

FIG. 15 is a view showing a configuration of a DNA detection cell formed on a DNA cell lower substrate by connecting counter electrodes by matrix wiring in the fifth embodiment of the present invention. FIG. 16 is a view for explaining selection of a section in which electrochemiluminescence is induced by selection of a gate and a conductor in the fifth embodiment of the present invention.

FIG. 17 is a diagram for explaining an example of voltage application for repeatedly generating electrochemiluminescence in a selected section in the sixth embodiment of the present invention.

FIG. 18 shows a DNA probe used as a DNA probe in the seventh embodiment of the present invention.

FIG. 3 is a diagram illustrating an oligonucleotide having a phosphorothioate (phosphotrothiothiate) bond between 2′-deoxyoligonucleosides.

FIG. 19 is an eighth embodiment of the present invention, and is a view showing a configuration of an inspection apparatus using a DNA detection cell.

FIG. 20 is a plan view of a DNA detection cell used in the eighth embodiment of the present invention.

FIG. 21 is a view for explaining an example of voltage application for repeatedly generating electrochemiluminescence in the eighth embodiment of the present invention.

FIG. 22 is a view showing a DNA probe that is complementary to a target polynucleotide to which an electrochemiluminescence-labeled oligonucleotide is bound in the ninth embodiment of the present invention and that is bound to a complementary strand.

FIG. 23 is a view showing a DNA probe which is complementary to a target polynucleotide labeled with electrochemiluminescence in the tenth embodiment of the present invention.

FIG. 24 is an eleventh embodiment of the present invention, and is a view for explaining the procedure of an inspection using the inspection apparatus of the present invention.

FIG. 25 and FIG. 26 are diagrams showing the structure of an electrochemiluminescent label usable in each embodiment of the present invention and an example of an electrochemiluminescent reaction. BEST MODE FOR CARRYING OUT THE INVENTION

Figures 25 and 26 show examples of the structure of the electrochemiluminescent label that can be used in each embodiment of the present invention described below, and an example of the use of a ruthenium complex (two types) as an example of the electrochemiluminescent reaction. Is shown. Fig. 25 shows a ruthenium-tribibiridyl complex (ruthenium (ID trisb ipyri dy l) (hereinafter abbreviated as Ru (bpy) ₃ )) and tripropyl amine (TPA) as a reducing agent. It is a figure explaining the example of the electrochemiluminescence reaction used (refer to Clinica 1 Chemistry 37, No. 9, 1534-1539 (1991).) Ruthenium-tribibiridyl complex (Ru (b py ) 3), in neutral solution stably exist in the +2 state (the ground state) (reference number _{20 1, Ru (bpy) 3} 2 +). TPA202 is almost stable in neutral solution When a voltage is applied between the working electrode and one of the counter electrodes so that the potential of the working electrode 203 with respect to the solution is about +1.1.1 V or more, the ruthenium-tribibiridyl complex in the +2 valence state is obtained. is oxidized at the surface and in the vicinity of the working electrode, a + 3 valence state (reference numeral ^{_{204, Ru (bpy) 3 3}} +). TPA20 2 also It is oxidized on and near the surface of the test electrode to form a TPA in the +1 monovalent excited state (reference number 205.) The asterisk in Fig. 25 and Fig. 26 indicates the excited state. The excited state of TPA (Ref. 205) becomes a neutral excited state of TPA (Ref. 206) by deprotonation in the excited state, and the +3 valence state (Ref. 204, Ru (bpy) ₃ ) Acts as a reducing agent for ^{3 +} ).

+ Trivalent state (Ref. 204, Ru (bp y) ₃ ^{3 +} ) is reduced by the excited state of TPA (Ref. 206) to become + divalent excited state (Ref. 207), and electrochemiluminescence (emission center wavelength of distribution of about 62 O nm is) Ru back to the Te Bantsu +2 state (the ground state) (reference number _{20 1, Ru (bpy) 3} 2 +). In the electrochemiluminescence reaction shown in Fig. 25, the ruthenium-tribibiridyl complex is not consumed and participates in luminescence repeatedly.

Figure 26, uses a ruthenium one triflumizole Henin Toro phosphorus complex (ruthenium II) tris- phen an thro 1 ine) Ru (phen) 3 2 + abbreviated) (reference number 2 1 1), the TP A as a reducing agent FIG. 5 is a diagram illustrating an example of an electrochemiluminescent reaction (Clinical Chemistry 37, No. 9, 1534-1539 (1991)). Electrochemiluminescence (the center wavelength of the luminescence distribution is about 590 nm) is generated by the same electrochemiluminescence reaction pathway.

 In addition to the examples of the electrochemiluminescent labels shown in FIGS. 25 and 26, JP-A-7-173185, JP-A-7-309836, and J. E1 ectroch em. Soc , Vol. 132, No. 4, 842-849 (1989) can be used in the detection device of the present invention.

 (First embodiment)

 FIG. 1 is a diagram showing a configuration of a DNA detection cell according to a first embodiment of the present invention. The DNA detection cell of the first embodiment is constructed by laminating a substrate 11 below the DNA detection cell and a substrate 12 above the DNA detection cell via the gasket 112 in the z direction shown in Fig. 1. Is done. The space between the substrate 11 below the DNA detection cell and the substrate 12 above the DNA detection cell constitutes the DNA detection cell used to hold the reagent solution involved in the chemical and electrochemiluminescence reactions used in the following description. . A working electrode 111 of a predetermined shape made of Au is formed on the upper surface of the lower substrate 11 of the DNA detection cell. The upper substrate 12 of the DNA detection cell is made of a light-transmitting material, and has a counter-electrode 1 13-1, 1 13-2 with a slender shape on the lower surface. . The counter electrode 1 1 13-1 faces sections 4 and 6, and the counter electrode 113 2 faces counter sections 3 and 5.

 Although the outer shape of the DNA detection cell lower substrate 11 and the DNA detection cell upper substrate 12 shown in FIG. 1 is circular, the external shape is not limited to a circle, but may be any shape such as a square, a rectangle, or a polygon. The shape of the working electrode 111 shown in Fig. 1 is square, but any shape is acceptable.

Multiple types of DNA probes (oligomers) Pre-fixed to the surface of the working electrode 11i. As shown by the dashed line in FIG. 1, the surface of the working electrode 111 is divided into a plurality of compartments, and the compartment a has a DNA probe b, the compartment a has a DNA probe b, and so on. In each compartment, different types of DNA probes are fixed. The shape of the section of the working electrode 111 shown in Fig. 1 is square. Is optional. Counter electrode 113-1-1 faces sections 3 and 5 of working electrode 111, and counter electrode 113-2-2 faces sections 4 and 6 of working electrode 111. In Fig. 1, the working electrode 1 1 1 and the counter electrode 1 1 1

The voltage application lines 3-1 and 1 1 3-2 are omitted. In order to detect electrochemiluminescence efficiently, it is desirable that the counter electrodes 1 13-1 and 1 13-2 be transparent electrodes. In Fig. 1, a transparent counter electrode 113 with the same area as the working electrode 111 is replaced by a counter electrode 113-1-1, 1

It may be used in place of 13-2 and placed opposite the working electrode 1 1 1. In the first example, 8.7 0 NA selected from a human-derived DNA library was used as a sample, and DNA fragments were cut with the restriction enzyme N1aIII. I do. In the following, the first DNA probe 13 having the nucleotide sequence of SEQ ID NO: 1 and the second DNA probe 14 having the nucleotide sequence of SEQ ID NO: 2 are used as the first and second DNA probes. An example in which a DNA fragment that binds to a complementary strand is detected will be described below.

The first DNA probe (SEQ ID NO: 1) The second DNA probe (SEQ ID NO: 2) The first DNA probe is composed of the nucleotide sequence between bases 1383 and 1927 of the sample DNA described above. The probe that complementarily binds to the DNA fragment (first target DNA fragment) having the same base sequence and the second DNA probe are the same as the base sequence between 199 bases and 558 bases of the above sample DNA. It is a probe that complementarily binds to a DNA fragment (second target DNA fragment) having the same base sequence. The third DNA probe 15 and the fourth DNA probe 16 are examples of a DNA probe that does not complementarily bind to any site in the nucleotide sequence of the sample DNA.

The first thiol group introduced at the 5 'end of each DNA probe allows the first probe to be used according to the method described in the literature (Biophysical Journal 171, 1079-106 (1996)). Of DNA probe 13 In Figure 3, the second DNA probe 14 is in section 4 of the working electrode 111, the third DNA probe 15 is in section 11 of the working electrode 11, and the fourth DNA probe 16 is in section 4. Fix each to section 6 of the working electrode.

 A sample solution containing a DNA fragment group to be measured is placed between the lower substrate 11 of the DNA detection cell and the upper substrate 12 of the DNA detection cell (DNA detection cell) shown in FIG. Are bound by complementary strands.

 Figure 2 shows the hybrid due to complementary strand binding between the first DNA probe 13 immobilized in section 3 of the DNA detection cell and a partial base sequence of the first target DNA fragment 21. It is. A hybrid is formed by complementary strand binding between the second DNA probe 14 fixed to the section 4 of the DNA detection cell not shown in FIG. 2 and a partial base sequence of the second target DNA fragment 22. After formation of the hybrid, the unbound DNA fragments are washed out of the DNA detection cell using a washing solution.

Next, an extension reaction of the first DNA probe 13 that is complementary to the target DNA fragment 21 is performed. In the elongation reaction, a substrate mixture containing at least one dNTP (N = A, C, G, T) (Ru: abbreviated as dNTP) to which a ruthenium complex is bound via a linker is used. As shown in Fig. 3, in Ru: dATP, a ruthenium complex is bonded to the nitrogen atom at position 7 of adenine via a linker and a peptide bond. As shown in Fig. 4, in Ru: dCTP, a ruthenium complex is bonded to the 5-position carbon atom of cytosine via a linker and a peptide bond. As shown in Fig. 5, in Ru: dGTP, a ruthenium complex is bonded to the nitrogen atom at position 7 of guanine via a linker and a peptide bond. As shown in Fig. 6, in Ru _: dTTP, a ruthenium complex is bonded to the 5-position carbon atom of thymine via a linker and a peptide bond. The linker is 1 (CH ₂ ) _n —, where n = 2 to 20. As an example of the composition of the substrate mixture used for the extension reaction, any of the following compositions is possible.

 (1) Ru: dATP + dCTP + dGTP dTTP

(2) dATP + Ru: dCT + dGTP + P dTTP 3) dATP + dCTP + Ru: dGTP + dTTP

 ) dATP + dCTP + dGTP + Ru: dTTP

 5) Ru: dATP + Ru: dCTP + dGTP + dTTP

 6) Ru: dATP + dCTP + Ru: dGTP + dTTP

 7) Ru: dATP, dCTP + dGTP + Ru: dTTP

 8) dATP + Ru: dCTP + Ru: dGTP + dTTP

 9) dATP + Ru: dCTP + dGTP + Ru: dTTP

 10) d ATP + dCTP + Ru: dGTP + Ru: dTTP

 1 1) dATP + Ru: dCTP + Ru: dGTP + Ru: dTTP

1 2) Ru: dATP + dCTP + Ru: dGTP + Ru: dTTP

1 3) Ru: dATP + Ru: dCTP + dGTP + Ru: dTTP

14) Ru: dATP + Ru: dCTP + Ru: dGTP + dTTP

1 5) Ru: dATP + Ru: dTTP + Ru: dGTP

 + Ru: dCTP

 In the first example, the above composition (4) was used, and 2 L (microliter) of a substrate mixture containing 2.5 mM dATP, dCTP, dGTP, and Ru: dTTP was added to the DNA detection cell. Then, the denaturation reaction at 94 ° C (for 10 sec) and the anneal reaction at 66 ° C (for 20 sec) are repeated once to several times, and then the extension reaction is performed at 72 ° C.

FIG. 7 is a diagram for explaining the extension reaction of the first DNA probe 13 that has been complementarily bound to the first target DNA fragment 21. Although not shown in FIG. 7, the extension reaction of the second DNA probe 14 complementary to the second target DNA fragment 22 similarly occurs. 24 is unreacted Ru: dTTP, and 25 is unreacted dATP, dCTP or dGTP. As a result of extension of the first DNA probe 13 complementary to the first target DNA fragment 21 by the extension reaction, Ru: dTTP was not incorporated in the extended strand, and dNTP was incorporated in the extended strand. An extended chain consisting of portion 27 and extended portion 26 in which Ru: dTTP is incorporated into the extended chain is formed. Therefore, by extension reaction, at least one molecule Ru: dTTP force Incorporated into the extended strands of the first and second DNA probes, and at least one ruthenium complex 23 is captured in compartments 3 and 4. After the extension reaction, wash with a washing solution to remove unreacted substrate.

 Ru: dTTP is a macromolecule compared to dNTP (N = A, C, G, T), and the reaction rate of incorporation into extended chains is lower than that of dTTP, but at least the first one molecule An elongation reaction occurs until the Ru: dTTP is incorporated into the extended strand. Since the third and fourth DNA probes 15 and 16 do not bind to the target DNA fragments 21 and 22, the elongation reaction does not occur, and the incorporation of Ru: dTTP into the elongate strand does not occur. Therefore, ruthenium complex 23 is not trapped in compartments 5 and 6. That is, in the first embodiment, a target DNA fragment having a specific base sequence binds to a DNA probe immobilized on a DNA detection cell in a complementary manner, and a riltenium complex is formed by a DNA probe elongation reaction. The bound dNTP is incorporated into the extended chain, and the ruthenium complex is indirectly captured in a specific compartment. In the first embodiment, the amount of the ruthenium complex 23 indirectly captured in the compartments 3 and 4 is measured to detect the presence or absence of the target DNA fragment 21 in the solution containing the DNA fragment group.

 In the conventional DNA probe method using a DNA probe labeled with a ruthenium complex, there is a problem that the labeled DNA probe is specifically adsorbed inside the DNA detection cell, but the Ru: dTTP used in the present invention is a ruthenium complex. Compared with DNA probes labeled with, they have the advantage of having a smaller molecular weight, less nonspecific adsorption, and a smaller background derived from nonspecific adsorption.

Next, the method for measuring the amount of ruthenium complex 23 indirectly captured in compartments 3 and 4 is described. First, the DNA detection cell was replaced with a buffer solution containing an amine-based reducing agent, and the working electrode 1 1 1 and the counter 1 electrode (1 13-1, 1 13-2; or 1 13) were formed. Wash the inner surface of the detection cell. In the first embodiment, a 0.3 Omo 1 ZL phosphate buffer (ρΗ6.8) containing 0.18 mol 1 / L (liter) of tripropylamine (TPA) was used as the reducing agent, and the temperature was 28 ° C. ° C. Next, a voltage is applied between the working electrode 1 1 1 and the counter electrode (1 1 1 3-1, 1 1 3-2; or 1 1 3) so that the working electrode 1 1 1 side is positive. The optimum value of the applied voltage differs depending on the type of reducing agent used, the type of buffer solution, and the like. In the first embodiment, voltage was applied so that the potential difference was 1.35 V. The electrochemiluminescence according to the electrochemiluminescence reaction by applying voltage (the electrochemiluminescence reaction involving the ruthenium complex used in the first embodiment (Ru: the part of the ruthenium complex of dTTP The emission wavelength at which the intensity of the electrochemiluminescence generated by) is maximized is 620 nm.

 The intensity of electrochemiluminescence is proportional to the amount of ruthenium complex present near the working electrode. The presence or absence of a hybrid between the DNA probe and the target DNA fragment can be determined by measuring the intensity of the electrochemiluminescence.

 In the above-mentioned examples of the composition of the substrate mixture used for the extension reaction (1) to (15), instead of Ru: dNTP (N = A, T, G, C), the linker Example of the composition of a substrate mixture using d dNTP (N = A, T, G, C) (Ru: abbreviated as d dNT P) to which a ruthenium complex is bound via () to (15 ′) Either one may be used.

 (1 ') Ru: d dATP + dCTP + dGTP + dTTP

 (2 ') dATP + Ru: d dCT + dGTP + P dTTP

 (3 ') dATP + dCTP + Ru: d dGTP + dTTP

 (4 ') dATP + dCTP + dGTP + Ru: d dTTP

 (5 ') Ru: d d ATP + Ru: d d C T P + d G T P + d T T P

 (6 ') Ru: d dATP + dCTP + Ru: d dGTP + dTTP

 (7 ') Ru: d dATP + dCTP + d dGTP + Ru: dTTP

 (8 ') dATP + Ru: d dCTP + Ru: d dGTP + dTTP

 (9 ') dATP + Ru: d dCTP + dGTP + Ru: d dTTP

 (10 ') dATP + dCTP + Ru: d dGTP + Ru: d dTTP (11') dATP + Ru: d dCTP + Ru: d dGTP

+ Ru: d dTTP (1 2 ') Ru d dATP + dCTP + Ru: d dGTP

 + Ru d dTTP

 (1 3 ') Ru d d ATP + Ru: d dCTP + dGTP

 + Ru d dTTP

 (14 ') Ru d dATP + Ru d dCTP + Ru: d dGTP

 + dTT P

 (1 5 ') Ru: d dATP + Ru: d dTTP + Ru: d dGTP

 + Ru: d dCTP

 When the composition of the substrate mixture (1 ';) to (15') is used, only one molecule of Ru: d dNTP is incorporated into the extended strand in the extension reaction of the DNA probe. Therefore, the amount of the hybrid between the DNA probe and the target DNA fragment can be quantitatively determined by measuring the intensity of the electrochemiluminescence.

 For the detection of the above-mentioned electrochemiluminescence, a photodetection system having a spatial resolution capable of detecting the electrochemiluminescence intensity separately for each section of the working electrode 111 is used.

FIG. 8 is a diagram showing an example of an electrochemiluminescence detection system in the first embodiment. One end of each of the optical fibers 3a, 3b, 3c, 3d is arranged in one-to-one correspondence with each of the sections 3, 4, 5, and 6, and the optical fibers 3a, 3b, 3c are arranged. , 3d, the other end can be connected to a high-sensitivity solid-state detector 33, 34, 35, 36 such as an avalanche photodiode (APD). The electrochemiluminescence generated in sections 3, 4, 5, and 6 passes through optical fibers 3a, 3b, 3c, and 3d, and is photoelectrically converted and detected in APDs 33, 34, 35, and 36. . The output of the APD is converted to a digital signal by the A / D converter 38, processed by the data processing device 39, and present in the DNA fragment group based on the above-described principle based on the detected intensity of electrochemiluminescence in each section. The type of the target DNA fragment can be determined. The determined result is displayed on the display unit (display) of the data processing device 39. Fig. 9 shows an example of the display screen displayed on the display. The screen shows the number of the used DNA detection cells and the number of types of DNA probes fixed to the DNA detection cells. Number) and the arrangement of DNA probes fixed in each compartment. Column No. and test result of the reaction between the DNA probe and the DNA in the sample {+ (Positive: DNA that forms a complementary strand with the DNA probe exists in the sample),-

(Negative: DNA that forms a complementary strand with the DNA probe does not exist in the sample.)} Is displayed. If the test result is positive, the nucleotide sequence of the DNA probe is displayed. The example shown in Fig. 9 shows that DNA having a base sequence complementary to the DNA probes of SEQ ID NO: 1 and SEQ ID NO: 2 was detected in the sample. In the description of the first embodiment, for simplicity, a DNA detection cell having four sections is taken as an example. However, the number of sections of the DNA detection cell used in an actual inspection device is, for example, As described in the second embodiment, 100 × 100 = 10000. The number of sections is appropriately selected according to the purpose of the inspection.

 Instead of the ruthenium complex label, it is also possible to use a label used for other electrochemiluminescence reactions, such as an osmium complex. For example, in the first embodiment, Os: dTTP and 0s: ddTTP are used instead of Ru: dTTP and Ru: ddTTP.

 (Second embodiment)

 If the DNA detection cell is made highly integrated and the number of DNA probes handled at one time is increased (that is, if the number of sections of the DNA detection cell is increased), electrochemiluminescence can be performed using a high-sensitivity TV camera. A method of detecting with a two-dimensional imaging device is effective. The distribution of electrochemiluminescence near the working electrode of the DNA detection cell

A large amount of data can be processed at once by capturing as a two-dimensional image and performing image processing.

Fig. 10 shows an explanation of an inspection device that measures the electrochemical luminescence in the vicinity of the working electrode 111 of the DNA detection cell 41 using an optical system 42 and a TV camera 43 having a plurality of imaging devices 40. FIG. A voltage applied by the power supply 44 between the working electrode 1 1 1 and a transparent counter electrode 1 1 3 disposed opposite the working electrode 1 1 1 and having the same area as the working electrode 1 1 1; The duration of the voltage application (0.4 sec in the second embodiment) is controlled by the power supply controller 45. As the optical system 42, a normal optical lens may be used. It is effective to increase the photodetection sensitivity using a sifier (I.I.) or a microchannel plate (MCP). If a higher spatial resolution is required as the DNA detection cell becomes more integrated, an optical system that directly connects a bundle of optical fibers to the imaging surface of the TV camera 43 is used.

 In the DNA detection cell of the second embodiment, the surface of the working electrode of 20 mm × 20 mm is arranged in the x and y directions with an area of 200 μπππ200 m. = 100 000 sections, and different DNA probes are fixed in each section. Assuming that one molecule of the DNA probe is fixed to an area of about 50 nm x 50 nm, each section having an area of 200 ^ mX 200 μπι has about 1,600,000 molecules (0.027 The DNA probe of f mo 1) is fixed.

Here, by extension reaction of the DNA probe hybridized with the target DNA fragment, only one molecule of the dNTP or ddNTP bound to one molecule of the DNA probe to the electrochemiluminescent label is incorporated into the extended strand. Let's take an example. Assuming that the distance between the working electrode and the substrate on the DNA cell is 200 μπι, an electrochemiluminescent label with a concentration of about 3.3 nmol / L is indirectly in one section (200 μππ cubic). is capturing捉(0. 0 2 7 X 1 0- 15 mo 1 (2 0 0 X 1 0- 4 cm) 3 = 3. 3 nm 0 I / L).

According to the literature (Clinical Chemistry 37, No. 9, 1534-1539 (1991)), a ruthenium tribibiridyl complex and TPA are used. In the case of electrochemiluminescence, the detection limit is 200 fmo 1 ZL. Therefore, in the second embodiment, the detection limit is about 1650 times the detection limit (200 fmo 1 / L) described in the literature. The electrochemiluminescent label will be present in one compartment (3.3 x 10-9 + (200 x 10 " ¹⁵ ) = 16500).

When the working electrode area is 4 mm X 5 mm, the electrochemiluminescence in a solution with a ruthenium tribibiridyl complex concentration of 10 nmo and 1 L was measured for 0.4 sec. number of photons is about 40 0 0 Huo tons have enough those working electrode ^{1 mm 2 (CV = 0. 5} %). However, the apparatus used in the experiment did not use an optical system for focusing electrochemical luminescence, The light utilization efficiency is the ratio of the solid angle formed by the area of each section to the area of the light-receiving surface of the PMT, which is the photodetector, and 2π (str), and the quantum efficiency of the PMT (here, 5%). And about 0.6%.

 Therefore, in the second embodiment, using a DNA detection cell in which the working electrode area 2 Ommx 20 mm is divided into 100000 sections having an area of 2 OO ^ mX SOO ^ m, Assuming that the F value of the lens that collects the electrochemiluminescence is 0.65 and the quantum efficiency of the cooled CCD camera is 10%, the utilization efficiency of the electrochemiluminescence is about 0.7%. Photons detected in 0.4 sec

(Electrochemiluminescence S) becomes about 50 photons (400 000 / {(100 000 fim) ² x (10 mn o 1 / L)) x {(({200, πι ) ² X (3.3 nm o

1 ZL)} ½5 2. 8). Assuming that the amount of electrochemiluminescence is proportional to the concentration of the electrochemiluminescence label (complex) and that the S / N is proportional to the square root of the amount of electrochemiluminescence (S), the SZN in the measurement in the second embodiment is approximately It becomes 7. The configuration of the DNA detection cell used in the experiment was as follows: the working electrode area was 1 Ommx10 mm, and the area was 200 × 111 × 200 × 111 with 250 sections. When using a lens and a cooled CCD camera, the collection efficiency of electrochemiluminescence doubles, and the number of photons (electrochemiluminescence S) detected within 0.4 sec per section is: The number of photons becomes about 100, and the S / N ratio is improved to about 10. In the above explanation, the S / N for the case where a molecule of dNTP or ddNTP to which the electrochemiluminescent label is bound is incorporated into the extension chain of one molecule of DNA probe is used. If n molecules of the bound dNTP are incorporated into the extended strand of one DNA probe, SZN will be ri times the above value.

The MCP is used for the optical system 42, and the distribution of light emission at the working electrode is photographed using a cooled CCD camera with 100,000 pixels as a two-dimensional detector. The signal charge stored in the element 40 is converted into a current or a voltage, and is digitized by the AZD converter 38 to obtain a two-dimensional digital image. The obtained two-dimensional digital image is binarized by a data processor 39, and light emission occurs. A distinction is made between a section and a section where no light emission occurs. The probe information for each section, which kind of DNA probe is fixed in which section, and the luminescence information (presence / absence of luminescence, intensity of luminescence) obtained as a result of the measurement are compared in the sample. The type of existing DNA fragments can be identified.

 As described above, using a cooled CCD camera with 10000 pixels, the presence or absence of binding of the complementary strand between 10,000 types of DNA probes and the target DNA fragment in the sample was measured for a measurement time of 0. It can be detected in 4 sec. From the intensity of electrochemiluminescence detected in each compartment, the type and amount of the target DNA fragment present in the DNA fragment group can be determined based on the principle described in the first embodiment.

 (Third embodiment)

 Fig. 11 is a diagram showing the configuration of a DNA detection cell in which a comb-shaped working electrode and a comb-shaped counter electrode are formed on the same plane. Fig. 11 is a diagram of the working electrode viewed from the side of the light detection means. In the third embodiment, the working electrode 52 and the counter electrode 53 are formed on the same plane to increase the use efficiency of electrochemiluminescence. The comb-shaped working electrode 52 is provided with a plurality of partitions separated by broken lines 51-1-1 to 51-7. The total number of blocks (size 200 μπιΧ 200 μιη) is 10,000 as in the second embodiment. The comb-shaped working electrode 52 and the comb-shaped counter electrode 53 are connected to the substrate under the DN cell so that each tooth of the comb-shaped working electrode 52 and each tooth of the comb-shaped force center electrode 53 are alternately opposed in one direction. Formed on the surface.

 That is, the working electrode 52 in each section is arranged with the counter electrode 53 (width 5 μπι) in one direction with a gap of 5 μιη. With this arrangement, a substantially equal voltage can be applied to each section, so that there is no difference in the total intensity of electrochemiluminescence generated from each section. In the third embodiment, since it is not necessary to provide a counter electrode on the upper substrate of the DNA cell corresponding to the lower substrate of the DNA cell, the propagation of the electrochemiluminescence is not interrupted, so that the utilization efficiency of the electrochemiluminescence can be improved. .

In the third embodiment, a voltage is applied between the working electrode 52 and the force counter electrode 53 under the same conditions as in the second embodiment. When it takes to measure electrochemiluminescence The interval is 0.4 sec.

 (Fourth embodiment)

 Fig. 12 is a diagram showing the configuration of a DNA detection cell in which the working electrode and a plurality of independent counter electrodes are formed on the same plane. In the fourth embodiment, the configuration of a DNA detection cell in which sections are integrated beyond the spatial resolution of the light detection means will be described. The configuration of the electrodes of the DNA detection cell of the fourth embodiment is similar to that of the third embodiment. The total number of sections (size 200 μπιΧ 200 μιη) separated by force dashed lines is the same as in the second embodiment. The working electrode 60 of 10000 is shown in Fig. 12 and only 6 sections are shown.) The counter electrode (width: 5 m) 62-1, 62-2, 62-3 in one direction The configuration in which a gap of 5 μπι is interposed and the voltage can be applied to each of the counter electrodes 62-1, 62-2 and 62-3 independently of each other is different from the configuration of the third embodiment. The working electrode 60 is always at the ground potential, and the power is supplied independently to the counter electrodes 62-1, 62-2, and 62-3 using the switch 62-1S, 62-2S, 62-3S. Voltage can be applied by the controller 45 and the power supply 44.

The working electrode 60 is provided with sections 6 1-1-6 1-1 6 separated by broken lines. When the potential of all counter electrodes is 0 V, the potential of the solution in the DNΑ detection cell is uniformly 0 V. When the potential of the counter electrode 62-2 is changed from 0 V potential to 11.4 V potential, the uniformity of the solution potential is lost, and the solution potential near the counter electrode 62-2 changes from 0 V to a negative potential. To be reduced to The negative potential of this solution spreads radially from the counter electrode 62-2 with time. When the solution potential on the working electrode surface becomes negative, the potential distribution of the electric double layer formed near the working electrode surface changes to reflect the potential difference between the solution and the working electrode. This change propagates in the direction of arrows 63 and 64 at the velocity V _T with the spread of the negative potential of the solution. The propagation velocity V _T depends on the ionic concentration of the solution, the temperature of the solution, the potential difference between the solution and the working electrode, etc., for example, including 0.1 I Smol ZL tripropylamine (TPA) as a reducing agent. The value was about 15 mm / sec in a phosphate buffer (pH 6.8) of 30 m 01 ZL. When the potential distribution of the electric double layer is formed so that the potential between the solution and the working electrode is 11.1 V or higher, an electrochemiluminescent reaction occurs between the Ru complex and TPA. The light emitting region expands in the directions of arrows 63 and 64 at a speed of about 15 mm / sec from the counter electrode 62-2.

The distance between the center of the counter electrode and the boundary of the section is d. After the time T = dZV _T elapses after setting the counter one electrode 62_2 to the negative potential, the electric double layer formed near the working electrode surface due to the change in the potential of the counter electrode 62-2 when the time T = dZV _T elapses. The change in the potential distribution reaches the boundaries of the compartments. As a result, of the compartments 6 1–3 and 6 1–4, in the compartment where the electrochemiluminescent label is captured, an electrochemical reaction occurs and electrochemiluminescence occurs. When the potential of the counter electrode 62-2 is returned to ground when the time T = dV _τ has elapsed after the counter electrode 62-2 is set to a negative potential, the propagation of the potential difference between the solution and the electrode disappears. However, no electrochemiluminescence occurs in the other compartments under the influence of the counter electrode 62-2. Similarly, if any other counter electrode is selected and a potential is applied for a time Τ, the electrochemiluminescence reaction can be excited only in the section containing the selected counter electrode. In the fourth embodiment, a light detection system using three sections as one light detection unit is used.

FIG. 13 is a diagram illustrating an optical system that collects and detects electrochemiluminescence from a plurality of sections. The total number of partitions is 10,000 as in the second embodiment, and Fig. 13 shows only six partitions. In Fig. 13, the electric light emitted from the sections 6 1-1, 6 1-3, 6 1-5 is collected on the APD 7-1 by the optical fiber 7-1-1, and the section 6 1- Electrochemiluminescence from 2, 6 1 -4, 6 1-6 is condensed on APD 72-2 by optical fiber 1 7-2. (To detect electrochemiluminescence from 100 000 sections, , 100 APDs are required). A switch selects one electrode of the counter one by one to apply a negative potential, and reads out the photodetection signal from the two APDs in synchronization with the selection of the one electrode of the counter. Measurement of electrochemiluminescence from the compartment is possible. According to the fourth embodiment, according to the fourth embodiment, the light receiving surfaces of the optical fibers 71-1 and 72-2 are selected by a force counter electrode which is larger than the area of each section. The advantage is that it is possible to measure the electrochemiluminescence from each compartment sequentially with one photodetection system, and to evaluate DNA detection cells with more types of probes than the total number of photodetection systems. There is.

In the fourth embodiment, after the counter electrodes are sequentially selected and switched, a voltage (for example, 1 1 1) is applied between the working electrode 60 and the counter electrodes 62-1 to 62-3,. Apply 4V). Assuming that the size of the section is 200 ^ 111X200 ^ 111 and the propagation velocity V _T is about 15 mm / sec, as in the second embodiment, the duration of the above voltage application T is (0.2 mm 2) 15 = 0.0067 sec = 6.7 ms ec or less, and the time required to detect electrochemiluminescence from 10,000 sections is 6.7 msec X 100 = 0.67 sec.

 (Fifth embodiment)

Fig. 14 shows the relationship between the size of the compartment observed on the imaging surface of the TV camera and the size of the image sensor when the electrochemiluminescence from the compartment of the integrated DNA detection cell is collected and detected by the TV camera. FIG. Using a TV camera as the light detection means is effective when using a DNA detection cell with many fractions that uses many types of DNA probes at once. In FIG. 14, a working electrode 111 (formed on the substrate 11 below the DNA cell), which is indicated by oblique lines, has a plurality of sections (200 ^ 111X200 m) defined by broken lines in the X and y directions. With a counter electrode (having a square outer shape with a side of 5 μm to 10 im) with a power of several μm at the center of each section. It is surrounded by 1 (shaded area) and arranged opposite the working electrode. That is, the counter electrode is separated from the working electrode 1 1 1 (shaded area) and formed on the substrate 12 below the DNA cell. The distance between the center of each counter electrode and the boundary of each section is defined as h (= 100 ^ m). As in the second embodiment, the total number of sections is 10,000 and the outer dimensions of the working electrode are 2 Omm x 2 Omm, and Fig. 14 shows only 16 sections. In the following, the light-receiving area of the first detection (imaging) element (specifying the spatial resolution) is defined as the light detection means, for example, the image observed on the imaging surface of the TV camera. 8 1—1 Consider the case where the electrochemical emission from the four sections 82—1 to 82-4—total enters.

 FIG. 15 is a diagram showing a configuration of a DNA detection cell in which counter electrodes are connected by matrix wiring and formed on a substrate under the DNA cell. The wiring and gate shown in Fig. 15 are formed on the surface of the substrate (electrically insulating material) under the DNA cell, and then the working electrode and the power electrode shown in Fig. 14 are formed via the insulating layer. The gate is electrically connected to the electrode. The total number of counter electrodes is 10000. As in Fig. 14, only 16 counter electrodes are shown in Fig. 15. As shown in Fig. 15, the matrix wiring consists of TFT gates 91-1 to 91-4 corresponding to each counter electrode 83-1 to 83-4, gates 91-1 and 91, respectively. — Conductor connected to 3 92 — 1, Conductor 92-1 connected to gates 9 11 and 9 1 — 4, Gate wire that controls ONZOF F of gates 9 11 1 and 9 1 1 2 93 — 1, Gates 91 and 3 and 9 1 and 4 are used to control the ONZOFF of the gate lines.

Fig. 16 is a diagram illustrating the selection of the counter electrode, that is, the section that induces electrochemiluminescence, by selecting the gate line (93-1, 93-2) and the conductor (92-1, 92-12). is there. In the DNA detection cell shown in Figs. 14 and 15, the conductors except for conductor 92-1 were set to 0 potential, and a negative potential was applied to conductor 92-1. Is set to the OFF potential (for example, 0 potential) and the gate line 93_1 is set to the ON potential (for example, 10 V or more) for the interval of time T == hV _T. Gate 911 turns on, and conductor 92-1 and counter electrode 83-1 conduct. As a result, the electric double layer can be formed so that the counter electrode 83-1 has a negative potential only for the time T and the potential difference between the solution and the electrode is 11.4 V only in the section 82-1. If an electrochemiluminescent label is captured in Section 82-1, an electrochemical emission reaction will occur. Note that the upper left section of the light-receiving areas 81-1-1 to 81-4 of the first to fourth imaging elements (in Fig. 14, only reference numerals 82-11 are assigned for simplicity). A negative potential is applied to all conductors connected to the counter electrode When the ON potentials of all the gate lines connected to the gates in the upper left section are applied, electrochemiluminescence can be induced selectively only in the upper left section in the light receiving area 81-1-1 to 81-4. it can.

 In the following, as shown in Fig. 16, by selecting the gate line and the conductor, the selected counter electrode, that is, the surface (section) that induces electrochemiluminescence is selected, and the light receiving area 8 1-1 to 8 1- Electrochemiluminescence from each section belonging to each of 4 can be sequentially detected for each section. For example, the electrochemiluminescence from the four sections 82-1 to 82-4 belonging to the light receiving area 81-1 of the first image sensor can be separated for each section and detected sequentially. As a result, in the fifth embodiment, the number of sections that can be detected by one detection element is larger than the area of one section of the DNA detection cell, even though the light receiving area of one detection element of the light detection means is larger than the area of one section of the DNA detection cell. Becomes 4.

In the fifth embodiment, one detection (imaging) element of a TV camera detects the electrochemiluminescence from four different sections by changing the time. That is, the electrochemiluminescence from the 10,000 sections is detected in four times. Apply a voltage (for example, 1.1 V) between the working electrode 1 1 1 and the counter electrode 83-1 to 83-2,. Assuming that the size of the compartment is SOO mx SOO / ^ m, as in the second embodiment, and the propagation velocity V _T is about 15 mmZs ec, as in the fourth embodiment, The duration T of the voltage application is less than 6.7 ms ec, and the time required to detect electrochemiluminescence from the 10,000 compartments is 6.7 ms ec X 4 = 26.8 ms ec.

In the above description of the fifth embodiment, an example in which a cooled CCD camera is used as a TV camera will be described below. Here, a one-inch cooled CCD camera consisting of one million elements is used (the size of the CCD element on the light-receiving surface of the CCD camera is 18 m square). The reduction ratio of the optical system placed between the CCD camera and the DNA detection cell is about 1-11. When a TV camera such as a CCD camera is used, the spatial resolution is about 2 L when the size of the photodetector on the light receiving surface is L, from a practical point of view. When L = 18 μπι, four CCD elements detect electrochemiluminescence from four sections of the DΑ detection cell In this case, the electrochemiluminescence from the four compartments is separated and detected by the same method as described above. The four elements of the CCD element form the first light detection aperture (used to mean that four elements form an effective one element; corresponding to 81-1 shown in Fig. 14). Similarly, the second to fourth light detection apertures 81-2 to 81-4 are formed by the other four elements. Since the light detection aperture is formed by four CCD elements, if a 1-inch CCD camera with 100,000 elements is used, a total of 250,000 light detection apertures will be formed. The size of the working electrode of the DNA detection cell described above was increased from 20 mm to 200 mm, and the number of compartments was formed to 1,000,000, and different probes were fixed in each compartment. The time required to detect electrochemical emission emitted from a DNA detection cell having 1 million sections using a 1-inch CCD camera consisting of 100,000 elements is the same as in the case described above. 8ms ec.

 According to the configuration of the fifth embodiment, measurement can be performed in a short time of 26.8 ms ec, independently of the other sections, regardless of the number of sections formed in the DNA detection cell. In addition, by forming one light detection aperture with multiple CCD elements, it is possible to independently detect, with high spatial resolution, electrochemiluminescence from a plurality of sections that one light detection opening expects.

 (Sixth embodiment)

FIG. 17 is a diagram illustrating an example of voltage application for repeatedly generating electrochemiluminescence in one selected section. In the sixth embodiment, in the fourth and fifth embodiments, after a predetermined relaxation time, a negative potential is repeatedly applied to one electrode of the counter, and the electrochemiluminescence reaction is restarted. Induce. If the intensity of the electrochemiluminescence is not sufficient due to the time T during which the negative potential is applied to the counter electrode and only one cycle of the period t and the detection sensitivity of the detection element of the light detection means is not reached, the specified relaxation time is set. After that, a negative potential is repeatedly applied to the counter electrode to re-induce the electrochemiluminescence reaction. For example, as shown in Fig. 17, a voltage is applied to one counter electrode multiple times at period t. Repeatedly applying the detection element The light is stored in the cell to detect the electrochemiluminescence. The voltage application is controlled by controlling the power supply 44 by the power supply control device 45.

For example, in the fifth embodiment, if h ^ OO m, V _T = 15 mm / sec, the voltage application time is T = 6.7 m sec. Assuming that the period is t == 2 3. = 13.4 ms ec, the applied voltage can be controlled by a 75 Hz rectangular wave (the above relaxation time is 6.7 msec). As a result, the integrated intensity of electrochemiluminescence obtained by repeating the electrochemiluminescence reaction is obtained, and the problem of insufficient intensity of electrochemiluminescence in only one cycle (low detection sensitivity, low SZN) can be solved. When the voltage detection shown in Fig. 17 is repeated n cycles using a DNA detection cell with 10,000 compartments, the measurement time required for the detection of electrochemiluminescence is as shown in the fourth embodiment. Is 0.67 X (2 n) sec, and in the fifth embodiment it is 26.8 X (2 n) msec, which is n times the intensity of electrochemiluminescence in only one cycle. n times better. For example, if n = 60, the measurement time is 80.4 sec (fourth embodiment) and 3.2 sec (fifth embodiment).

 In the third to sixth embodiments, the signal detected by the light detecting means is converted into a current or a voltage, is converted into a digital signal by the A / D converter 38, and is processed by the data processing device 39. , The first and the second embodiments. (Seventh embodiment)

In the first to sixth embodiments, the DNA probes 13, 14, 15, and 16 immobilized on the DNA detection cell are phosphates between the 2, -deoxy oligonucleosides. Oligonucleotides having diester bonds. In the ninth embodiment, as shown in FIG. 18, in the first to sixth embodiments, a phosphorothioate bond is formed between 2'-deoxyoligonucleosides. Oligonucleotides (reference number 231) are used as DNA probes 13, 14, 15, 15 and 16 and fixed to DNA detection cells. B in FIG. 18 represents a nucleic acid base (A, T, G, or C). DNA probes with phosphoroate bonds are degraded by S1 nuclease. Absent.

 (Eighth embodiment)

 FIG. 19 is a diagram showing a configuration of an inspection device using a DNA detection cell. The DNA detection cell has a lower substrate 241 having a concave portion and a transparent upper substrate 243, and the bottom surface 242 of the concave portion of the lower substrate 241 is described in the third embodiment (FIG. 11). As described above, the working electrode and the counter electrode are arranged on the same plane. The DNA detection cell is inserted and fixed in an optically opaque cell holder 244. An imaging lens 245 and a cooled CCD camera 246 are arranged at an imaging position of the imaging lens 245 so as to face the upper substrate 243. The camera head 247 that fixes the CCD camera 246 is optically opaque and is connected to the cell holder 24 to block external light from entering the DNA detection cell and the optical system. A power supply 44 is connected to the working electrode and the counter electrode of the DNA detection cell. The application of a voltage by the power supply 44 and the reading of signals stored in the CCD camera 246 are controlled by a controller 248. The read signal is digitally converted by the AZD converter 38, processed by the data processing device 39, and stored as a two-dimensional digital image in the memory. In the configuration of FIG. 19, as the configuration of the counter electrode, FIG. 1 (first embodiment), FIG. 10 (second embodiment), and FIG. 12 (fourth embodiment) For example, the configurations shown in FIGS. 14 and 14 (fifth embodiment) may be used.

 FIG. 20 is a plan view of the DNA detection cell shown in FIG. 19 as viewed from the imaging lens 245 side. The DNA detection cell according to the eighth embodiment has the same configuration as the DNA detection cell according to the second embodiment. The surface of the working electrode of 20 mm × 20 mm has an area of 200 mx 200 μπι. Each DN is divided into 100 = 10000 sections, and a different DN II probe is fixed in each section.

 Note that, in the eighth embodiment having the above configuration, the time required for the measurement of electrochemical luminescence is 0.4 sec, as in the second embodiment.

When electrochemiluminescence is obtained as a two-dimensional digital image as described in the second embodiment, or as described in the fourth, fifth, and sixth embodiments, DNA detection is performed. If the degree of integration of the cell exceeds the spatial resolution of the photodetector, the arrangement of the sections of the DNA detection cell and the arrangement of the detector elements of the photodetector should be matched accurately, or each of the light emitted after the measurement of electrochemical luminescence. There is a need to accurately determine the position of the section.

 In order to facilitate alignment between the arrangement of the sections of the DNA detection cell and the arrangement of the detection elements of the photodetector, a plurality of markers are provided in the DNA detection cell, and the position of the marker is used to identify the DNA detection cell. Adjust the position of the photodetector. Alternatively, the position of the optical marker is measured at the same time as the measurement of the electric light, and the positional relationship between the DNA detection cell and the photodetector is detected during data processing, so that the position of each light-emitting section can be accurately determined. Ask for. Either place a light emitting diode with a small area such as a light emitting diode in the DNA detection cell and use it as a marker, or place a pinhole in the DNA detection cell and use the light passing through the pinhole as a marker. A section at a specific position of the DNA detection cell can also be used as a marker.

 The four compartments 251, 252, 253, and 254 shown in Fig. 20 are specially prepared compartments that are used as markers for DNA detection cells. As in the second embodiment, when the amount of the DNA probe immobilized in one compartment is 0.027 fmo1, 0.0270 fmol in compartment 25 1 and 0.002 03 fm in compartment 252. 0 1, 0.135 fmo in section 253 and 0.0 068 fm 0 1, Ru complex in section 254 with known concentration. Electrochemiluminescence from a compartment in which a known concentration of Ru complex is immobilized can be used as a scale of emission intensity.

To fix a known concentration of Ru complex, there are two methods: first, fix it when preparing a DNA detection cell, and the other method is described below. For example, a probe having a base sequence different from that of all other DNA probes (referred to as a marker probe) is fixed in advance at sections 251, 252, 253, and 254 at the above known concentration, and the target DNA fragment When complementarily binding to the DNA probe of each compartment, a marker DNA that complements only the marker-probe is added, and the marker probe and the marker DNA are complementarily bound to each other, and the DNA described in the first embodiment is added. Perform the marker probe extension reaction simultaneously with the probe extension reaction. using this method By measuring the luminescence based on Ru: dNTP or Ru: d dNTP incorporated in the extended chain of the marker probe, it can be confirmed that the complementary binding and extension reactions were performed smoothly. .

 The position of the luminescent compartment in the DNA detection cell is determined by the following method. Electrochemical luminescence is always detected from the four sections 25 1, 252, 253, and 254 shown in FIG. The method of analyzing the detected two-dimensional emission image will be described below. The two-dimensional luminescence image including the luminescence from the sections 25 1, 252, 253, 254 is represented by the xy coordinates with the reference number 255 as the origin, forming a two-dimensional luminescence image. Find the number of P y pixels in the y direction. Since the DNA detection cell is composed of 100 x 10 ° sections, the number of pixels per section is Px / 100 = Qx and Py / 100 = Qy. The coordinates of the corner point 257 close to the origin of the light-emitting section 256 are determined as (Mx, My) (the number of pixels Mx, My is the number of pixels), and I = [MxZQx10 1], J = [MyZQy + l] ([] Means round the first decimal place of the value in [], and the resulting I and J are integers). As a result, it can be seen that the section 256 that emits light is a section of the J row and column I of the DNA detection cell.

 In the second embodiment, the electrochemiluminescence is measured for 0.4 sec, but in the eighth embodiment, the electrochemiluminescence from the same section is repeatedly measured.

FIG. 21 is a diagram illustrating an example of voltage application for generating electrochemiluminescence repeatedly. In Fig. 21, the horizontal axis represents time, reference numeral 261 is the voltage applied between the working electrode and the counter electrode of the DNA detection cell, and reference numeral 262 is the amount of luminescence from the electrochemiluminescent label. Is shown. When a voltage is applied for 0.4 sec, electrochemiluminescence occurs at the same time as the voltage is applied. However, the reducing agent (TPA in the eighth embodiment) is rapidly consumed on and near the working electrode surface. The light emission decreases rapidly. Therefore, even if the voltage application time is prolonged, an increase in light emission cannot be expected. However, if the application of voltage is stopped and a relaxation time of 9.6 sec is set, the reducing agent in the solution is supplied to the surface of the working electrode and its vicinity by diffusion. When voltage is applied again for 0.4 sec, electrochemiluminescence occurs. 0. By repeating the application of the voltage for 4 sec and the stop of the application of the voltage for 9.6 sec (period 10 sec), the total amount of electrochemiluminescence can be increased and the detection sensitivity can be improved. It is valid. Note that the voltage application method shown in FIG. 21 is not limited to the second embodiment, but can be applied to each of the first to seventh embodiments, and a similar effect is obtained. Can be

 (Ninth embodiment)

 In the first embodiment, Ru: dNTP or Ru: d dNTP is used to carry out the extension reaction of a DNA probe complementary to the target DNA fragment. In the seventh embodiment, the extension reaction is carried out. Use a method that does not execute. An electrochemiluminescent-labeled oligonucleotide 28 is bound to the target polynucleotide (target DNA fragment) 21 in advance.

 FIG. 22 shows a DNA probe complementarily bound to a target polynucleotide bound to an electrochemiluminescent labeled oligonucleotide. Electrochemiluminescence is detected by the method described in each of the first to sixth embodiments.

 (Tenth embodiment)

 In the ninth embodiment, an electrochemiluminescent label may be bound to the 5 ′ end of the target polynucleotide (target DNA fragment) 21 using an oligonucleotide labeled with an electrochemiluminescent label.

 Fig. 23 shows a DNA probe that is complementary to the target polynucleotide labeled with electrochemiluminescence. Electrochemiluminescence is detected by the method described in each of the first to sixth embodiments.

 (First Embodiment)

FIG. 24 is a diagram for explaining the inspection procedure of the polynucleotide inspection device. The DNA detection cell described in each embodiment (for example, the cell formed by the DNA lower substrate 11 and the DNA cell substrate 12 shown in Fig. 1) contains the DNA fragment group to be measured. Add sample solution. Next, the temperature of the solution is set to a temperature suitable for hybridization, and the DNA probe and the DNA fragment are combined with each other by complementary strand binding. Best binding efficiency between DNA probe and DNA fragment and non-specific A temperature condition that does not cause strong binding is determined experimentally in advance within the range of 55 to 65 ° C, and the temperature of the solution is set. After the complementary strand binding reaction, use 2 OmM phosphate buffer (pH 7.0) supplemented with 0.05% Tween 20 as a washing solution, and remove unbound DNA fragments at normal temperature from the DNA detection cell. To the outside of the Next, an extension reaction is performed on the DNA probe complementary to the target DNA fragment. After the extension reaction, the cell is washed with a washing solution to remove unreacted substrate, an electrochemiluminescent reagent is injected into the cell, and a voltage is applied between the working electrode and the counter electrode to generate the electrochemical reaction. Light emission is detected. After completion of the electrochemiluminescence detection, the DNA probe fixed in each compartment of the DNA detection cell is released, and the DNA detection cell is regenerated. The DNA detection cell can be played back in the following three typical methods.

 In the first regeneration method, the DNA probe immobilized in each compartment of the DNA detection cell is released and completely removed, and a new DNA probe is immobilized. In the first method, false positives are unlikely to occur because there is almost no residual sample.

 In the second regeneration method, the DNA detection cell is washed with pure water at 95 ° C to release the target DNA fragment from the DNA probe, and only the target DNA fragment is removed from the detection cell. The second method can easily regenerate the DNA detection cell in a short time and is effective in the ninth and tenth embodiments.

The third reproduction method is effective in the seventh embodiment. In the third regeneration method, first, the DNA detection cell is washed with pure water at 95 ° C to release the target DNA fragment from the DNA probe and removed from the DNA detection cell. As a result, a DNA probe (15 shown in Fig. 7) and a single-stranded DNA probe having an extended portion (26 and 27 shown in Fig. 7) by complementary strand synthesis were found in the DNA detection cell. Remains. Next, when S1 nuclease is injected into the DNA detection cell, the extended portions 26 and 27 are decomposed into mononucleotides by S1 nuclease. The DNA probe remains immobilized on the DNA detection cell without being degraded by S1 nuclease, and the DNA detection cell is regenerated to the state before use. In the third regeneration method, the DNA probe can be reused and may remain in the DNA detection cell. The characteristic feature is that even target DNA fragments that have no residue can be degraded by S1 nuclease, and no sample remains.

 (Sequence listing)

SEQ ID NO: 1

Array length: 30

Sequence type: nucleic acid

Number of chains: 1 strand

 Topology: linear

Sequence type: Other nucleic acids Synthetic DNA

Array:

TC

SEQ ID NO: 2

Array length: 30

Sequence type: nucleic acid

Number of chains: 1 strand

 Topology: linear

Sequence type: synthetic DNA

Array:

Claims

The scope of the claims

1. Different DNA probes (13,14,15,16) have different types (3,4,5,6,61-1-6-1-6,82-1-8-2). — The first electrode (111, 52, 60) fixed to (4) and the second electrode (113-1-1, 113-2-2, 53, 62) facing the first electrode. And a voltage detection means for applying a voltage between the first electrode and the second electrode. 44), the target polynucleotide is captured by complementary strand binding between the DNA probe fixed to the compartment and the target polynucleotide (21), and the electrochemiluminescent labeled base is captured.

A photodetection means (33, 34, 35, 36, 43, 72) for performing an extension reaction using (24) to extend the DNA probe with the complementary strand bound, and detecting the electrochemiluminescence generated by the application of the voltage. -1, 7, 2-2, 246), and detecting the presence or absence of an extended chain (26) generated by the extension reaction.

 2. The polynucleotide testing device according to claim 1, wherein the electrochemiluminescent label is a ruthenium complex or an osmium complex.

3. The polynucleotide according to claim 1, wherein the light detection means is an imaging means (43, 266) for detecting the electrochemiluminescence from the plurality of sections as a two-dimensional image. Inspection equipment.

 4. The second electrode is composed of a plurality of electrodes, and an electrode selecting means (62-1S to 3S, 91-1 to 91-4) for selecting a predetermined electrode from the plurality of electrodes is provided. And applying the voltage between the electrode selected by the electrode selecting means and the first electrode, and performing electrochemiluminescence from a predetermined section selected from a plurality of the sections. 2. The polynucleotide testing device according to claim 1, wherein the polynucleotide is detected.

5. The electrode selecting means according to claim 4, wherein said electrode selecting means comprises a TFT gate (91-1 to 91-14) connected to each of said plurality of electrodes. Polynucleotide testing device.

 6. The first electrode and the second electrode are alternately and repeatedly arranged on the same surface in parallel in one direction, and alternately and alternately arranged with the expanding speed of the region where the electrochemiluminescence occurs. Controlling the voltage application time based on the distance between the center line of the first electrode in the one direction and the center line of the second electrode in the one direction. The polynucleotide testing device according to claim 1, further comprising means (45).

 7. The polynucleotide testing apparatus according to claim 6, wherein the voltage is repeatedly applied.

 8. Different DNA probes (13, 14, 15, 15 and 16) have different types (3, 4, 5, 6, 61-1 to 61-6, 82-1 to 82- The first electrode (111, 52, 60) fixed to 4) and the second electrode (113-1, 1, 113, 2, 53, 62) opposed to the first electrode are fixed. A polynucleotide detection cell comprising: 1 to 62-3, 83-1 to 83-3-4), and voltage applying means for applying a voltage between the first electrode and the second electrode. And the DNA probe fixed to the compartment and an oligonucleotide labeled with electrochemiluminescence.

A light detection means (33, 34, 35) for capturing the target polynucleotide by complementary strand binding with the target polynucleotide (21) to which (28) is bound, and detecting the electrochemical emission generated by the application of the voltage. , 36, 43, 72-1, 72-1, 246).

9. Different DNA probes (13,14,15,16) have different types (3,4,5,6,61-1-6-1-6,82-1-82- 4) A first electrode (111, 52, 60) fixed to the first electrode and a second electrode (113-13-1, 11, 13-2, 53, 62) opposed to the first electrode. -1 to 62—3, 8 3—1 to 8

3-4); a voltage application means (44) for applying a voltage between the first electrode and the second electrode; and the DNA fixed to the compartment. Capturing the target polynucleotide by complementary strand binding between the probe and an electrochemiluminescent-labeled target polynucleotide (21); A polynucleotide inspection device comprising: light detection means (33, 34, 35, 36, 43, 72-1, 72-2, 246) for detecting electrochemiluminescence generated by application of a voltage.

 10. Different compartments (3, 4, 5, 6, 6, 6 1—1 to 6 1—6, 82—1 to 82—4 for different types of different DNA probes (13, 14, 15 and 16) )) And a plurality of second electrodes (113-1, 2, 113-3, 53, 53) opposed to the first electrode. 62-1 to 62-3, 83-1 to 83-4), and an electrode selecting means (62-1S-3) for selecting an electrode from the plurality of second electrodes. S, 91-1 to 91_4) and voltage applying means (44) for applying a voltage between the first electrode and the selected electrode, wherein the electrochemiluminescent label is modified. The plurality of compartments are generated by causing the target polynucleotide captured by complementary strand binding between the target polynucleotide and the DNA probe to generate electrochemiluminescence from the electrochemiluminescence label by applying the voltage. Selected from Polynucleotide inspection apparatus characterized that you detected for each zone.

 1 1. Different compartments (3, 4, 5, 6, 6 1—1 to 6 1—6, 82—1 to 8 2— for different types of different DNA probes (13, 14, 15 and 16) 4) a first electrode (111, 52, 60) fixed to the first electrode and arranged on the same surface as the first electrode, separated from the first electrode, and arranged at the center of each section. A polynucleotide detection cell comprising a plurality of second electrodes (83-1 to 83-4) thus selected; and an electrode selecting means (91-1-9) for selecting an electrode from the plurality of second electrodes.

1-4), voltage applying means (44) for applying a voltage between the first electrode and the selected electrode, and detecting electrochemiluminescence generated from the electrochemiluminescence label by the application of the voltage. Light detection means (33, 34, 35, 36, 43, 7

2-1, 7 2 -2, 246), a center of the selected second electrode, and a boundary with the section adjacent to the section in which the selected second electrode is arranged. And means for controlling the time for applying the voltage based on the distance between the section and the area in which the electrochemiluminescence occurs. Detecting the target polynucleotide captured by the polynucleotide

12. The polynucleotide testing apparatus according to claim 11, wherein the plurality of second electrodes are arranged at equal intervals in two directions.

 1 3. Different DNA probes (13,14,15,16) have different compartments for each type (3,4,5,6,61-1-1 to 61--6,82_1-82 — 4) fixed to the first electrode (111, 52, 60) and a plurality of second electrodes (53, 62−1 to 62) arranged on the same surface as the first electrode. —3, 83—1 to 83—4), and a polynucleotide detection cell, and an electrode selection means (62−1S ，, 62—3S, for selecting an electrode from the plurality of second electrodes). 9 1— 1 to 9

1-4), voltage applying means (44) for applying a voltage between the first electrode and the selected electrode, and electrodynamic emission generated from the electrochemiluminescence label by the application of the voltage. Detection means (33, 34, 35, 36, 43, 7)

2-1, 72-2, 246) and means (45) for controlling the time for applying the voltage based on the expanding speed of the region where the electrochemiluminescence occurs. A polynucleotide testing device for detecting the target polynucleotide.

 14. Different DNA probes (13,14,15,16) have different sections for each type (3,4,5,6,61-1 to 61-6, 82-1 to 82- The first electrode (111, 52, 60) fixed to 4) and a plurality of second electrodes (113-1-1, 113-2-2, 53) facing the first electrode. , 6 2—1 to 6 2—3, 8

3-1 to 83-4), and an electrode selection means (62-1S ~, 62-3S,) for selecting an electrode from the plurality of second electrodes.

9 11-1 to 9 11 4), and voltage applying means (44) for applying a voltage between the first electrode and the selected electrode, wherein the application of the voltage causes an electrochemical reaction. A polynucleotide testing apparatus, comprising: detecting electrochemiluminescence generated from a luminescent label for each of the sections selected from the plurality of sections to detect the target polynucleotide captured for each of the sections.

1 5. Different DNA probes (13, 14, 15 and 16) Different compartments for each force type (3, 4, 5, 6, 6 1—1 to 6 1—6, 82—1 to 82—4 ) Is fixed to the first electrode (111, 52, 60) and the second electrode (113-1-1, 113--2, 53, 62-1) facing the first electrode. -62-3, 83-1 to 83-4), the DNA probe immobilized in the compartment of the polynucleotide detection cell comprising the target polynucleotide (21) and the target polynucleotide (21) in a complementary strand bond. A step of capturing a polynucleotide, a step of performing an extension reaction using an electrochemiluminescent-labeled base (24) to extend the DNA probe having a complementary strand bound thereto, and a step of: Applying a voltage between the electrodes, and detecting the presence or absence of electrochemiluminescence caused by the application of the voltage to detect the presence or absence of an extended chain (26) generated by the extension reaction A method for testing a polynucleotide.

 1 6. Different DNA probes (13, 14, 15 and 16) Different compartments for each type of force (3, 4, 5, 6, 6 1—1 to 6 1—6, 82—1 to 82—4 ) And a second electrode (113-1-1, 113--2, 53, 62-1) facing the first electrode (111, 52, 60) fixed to the first electrode. ~ 62- 3, 83- 1 ~

83-4) complementary strand binding between the DNA probe immobilized in the compartment of the polynucleotide detection cell comprising: and the target polynucleotide (21) to which the oligonucleotide (28) labeled with electrochemiluminescence is bound. And capturing a target polynucleotide; and applying a voltage between the first electrode and the second electrode to detect electrochemiluminescence generated by the application of the voltage. A method for testing a polynucleotide, comprising:

1 7. Different DNA probes (13,14,15,16) Different compartments for each type of force (3,4,5,6,61-1-1 to 6 1-6,82-1-1 to 82- 4) A first electrode (111, 52, 60) fixed to the first electrode and a second electrode (113--1, 1, 113-3, 53, 62) opposite to the first electrode. 1 to 62-3, 83-1 to 83-4), the DNA probe immobilized in the compartment of the polynucleotide detection cell, and an electrochemiluminescent-labeled target polynucleotide (2 1) capturing the target polynucleotide by complementary strand bonding, and applying a voltage between the first electrode and the second electrode to generate electricity generated by the application of the voltage. A step of detecting chemiluminescence.

 1 8. Different DNA probes (13, 14, 15 and 16) Different compartments for each force type (3, 4, 5, 6, 61-1 to 61 to 6, 82 to 1 to 82-4) ) And a plurality of second electrodes (53, 62-1 to 62-3) arranged on the same surface as the first electrode. , 83-1 to 83-4), wherein a step of selecting an electrode from the plurality of second electrodes is performed between the first electrode and the selected electrode. Applying a voltage, detecting electrochemiluminescence generated from the electrochemiluminescence label by applying the voltage, and applying the voltage based on a speed at which the region where the electrochemiluminescence occurs expands. Controlling the length of time for which the target polynucleotide is retained as a target, and detecting the target polynucleotide captured in each of the compartments. Method.

 1 9. The area where the electrochemiluminescence occurs is enlarged to be approximately equal to the time required to reach the section adjacent to the section where the electrode selected from the plurality of second electrodes is located. 19. The method for testing a polynucleotide according to claim 18, wherein the voltage is applied and held for a length of time.