JP2008157916A - Water-bearing sheet used in specific detection of substance to be inspected using photocurrent, detection method using the sheet, sensor unit and measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrolyte medium which significantly simplifies apparatus structure and detection procedure in the specific detection of a substance to be inspected, using the photocurrent produced by the optical excitation of a sensitizing dye and has detection sensitivity equal to that when an electrolytic liquid is used. <P>SOLUTION: A water-bearing sheet used as the electrolyte medium in a specific detection of the substance to be inspected by using the photocurrent produced by the optical excitation of the sensitizing dye is disclosed. This water-bearing sheet contains a water-bearing base material and the electrolyte contained in the water-bearing base material. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

FIELD OF THE INVENTION The present invention is used in place of an electrolytic solution as an electrolyte medium when specifically detecting a test substance having specific binding properties such as a nucleic acid, an exogenous endocrine disrupting substance, and an antigen using a photocurrent. The present invention relates to a possible water absorbent sheet, a detection method using the same, a sensor unit, and a measuring apparatus.

  Gene diagnosis methods for analyzing DNA in biological samples are promising as new prevention and diagnosis methods for various diseases. As a technique for performing such DNA analysis simply and accurately, a sample DNA is hybridized with a DNA probe having a complementary base sequence and a fluorescent substance and labeled with a fluorescent signal. Methods for analyzing DNA to be detected are known (see, for example, Patent Document 1 and Patent Document 2). In this method, the formation of double-stranded DNA by hybridization is detected by fluorescence of the dye.

  In addition, a method for specifically detecting a test substance (biomolecule such as DNA or protein) using a photocurrent generated by photoexcitation of a sensitizing dye has also been proposed (see, for example, Patent Document 3 and Non-Patent Document 1). ). Such a detection method is performed using a sensor unit filled with an electrolytic solution.

  On the other hand, in a micro biosensor using an oxygen electrode that converts an increase / decrease of an enzyme into an electrical signal, it is known to use a gel such as agarose as an electrolyte solution-containing body (for example, see Patent Document 4).

JP-A-7-107999 Japanese Patent Laid-Open No. 11-315095 JP 2006-119111 A Japanese Patent Publication No. 5-84860 Nakamura et al. "New DNA double strand detection method by photoelectric conversion" (Proceedings of the Chemical Society of Japan Vol.81ST nO.2 (2002), p. 947)

As described above, when a test substance (biomolecules such as DNA and protein) is specifically detected using a photocurrent, it has so far been performed using a sensor unit filled with an electrolytic solution. However, in this case, the sensor unit leaks liquid, it takes time to feed liquid into the sensor unit, and it is necessary to hold the liquid feeding mechanism having a complicated structure in the detection device. There was a problem of becoming large.

  In the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye, the present invention greatly simplifies the device structure and detection procedure, and can also detect the photocurrent with high accuracy. And it aims at providing the detection method.

  The present inventors can now use a water-absorbent sheet instead of an electrolyte as an electrolyte medium in the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye, whereby the device structure And we have found that the detection procedure can be greatly simplified. Moreover, the knowledge that the detection sensitivity and detection accuracy equivalent to the case where an electrolyte solution was used was obtained when a water absorbing sheet was used was also obtained.

That is, the water absorbent sheet according to the present invention is a water absorbent sheet used as an electrolyte medium in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A water absorbent substrate,
And an electrolyte contained in the water-absorbing substrate.

Further, the specific detection method of a test substance using photocurrent according to the present invention is as follows:
A working electrode comprising a probe substance to which a test substance is directly or indirectly specifically bound, and a sensitizing dye fixed by the binding, and a counter electrode are brought into contact with the water absorbent sheet, Light is applied to the working electrode to photoexcite the sensitizing dye, and a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode is generated. Detecting.

Furthermore, according to the first aspect of the present invention, a sensor unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A working electrode;
A counter electrode provided opposite to the electrode;
The water-absorbing sheet is sandwiched between the working electrode and the counter electrode, and each electrode surface of the working electrode and the counter electrode is in contact with the water-absorbing sheet.

Further, according to the second aspect of the present invention, a sensor unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
An electrode unit in which a working electrode and a counter electrode are patterned;
A facing member provided facing the electrode unit;
The water-absorbing sheet is sandwiched between the working electrode and the counter electrode, and each electrode surface of the working electrode and the counter electrode is in contact with the water-absorbing sheet.

Furthermore, the measuring apparatus used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye according to the present invention,
The sensor unit;
A light source for irradiating the working electrode with light;
And an ammeter for measuring a current flowing between the working electrode and the counter electrode.

  According to the present invention, the apparatus structure and detection procedure can be greatly simplified in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye.

Water-absorbing sheet The water-absorbing sheet according to the present invention is a sheet-like water-absorbing substrate used as an electrolyte medium in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. And this water absorbing sheet comprises a water absorbing substrate and an electrolyte contained in the water absorbing substrate. The electrolyte can freely move in the water-absorbing substrate and participate in the transfer of electrons between the sensitizing dye, the working electrode, and the counter electrode. Therefore, light generated by photoexcitation of the sensitizing dye between the working electrode and the counter electrode can be obtained by sandwiching the water absorbing sheet between the working electrode and the counter electrode and keeping the surface of each electrode in contact with the water absorbing sheet. A current can flow. This water-absorbing sheet is the same as the electrolytic solution conventionally used in terms of the electrolyte medium, but because it is a sheet-like material that can be handled alone, it can be easily sandwiched between the working electrode and the counter electrode, It can be removed or carried, so that the device structure and detection procedure can be greatly simplified. This is a conventional method in which an electrolytic solution is filled between a working electrode and a counter electrode, and a mechanism for feeding the electrolytic solution (for example, a pump, a valve, and a control mechanism thereof), a liquid leakage prevention mechanism (for example, In light of the fact that complicated mechanisms or processes such as packing) and waste solution treatment of the electrolyte are required, this has led to an increase in cost and an increase in the size of the apparatus. In addition, the use of a water-absorbing sheet eliminates the need for time to send the electrolyte solution, so that the measurement can be immediately performed by simply sandwiching the water-absorbing sheet between the working electrode and the counter electrode. Measurement time is also shortened.

According to a preferred embodiment of the present invention, the water absorbent sheet preferably has a solvent content of 20% by mass or more and 300% by mass or less, more preferably 30% by mass or more and 200% by mass, and further preferably 40% by mass. % To 200% by mass. When the solvent content is such, a high photocurrent can be detected when the photocurrent is detected, and the detection sensitivity is improved. The solvent content is determined from (solvent content per 1 mm ³ [g]) / (density of water-absorbing substrate [g / mm ³ ]). In addition, the solvent content rate said here is a solvent content rate of the water absorbing sheet at the time of detecting a photocurrent, Comprising: As mentioned later, the said solvent content rate does not need to be satisfy | filled at the time of storage.

  As a form of the water-absorbent sheet of the present invention, it is preferable that the contact portion with each electrode is a smooth plane so as to ensure good adhesion with the working electrode and the counter electrode. Therefore, when sandwiching and using between a working electrode and a counter electrode, it is preferable to set it as the form which has uniform thickness so that adhesiveness may not be affected. On the other hand, when using an electrode unit in which the working electrode and the counter electrode are patterned on the same plane, it is sufficient that at least one side contacting the electrode unit is a smooth plane, and the thickness and thickness are uniform. Sex is not a particular problem.

  According to a preferred embodiment of the present invention, the water absorbent sheet preferably has a thickness of 0.01 to 10 mm, more preferably 0.1 to 3 mm. With such a thickness, it is easy to obtain strength suitable for handling the water-absorbent sheet alone, so it can be easily sandwiched between the working electrode and the counter electrode, removed, or carried. As a result, the sensor unit structure and detection procedure can be greatly simplified. Moreover, it does not adversely affect the photocurrent measurement.

  In the present invention, the water-absorbing substrate includes natural fibers such as cotton, hemp, wool, silk and cellulose, pulp fibers used for filter paper and papermaking, regenerated fibers such as rayon, glass fibers used for filter paper, felt, sponge and the like. It is preferable to contain at least one kind of fiber selected from the synthetic fibers used in the present invention, and there is no limitation as long as it is a water-absorbing base material that exhibits moderate strength, moisture content, and adhesion to the electrode. In addition, the processing method of the fiber used for the water absorbing base material of the present invention is not limited to a specific processing method. In this invention, it has the process of making a water absorbing base material contact the electrode in operation before a measurement. The water-absorbing substrate is required to have such a property that it is not easily damaged during handling. Therefore, since the water-absorbing substrate comprising fibers has an appropriate strength, it can be suitably used.

  According to a preferred embodiment of the present invention, preferred examples of the water-absorbing substrate include filter paper, membrane filter, glass filter, filter cloth and the like, and more preferably filter paper and membrane filter.

In the present invention, the electrolyte is not limited as long as it can freely move in the fiber matrix and participate in the exchange of electrons between the sensitizing dye, the working electrode, and the counter electrode, and a wide variety of electrolytes can be used. It is. A preferable electrolyte is a substance that can function as a reducing agent (electron donor) for donating an electron to a dye excited by light irradiation. Examples of such a substance include sodium iodide (NaI), iodide Potassium (KI), calcium iodide (CaI ₂ ), lithium iodide (LiI), ammonium iodide (NH ₄ I), tetrapropylammonium iodide (NPr ₄ I), sodium thiosulfate (Na ₂ S ₂ O ₃₎ ), sodium sulfite _(Na 2 _SO 3), _{hydroquinone, K 4 [Fe (CN)} 6] · 3H 2 O, ferrocene-1,1'-dicarboxylic acid, ferrocene carboxylic acid, hydrogen sodium borohydride (NaBH _4), Triethylamine, ammonium thiocyanate, hydrazine (N ₂ H ₄ ), acetaldehyde (CH ₃ CHO ), N, N, N ′, N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), L-ascorbic acid, sodium tellurite (Na ₂ TeO ₃ ), iron (II) chloride tetrahydrate Product (FeCl ₂ .4H ₂ O), EDTA, cysteine, triethanolamine, tripropylamine, iodine-containing lithium iodide (I / LiI), tris (2-chloroethyl) phosphate (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethanol amine, thiourea _{dioxide, (COOH)} 2, HCHO, and include those combinations, more _{preferably, NaI, KI, CaI 2,} LiI, NH 4 I , tetrapropylammonium iodide _(NPr 4 I), sodium thiosulfate _{(Na 2 S 2 O 3)} , and nitrous Sodium acid _(Na 2 _SO 3), and a mixture thereof, more preferably tetrapropylammonium iodide _(NPr 4 I).

  According to a preferred aspect of the present invention, the water-absorbent sheet of the present invention is (1) processed into a desired sheet shape and immersed in an electrolytic solution before use, or (2) processed into a desired sheet shape, After immersing in an electrolytic solution and drying, a solvent may be added and used immediately before use.

The solvent used in the present invention is an aprotic solvent, a protic solvent, or a mixture thereof. That is, a polar solvent system mainly containing water and a buffer solution component or an aprotic polar solvent can be used. Examples of aprotic polar solvents include nitriles such as acetonitrile, carbonates such as propylene carbonate and ethylene carbonate, heterocyclic compounds such as 1,3-dimethylimidazolinone, 3-methyloxazolinone, and dialkylimidazolium salts. Dimethylformamide, dimethyl sulfoxide, sulfolane and the like can be used. A plurality of types of solvents contained in the electrolyte medium can be mixed and used, and in actual use, the solvent composition can be appropriately changed according to the detection target. For example, in particular, the electrolyte is made of a salt that can supply electrons to an oxidized sensitizing dye, specifically, an iodide compound that does not contain I ₂ , and the above aprotic polar solvent By using, it is possible to accurately detect a weak difference in current value, which is effective in determining the SNP. In protein measurement, the use of a buffer as a main component suppresses retention of protein binding and reduction in anion reduction ability, and enables accurate detection.

Sensor unit and measuring device By using the water-absorbent sheet of the present invention, an inexpensive and small-sized sensor unit or measuring device having a greatly simplified structure can be constructed. This is due to the use of a water-absorbing sheet, and a mechanism for feeding an electrolyte solution (for example, a pump, a valve, and the like) required in a conventional method performed by filling an electrolyte solution between a working electrode and a counter electrode. This is because complicated mechanisms or processes such as those control mechanisms, a liquid leakage prevention mechanism (for example, packing), and a waste liquid treatment of the electrolytic solution are unnecessary.

The sensor unit according to the first aspect of the present invention includes a working electrode, a counter electrode provided to face the electrode, a working electrode and a water absorbent sheet sandwiched between the counter electrode, and the working electrode and Each electrode surface of the counter electrode is in contact with the water absorbent sheet. That is, the front side of the working electrode (the side on which the test substance is immobilized) and the conductive surface (platinum or the like) of the counter electrode face each other and are in contact with the water absorbent sheet. FIG. 1 shows a sectional view and an exploded view of the sensor unit according to the first embodiment. In the sensor unit 10 shown in FIG. 1, the working electrode 11 is positioned above the counter electrode 12, and the water absorbent sheet 13 is sandwiched between the working electrode 11 and the counter electrode 12. The sensor unit 10 includes a support base material 14 that supports the counter electrode 12. On the uppermost part of the sensor unit 10, a pressing member 15 is provided that presses down the working electrode 11, the water absorbent sheet 13, and the counter electrode 12 toward each other toward the support base material 14. In the sensor unit of the present invention, the order in which the components are stacked is not limited to the illustrated example, and may be stacked so as to be upside down in the reverse order shown in FIG. May be configured to further include a supporting substrate that supports the working electrode and a pressing member that presses the counter electrode, the water-absorbing sheet, and the working electrode toward each other toward the supporting substrate.
Moreover, although each member is arrange | positioned horizontally in the example of illustration, you may arrange | position in the state which stood up each member. It is preferable that the pressing member 15 further includes an opening or a light transmitting portion for allowing light for light excitation to pass through.

  A sensor unit according to a second aspect of the present invention includes an electrode unit in which a working electrode and a counter electrode are patterned on the same plane, and a water absorbent sheet provided in contact with each electrode surface of the working electrode and the counter electrode. Prepare. 2 to 4 show a sectional view and an exploded view of the sensor unit according to the second embodiment. In the sensor unit 20 shown in FIG. 2, a facing member 22 is provided to face the electrode unit 21, and a water absorbent sheet 23 is sandwiched between the electrode unit 21 and the facing member 22. That is, the electrode unit 21 is disposed on the water absorbent sheet 23 placed on the facing member 22 so that the side on which the test substance is immobilized is on the bottom. The sensor unit 20 is provided with a pressing member 24 that presses the electrode unit 21 and the water absorbent sheet 23 toward each other toward the facing member 22. On the other hand, in the sensor unit 30 shown in FIG. 3, the electrode unit 31 is further provided with a support base material 34 that supports the electrode unit, and the opposing member 32 faces the support base material 34 so that the water-absorbent sheet is provided. 33 and the electrode unit 31 function as a pressing member that holds the electrode unit 31 in close contact with each other. That is, the electrode unit 31 is placed on the support base 34 so that the side on which the test substance is immobilized is on the upper side, and the water absorbent sheet 33 is placed on the electrode unit 31. In the sensor unit of the present invention, the order in which the components are stacked is not limited to the illustrated example, and may be stacked upside down in the order opposite to the order shown in FIGS. Moreover, although each member is arrange | positioned horizontally in the example of illustration, you may arrange | position in the state which stood up each member. It is preferable that the pressing members 24 and 32 further include one or more openings or light-transmitting portions for allowing light for light excitation to pass through. In the sensor unit 30 shown in FIG. 3, the irradiation light passes through the water-absorbent sheet. Therefore, when using an electrolyte having coloring properties, a light source or It is preferable to adjust the electrolyte concentration as appropriate. On the other hand, in the sensor unit 40 shown in FIG. 4, the electrode unit 41 is provided with a support base 44 that supports the electrode unit, and the electrode unit 41 is brought into close contact with the support base 44. A pressing member 42 for pressing is provided. The pressing member 42 is preferably configured to cover only the end portion of the electrode unit 41 or the vicinity thereof and secure a sufficient open portion on the electrode unit 41, whereby the electrode unit 41 of the water absorbent sheet 43 is configured. It becomes easy to place it on top. Further, it is preferable that the portion of the pressing member 42 that comes into contact with the electrode unit 41 is constituted by the contact probe 47. In this way, the water absorbent sheet 43 is placed on the electrode unit 41 held by the pressing member 42, preferably in an open portion that is not covered by the pressing member 42.

  In both the first and second embodiments, the sensor units 10, 20, 30, and 40 include light sources 16, 26, 36, and 46 for irradiating the working electrode with light, a working electrode, and a counter electrode. And an ammeter (not shown) for measuring a current flowing between them can be provided, and can be constructed as a measuring device. The ammeter is preferably capable of detecting nA level. In such a configuration, the light from the light source is applied to the surface of the working electrode. In the sensor units 10 and 40 shown in FIGS. 1 and 4, the light from the light sources 16 and 46 is irradiated from the back side of the working electrode 11 to the electrode unit 41 and passes through the transparent working electrode 11 to the electrode unit 41. It reaches the surface of the working electrode 11 or the electrode unit 41. On the other hand, in the sensor units 20 and 30 shown in FIGS. 2 and 3, the light from the light sources 26 and 36 passes through the openings of the pressing members 24 and 32 and reaches the surfaces of the electrode units 21 and 31. Thus, the photocurrent value generated by the photoexcitation of the sensitizing dye by the light reaching the working electrode can be detected by an ammeter. The means for connecting the working electrode and the counter electrode to the ammeter is not limited. For example, the lead wire is directly connected, or is connected via the contact probes 17, 27, 37, and 47 as in the illustrated example. The following means can be adopted. In particular, the working electrode that is attached and detached for each measurement has an advantage that the working electrode can be easily attached and detached by using a contact probe.

  According to the preferable aspect of this invention, a light source can be a light source which can irradiate the light of a different wavelength according to the kind of sensitizing dye. Alternatively, according to another preferred aspect of the present invention, a plurality of light sources are provided in advance, and the measuring apparatus can further include a mechanism for switching and irradiating the plurality of light sources.

  According to a preferred aspect of the present invention, an XY moving mechanism (not shown) is attached to the light source and / or sensor unit, and the light source moves over the working electrode by relatively moving the light source and sensor unit in the XY direction. It is preferable that irradiation can be performed while scanning in the XY directions. Thereby, the test substance fixed spot on a working electrode can be irradiated individually. In particular, since the sensor unit of the present invention does not require the feeding of the electrolytic solution, the structure is simplified, and therefore the XY movement of the sensor unit itself can be easily performed.

  According to a preferred aspect of the present invention, the XY movement mechanism is preferably configured so that the movement speed, the movement route, and the like can be designated by software incorporated in a computer or apparatus. In this case, it is preferable that the current value generated by the light irradiation is measured by an ammeter, and the result is sent to a computer or a memory in the apparatus so that data can be sequentially stored. The photocurrent value stored in the memory in this manner can be displayed on the display as a numerical value or a time-dependent graph of real time display. From the obtained data, the current value at the time of non-irradiation and at the time of light irradiation can be read from appropriate data points, and the difference between them can be used to determine the substance concentration in the sample and the presence or absence of SNPs. Also, from reading of these data to determination or determination of SNPs (significant difference test) can be automatically processed on software.

Specific detection of test substance using photocurrent As described above, the sensor unit of the present invention is used for specific detection of a test substance using photocurrent generated by photoexcitation of a sensitizing dye. A specific detection method for a test substance using a photocurrent generated by photoexcitation of the sensitizing dye will be specifically described below.

  In this method, first, a sample solution containing a test substance, a working electrode, and a counter electrode are prepared. The working electrode used in the present invention is an electrode provided on the surface with a probe substance capable of specifically binding directly or indirectly to a test substance. That is, the probe substance specifically binds not only to a substance that directly binds specifically to the test substance, but also to a conjugate obtained by specifically binding the test substance to a mediator such as a receptor protein molecule. It may be a possible substance. Next, in the presence of the sensitizing dye, the sample solution is brought into contact with the working electrode to specifically bind the test substance directly or indirectly to the probe substance, and the sensitizing dye is fixed to the working electrode by this binding. Sensitizing dyes are substances that can emit electrons to the working electrode in response to photoexcitation, and can be pre-labeled to the test substance or mediator, or intercalated to the conjugate of the test substance and the probe substance. When a sensitizing dye is used, it may be simply added to the sample solution.

  Then, after bringing the working electrode and counter electrode into contact with the electrolyte medium in the sensor unit, when the sensitizing dye is photoexcited by irradiating the working electrode with light, electron transfer from the photoexcited sensitizing dye to the electron acceptor is performed. Happens. By detecting the photocurrent flowing between the working electrode and the counter electrode due to this electron movement, the test substance can be detected with high sensitivity and accuracy. Further, since the detected current has a high correlation with the test sample concentration in the sample solution, the test sample can be quantitatively measured based on the measured current amount or electric quantity.

(1) Test substance and probe substance The test substance in the present invention is not limited as long as it has a specific binding property, and may be various substances. In such a test substance, the test substance is directly or indirectly attached to the probe substance by supporting on the surface of the working electrode a probe substance that can specifically or directly bind to the test substance. It becomes possible to detect it by specifically binding to.

  That is, in the present invention, substances that can specifically bind to each other can be selected as the test substance and the probe substance. That is, according to a preferred embodiment of the present invention, it is preferable that a substance having specific binding properties be a test substance and a substance that specifically binds to the test substance be carried on the working electrode as a probe substance. As a result, the test substance can be directly bound and detected on the working electrode. Preferable examples of the combination of the test substance and the probe substance in this embodiment include a single-stranded nucleic acid and a combination of single-stranded nucleic acids having complementarity to the nucleic acid, and a combination of antigen and antibody.

  According to a more preferred embodiment of the present invention, it is preferable that the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid having complementarity to the nucleic acid. More preferably, the probe substance has a complementary portion of 15 bp or more to the nucleic acid. The specific binding process of the test substance to the working electrode in this embodiment is shown in FIGS. As shown in these figures, a single-stranded nucleic acid 121 as a test substance is hybridized with a single-stranded nucleic acid 124 having complementarity as a probe substance carried on the working electrode 123, and A double-stranded nucleic acid 127 is formed.

  Sample liquids containing single-stranded nucleic acids as test substances are blood such as peripheral venous blood, white blood cells, serum, urine, feces, semen, saliva, cultured cells, tissue cells such as various organ cells, etc. Nucleic acids can be extracted from various specimen samples containing nucleic acids by a known method. At this time, destruction of the cells in the specimen sample can be performed, for example, by applying a physical action such as shaking or ultrasonic waves from the outside and vibrating the carrier. In addition, nucleic acid can be released from cells using a nucleic acid extraction solution. Examples of the nucleic acid elution solution include a solution containing a surfactant such as SDS, Triton-X, Tween-20, saponin, EDTA, protease and the like. When nucleic acids are eluted using these solutions, the reaction can be accelerated by incubating at a temperature of 37 ° C. or higher.

  According to a more preferred embodiment of the present invention, when the content of a gene as a test substance is very small, detection is preferably performed after the gene is amplified by a known method. A typical method for amplifying a gene would be a method using an enzyme such as polymerase chain reaction (PCR). Examples of enzymes used in the gene amplification method include DNA polymerases, DNA-dependent DNA polymerases such as Taq polymerase, and DNA-dependent RNA polymerases such as RNA polymerase I. And RNA-dependent RNA polymerases such as Qβ replicase, and the PCR method using Taq polymerase is preferred in that amplification can be repeated continuously only by adjusting the temperature.

  According to a preferred embodiment of the present invention, the nucleic acid can be specifically labeled with a sensitizing dye during the amplification. Generally, it can be carried out by incorporating aminoallyl-modified dUTP into DNA. This molecule is incorporated with the same efficiency as unmodified dUTP. In the next coupling step, a fluorescent dye activated by N-hydroxysuccinimide reacts specifically with the modified dUTP, and a test substance uniformly labeled with a sensitizing dye is obtained.

  According to a preferred embodiment of the present invention, the nucleic acid crude extract solution or purified nucleic acid solution obtained as described above is first subjected to heat denaturation at a temperature of 90 to 98 ° C., preferably 95 ° C. or higher, to obtain a single strand. Nucleic acids can be prepared.

  In the present invention, the test substance and the probe substance may indirectly bind specifically. That is, according to another preferred embodiment of the present invention, a substance having specific binding properties is used as a test substance, a substance that specifically binds to the test substance is used as a mediator, and It is preferable to carry a substance capable of binding to the working electrode as a probe substance. Thereby, even a substance that cannot specifically bind to the probe substance can be detected by specifically binding indirectly to the working electrode via the mediator substance. In this embodiment, preferred examples of the combination of the test substance, the mediator, and the probe substance include a ligand, a receptor protein molecule that can accept the ligand, and two molecules that can specifically bind to the receptor protein molecule. A combination of nucleic acids in a strand is mentioned. Preferable examples of the ligand include exogenous endocrine disrupting substances (environmental hormones). An exogenous endocrine disrupting substance is a substance that binds to DNA via a receptor protein molecule and produces toxicity by affecting its gene expression. According to the method of the present invention, the receptor produced by a test substance is used. It is possible to easily monitor the binding of a protein such as a body to DNA. The specific binding step of the test substance to the working electrode in this embodiment is shown in FIG. As shown in FIG. 6, a ligand 130 as a test substance first specifically binds to a receptor protein molecule 131 that is a mediator. The receptor protein molecule 133 to which the ligand is bound specifically binds to the double-stranded nucleic acid 134 as the probe substance.

  According to a preferred embodiment of the present invention, the test substance can be two or more kinds. According to the method of the present invention, by using a plurality of sensitizing dyes and irradiating light having different excitation wavelengths for each sensitizing dye, it is possible to individually detect a plurality of types of test substances. .

(2) Sensitizing dye In the present invention, in order to detect the presence of a test substance with a photocurrent, the test substance is directly or indirectly specifically bound to the probe substance in the presence of the sensitizing dye. Then, the sensitizing dye is fixed to the working electrode by the binding. Therefore, in the present invention, the test substance 121 or the mediator 131 can be labeled with the sensitizing dyes 122 and 132 in advance as shown in FIGS. In addition, when using a sensitizing dye 128 that can be intercalated with a conjugate 127 of a test substance and a probe substance (for example, a double-stranded nucleic acid after hybridization) as shown in FIG. By adding a sensitizing dye to the solution, the sensitizing dye can be fixed to the probe substance.

  The sensitizing dye used in the present invention is a substance that can emit electrons to the working electrode in response to photoexcitation, can be changed to the photoexcited state by irradiation with a light source, and can be injected into the working electrode from the excited state. Anything that can take a state may be used. Therefore, any sensitizing dye can be used as long as it can take the above-described electronic state with the working electrode, particularly the electron accepting layer. Therefore, various sensitizing dyes can be used, and expensive dyes can be used. There is no need to use it.

  Specific examples of the sensitizing dye include metal complexes and organic dyes. Preferred examples of metal complexes include metal phthalocyanines such as copper phthalocyanine and titanyl phthalocyanine; chlorophyll or derivatives thereof; hemin, ruthenium, osmium, iron described in JP-A-1-220380 and JP-A-5-504023, and Zinc complexes such as cis-dicyanate-bis (2,2′-bipyridyl-4,4′-dicarboxylate) ruthenium (II)) are mentioned. Preferred examples of the organic dye include metal-free phthalocyanine, 9-phenylxanthene dye, cyanine dye, methocyanine dye, xanthene dye, triphenylmethane dye, acridine dye, oxazine dye, coumarin dye, Examples include merocyanine dyes, rhodacyanine dyes, polymethine dyes, and indigo dyes.

  Preferable examples of the sensitizing dye capable of intercalating with a double-stranded nucleic acid include acridine orange and ethidium bromide. When such a sensitizing dye is used, a double-stranded nucleic acid labeled with a sensitizing dye is formed simply by adding to the sample solution after hybridization of the nucleic acid. Therefore, it is necessary to label the single-stranded nucleic acid in advance. No.

(3) Working electrode and production thereof The working electrode used in the present invention is an electrode having the probe substance on its surface, and can accept electrons emitted by a sensitizing dye immobilized via the probe substance in response to photoexcitation. Electrode. Accordingly, the configuration and material of the working electrode are not limited as long as the above-described electron transfer occurs with the sensitizing dye to be used, and may be various configurations and materials.

  According to a preferred embodiment of the present invention, the working electrode has an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, and a probe material is provided on the surface of the electron accepting layer. Is preferably provided. According to a more preferred aspect of the present invention, it is preferable that the working electrode further comprises a conductive substrate, and an electron accepting layer is formed on the conductive substrate. An electrode of this embodiment is shown in FIGS. The working electrode 123 shown in FIGS. 5 and 6 includes a conductive substrate 125 and an electron-accepting layer 126 formed on the conductive substrate and containing an electron-accepting material. A probe substance is carried on the surface of the electron accepting layer 126.

  The electron-accepting layer in the present invention comprises an electron-accepting material that can accept electrons emitted by a sensitizing dye immobilized via a probe material in response to photoexcitation. That is, the electron-accepting substance can be a substance that can take an energy level in which electrons can be injected from the photoexcited labeling dye. Here, the energy level (A) capable of injecting electrons from the photoexcited labeling dye means, for example, a conduction band (conduction band: CB) when a semiconductor is used as the electron-accepting material. When a metal is used as the electron-accepting material, it means the Fermi level. When an organic material or a molecular inorganic material such as C60 is used as the electron-accepting material, the lowest unoccupied molecular orbital (Lowest Unoccupied Molecular Orbital: LUMO). That is, in the electron acceptor used in the present invention, the level of A is lower than the LUMO energy level of the sensitizing dye, in other words, lower than the LUMO energy level of the sensitizing dye. Any material having an energy level may be used.

Preferred examples of the electron accepting material include simple semiconductors such as silicon and germanium; oxides such as titanium, tin, zinc, iron, tungsten, zirconium, hafnium, strontium, indium, cerium, yttrium, lanthanum, vanadium, niobium, and tantalum. Semiconductors: Perovskite type semiconductors such as strontium titanate, calcium titanate, sodium titanate, barium titanate, potassium niobate; sulfide semiconductors of cadmium, zinc, lead, silver, antimony, bismuth; cadmium, lead selenide semiconductors Cadmium telluride semiconductors; phosphide semiconductors such as zinc, gallium, indium and cadmium; gallium arsenide, copper-indium selenide, copper-indium-sulfide compound semiconductors; gold, platinum, silver, copper, aluminum Rhodium, indium, a metal such as nickel, polythiophene, polyaniline, polyacetylene, organic polymer polypyrrole; is C60, C70, etc. molecular inorganic, and more preferably include _{silicon, TiO 2, SnO 2, Fe} 2 O 3, WO ₃ , ZnO, Nb ₂ O ₅ , strontium titanate, indium oxide, CdS, ZnS, PbS, Bi ₂ S ₃ , CdSe, CdTe, GaP, InP, GaAs, CuInS ₂ , CuInSe, C60, more preferably _{, TiO 2, ZnO, SnO 2} , Fe 2 O 3, WO 3, Nb 2 O 5, strontium titanate, CdS, PbS, CdSe, InP , GaAs, a CuInS _2, CuInSe _2, and most preferably in TiO ₂ is there. Note that the semiconductors listed above may be either intrinsic semiconductors or impurity semiconductors.

  According to a preferred embodiment of the present invention, the electron acceptor is preferably a semiconductor, more preferably an oxide semiconductor, still more preferably a metal oxide semiconductor, and most preferably an n-type metal oxide semiconductor. . According to this aspect, electrons can be efficiently extracted from the dye by utilizing the band gap of the semiconductor. In addition, by using a semiconductor having a structure such as a porous body or an uneven surface shape, a working electrode having a large surface area can be produced, and the amount of immobilized probes can be increased.

  According to a preferred embodiment of the present invention, the potential of the conduction band of the semiconductor is preferably lower than the LUMO potential of the sensitizing dye, more preferably the LUMO of the sensitizing dye> the conduction band of the semiconductor> the redox of the electrolyte. Potential> potential satisfying the relationship of sensitizing dye HOMO. By having such a relationship, it becomes possible to efficiently extract electrons.

  According to a preferred embodiment of the present invention, when the electron accepting layer is made of a semiconductor, the layer surface may be cationized. By cationization, it becomes possible to adsorb the probe substance (DNA, protein, etc.) to the electron accepting layer with high efficiency. The cationization can be performed by, for example, allowing a silane coupling agent such as aminosilane, a cationic polymer, a quaternary ammonium compound, or the like to act on the surface of the electron accepting layer.

  According to another preferred embodiment of the present invention, indium-tin composite oxide (ITO) or fluorine-doped tin oxide (FTO) can be used as the electron accepting material. Since ITO and FTO have the property of functioning not only as an electron-accepting layer but also as a conductive substrate, by using these materials, only the electron-accepting layer can function as a working electrode without using a conductive substrate. Can do.

  When a semiconductor or metal is used as the electron-accepting substance, the semiconductor or metal may be single crystal or polycrystalline, but a polycrystalline body is preferable, and a porous material is more preferable than a dense material. As a result, the specific surface area is increased, and a large amount of the test substance and sensitizing dye can be adsorbed to detect the test substance with higher sensitivity and accuracy. Therefore, according to the preferable aspect of this invention, it is preferable that the electron-accepting layer has porosity, and the diameter of each hole is 3-1000 nm, More preferably, it is 10-100 nm.

  According to a preferred aspect of the present invention, the surface area in a state where the electron-accepting layer is formed on the conductive substrate is preferably 10 times or more, more preferably 100 times or more with respect to the projected area. . The upper limit of the surface area is not particularly limited, but will usually be about 1000 times. The particle diameter of the fine particles of the electron accepting material constituting the electron accepting layer is preferably 5 to 200 nm as a primary particle in terms of an average particle diameter using a diameter when the projected area is converted into a circle, and more preferably 8 to It is 100 nm, More preferably, it is 20-60 nm. The average particle diameter of the electron-accepting substance fine particles (secondary particles) in the dispersion is preferably 0.01 to 100 μm. In addition, for the purpose of improving the light capture rate by scattering incident light, an electron accepting layer may be formed by using fine particles of an electron accepting material having a large particle size, for example, about 300 nm.

  According to a preferred embodiment of the present invention, it is preferred that the working electrode further comprises a conductive substrate, and the electron accepting layer is formed on the conductive substrate. The conductive substrate that can be used in the present invention is not only a substrate having conductivity, such as a metal such as titanium, but also a substrate having a conductive material layer on the surface of a glass or plastic support. Good. When a conductive substrate having this conductive material layer is used, the electron accepting layer is formed on the conductive layer. Examples of the conductive material constituting the conductive material layer include metals such as platinum, gold, silver, copper, aluminum, rhodium, and indium; conductive ceramics such as carbon, carbide, and nitride; and indium-tin composite oxides, Examples include conductive metal oxides such as tin oxide doped with fluorine, tin oxide doped with antimony, zinc oxide doped with gallium, or zinc oxide doped with aluminum. Are indium-tin composite oxide (ITO) and metal oxide (FTO) in which tin oxide is doped with fluorine. However, as described above, when the electron-accepting layer itself also functions as a conductive substrate, the conductive substrate can be omitted. Further, in the present invention, the conductive substrate is not limited as long as it is a material that can ensure conductivity, and includes a thin film-like or spot-like conductive material layer that does not have strength as a support by itself. To do.

According to a preferred embodiment of the present invention, the conductive substrate is substantially transparent, specifically, the light transmittance is preferably 10% or more, more preferably 50% or more, and still more preferably. 70% or more. Moreover, according to the preferable aspect of this invention, it is preferable that the thickness of a electrically-conductive material layer is about 0.02-10 micrometers. Furthermore, according to the preferable aspect of this invention, it is preferable that the surface resistance of an electroconductive base material is 100 ohms / cm < ² > or less, More preferably, it is 40 ohms / cm < ² > or less. The lower limit of the surface resistance of the conductive substrate is not particularly limited, but will usually be about 0.1 Ω / cm ² .

  Examples of a preferable method for forming an electron-accepting layer on a conductive substrate include a method of coating a dispersion or colloidal solution of an electron-accepting material on a conductive support, and a precursor of semiconductor fine particles on the conductive support. And a method of obtaining a fine particle film by being hydrolyzed with moisture in the air (sol-gel method), a sputtering method, a CVD method, a PVD method, and a vapor deposition method. As a method for preparing a dispersion of semiconductor fine particles as an electron acceptor, in addition to the sol-gel method described above, a method of grinding with a mortar, a method of dispersing while grinding using a mill, or a semiconductor synthesis Examples thereof include a method in which it is precipitated as fine particles in a solvent and used as it is. Examples of the dispersion medium include water or various organic solvents (for example, methanol, ethanol, isopropyl alcohol, dichloromethane, acetone, acetonitrile, ethyl acetate, etc.). At the time of dispersion, a polymer, a surfactant, an acid, a chelating agent, or the like may be used as a dispersion aid as necessary.

  Preferred examples of the application method of the electron acceptor dispersion or colloidal solution include the roller method as the application system, the dip method, the air knife method as the metering system, the blade method, etc., and the application and metering in the same part. The wire bar method disclosed in Japanese Patent Publication No. 58-4589, the slide hopper method, the extrusion method, the curtain method, the spin method, and the spray method described in US Pat. Nos. 2,681,294, 2761419, 2761791, etc. Is mentioned.

According to a preferred aspect of the present invention, when the electron accepting layer is composed of semiconductor fine particles, the thickness of the electron accepting layer is preferably 0.1 to 200 μm, more preferably 0.1 to 100 μm, and still more preferably. Is 1-30 μm, most preferably 2-25 μm. As a result, the probe substance per unit projected area and the amount of sensitizing dye to be fixed can be increased to increase the photoelectric flow rate, and the loss of generated electrons due to charge recombination can also be reduced. The coating amount of the semiconductor fine particles per 1 m ² of the conductive base material is preferably 0.5 to 400 g, more preferably 5 to 100 g.

  According to a preferred embodiment of the present invention, when the electron-accepting material comprises indium-tin composite oxide (ITO) or metal oxide (FTO) doped with fluorine in tin oxide, the thickness of the electron-accepting layer is 1 nm. It is preferable that it is above, More preferably, it is 10 nm-1 micrometer.

  According to a preferred embodiment of the present invention, it is preferable to apply heat treatment after coating the semiconductor fine particles on the conductive substrate. Thereby, particle | grains can be made to contact electrically and the improvement of coating-film intensity | strength and adhesiveness with a support body can be improved. A preferable heat treatment temperature is 40 to 700 ° C, more preferably 100 to 600 ° C. Moreover, preferable heat processing time is about 10 minutes-10 hours.

  According to another preferred embodiment of the present invention, when a conductive base material having a low melting point or softening point such as a polymer film is used, the film is formed by a method that does not use high-temperature treatment in order to prevent deterioration due to heat. Preferably, the film is formed, and examples of such a film forming method include a method such as pressing, low-temperature heating, electron beam irradiation, microwave irradiation, electrophoresis, sputtering, CVD, PVD, and vapor deposition.

  A probe substance is supported on the surface of the electron accepting layer of the working electrode thus obtained. Loading of the probe substance on the working electrode can be performed according to a known method. According to a preferred embodiment of the present invention, when a single-stranded nucleic acid is used as a probe substance, an oxidized layer is formed on the surface of the working electrode, and the nucleic acid probe and the working electrode are connected via the oxidized layer. This can be done by bonding. At this time, the nucleic acid probe can be immobilized on the working electrode by introducing a functional group at the end of the nucleic acid. As a result, the nucleic acid probe into which the functional group has been introduced can be immobilized on the carrier as it is by an immobilization reaction. Introduction of a functional group at the end of a nucleic acid can be performed using an enzymatic reaction or a DNA synthesizer. Examples of the enzyme used in the enzymatic reaction include terminal deoxynucleotidyl transferase, poly A polymerase, polynucleotide kinase, DNA polymerase, polynucleotide adenyl transferase, RNA ligase. Can be mentioned. Moreover, a functional group can also be introduce | transduced by a polymerase chain reaction (PCR method), a nick translation, and a random primer method. The functional group may be introduced at any part of the nucleic acid, and can be introduced at the 3 ′ end, 5 ′ end or at a random position.

  According to a preferred embodiment of the present invention, amine, carboxylic acid, sulfonic acid, thiol, hydroxyl group, phosphoric acid and the like can be suitably used as the functional group for immobilizing the nucleic acid probe to the working electrode. Moreover, according to the preferable aspect of this invention, in order to fix | immobilize a diffusion probe firmly to a working electrode, it is also possible to use the material which bridge | crosslinks between a working electrode and a diffusion probe. Preferable examples of such a crosslinking material include silane coupling agents, titanate coupling agents, and conductive polymers such as polythiophene, polyacetylene, polypyrrole, and polyaniline.

  According to a preferred embodiment of the present invention, the nucleic acid probe can be immobilized efficiently by a simple operation called physical adsorption. The physical adsorption of the nucleic acid probe to the electrode surface can be performed, for example, as follows. First, the electrode surface is cleaned with distilled water and alcohol using an ultrasonic cleaner. Thereafter, the electrode is inserted into a buffer containing the nucleic acid probe to adsorb the nucleic acid probe onto the surface of the carrier.

  Moreover, nonspecific adsorption | suction can be suppressed by adding a blocking agent after adsorption | suction of a nucleic acid probe. The blocking agent that can be used is not limited as long as it can fill a site on the surface of the electron accepting layer on which the nucleic acid probe is not adsorbed and can adsorb the electron accepting material by chemical adsorption or physical adsorption. However, it is preferably a substance having a functional group that can be adsorbed via a chemical bond. For example, preferable examples of the blocking agent in the case of using titanium oxide as an electron-accepting layer include functional groups that can be adsorbed to titanium oxide such as carboxylic acid group, phosphoric acid group, sulfonic acid group, hydroxyl group, amino group, pyridyl group, and amide. Examples thereof include substances having a group.

  According to a preferred aspect of the present invention, it is preferable that the probe substance is divided and supported on each of the plurality of regions separated from each other on the working electrode, and the light irradiation by the light source is performed individually on each region. . Thereby, since a plurality of samples can be measured on one working electrode, integration of DNA chips and the like are possible. According to a more preferable aspect of the present invention, a plurality of regions separated from each other, on which a probe substance is supported on the working electrode, is patterned, and a sample in each region is scanned while scanning with light emitted from a light source. It is preferable that the detection or quantification of the test substance is continuously performed in one operation.

  According to a more preferred aspect of the present invention, a plurality of types of probe substances can be carried on each of a plurality of regions separated from each other on the working electrode. As a result, a large number of samples can be simultaneously measured by multiplying the number of regions by the number of types of probe substances for each region.

  According to a more preferable aspect of the present invention, a different probe substance can be carried in each of a plurality of regions separated from each other on the working electrode. As a result, the number of types of probe substances corresponding to the number of the divided areas can be carried, so that multiple types of analytes can be measured simultaneously. This aspect can be preferably used for multi-item analysis of single nucleotide polymorphism analysis (SNPs) because different test substances can be analyzed for each region.

(4) Counter electrode The counter electrode used in the present invention is not particularly limited as long as a current can flow between it and the working electrode when it is brought into contact with an electrolytic solution, and a metal or a conductive oxide is used. Vapor deposited glass, plastic, ceramics, etc. can be used. Moreover, the metal thin film as a counter electrode can be formed by a method such as vapor deposition or sputtering so as to have a film thickness of 5 μm or less, preferably 3 nm to 3 μm. Preferable examples of materials that can be used for the counter electrode include conductive polymers such as platinum, gold, palladium, nickel, carbon, and polythiophene, and conductive ceramics such as oxide, carbide, and nitride, and more preferably. Platinum and carbon, and most preferably platinum. These materials can be formed into a thin film by the same method as the electron accepting layer.

(5) Electrode unit According to the preferable aspect of this invention, you may use the electrode unit by which a working electrode and a counter electrode are patterned on the same plane. A preferred electrode unit includes an insulating substrate, and a working electrode that is provided locally on the insulating substrate and includes an electron-accepting layer that includes an electron-accepting material that can accept electrons emitted by a sensitizing dye in response to photoexcitation. And a counter electrode provided on the same plane as the working electrode of the insulating substrate and spaced apart from the working electrode. An example of such an electrode unit is shown in FIG. The electrode unit 71 shown in FIG. 7 includes an insulating substrate 72, a working electrode 73, and a counter electrode 74. The insulating substrate 72 is a substrate having an insulating property so as not to short-circuit the working electrode 72 and the counter electrode 73. The working electrode 73 is provided locally on the insulating substrate 72 and includes an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation. The counter electrode 74 is provided on the same surface as the working electrode 73 of the insulating substrate 72 and is separated from the working electrode 73. Lead wires 74 ′ are provided so as to extend from each of the working electrode 73 and the counter electrode 74.

  Thus, the electrode unit is an integrated electrode member having a working electrode and a counter electrode on the same plane, and by using this, the degree of freedom of sensor unit design and material selection is greatly expanded. The productivity, performance and ease of use of the sensor unit can be greatly improved. That is, since the electrode unit according to the present invention is an integral electrode member and does not require two electrodes to face each other, a configuration in which the light source faces the surface of the electrode unit can be easily adopted. As a result, the working electrode is not limited to a transparent material, and can be made of an opaque material such as ceramics or plastic, so that the degree of freedom in selecting an electrode material is expanded. In addition, the direct irradiation of the working electrode surface from the light source can eliminate the loss of light caused by the transparency of the transparent electrode material that occurs when irradiating from the back surface of the electrode, so that more accurate measurement can be expected. Furthermore, since the electrode unit according to the present invention is an integrated electrode member, the working electrode, the counter electrode, and the lead wire can be formed by one-step conductive patterning, which improves the productivity of the electrode. . In addition, since the material facing the electrode unit does not need to have conductivity, a commonly used material such as transparent plastic or glass can be used, and the productivity of the cell is improved.

(6) Measuring method In the measuring method using the sensor unit of the present invention, first, in the presence of a sensitizing dye, the sample solution is brought into contact with the working electrode, and the test substance is directly or indirectly applied to the probe substance. The sensitizing dye is fixed to the working electrode by this binding.

  According to a preferred embodiment of the present invention, when a single-stranded nucleic acid previously labeled with a sensitizing dye is used as a test substance, a hybridization reaction can be performed with a single-stranded nucleic acid that is a probe substance. . The preferred temperature for the hybridization reaction is in the range of 37-72 ° C., but the optimum temperature varies depending on the base sequence and length of the probe used.

  According to another preferred embodiment of the present invention, when a sensitizing dye that can be intercalated is used for a conjugate of a test substance and a probe substance (for example, a double-stranded nucleic acid after hybridization), it is added to the sample solution. By adding a sensitizing dye, the conjugate can be specifically labeled with a sensitizing dye.

  In this way, the working electrode on which the test substance is fixed together with the sensitizing dye is brought into contact with an absorbent sheet containing an electrolyte together with the counter electrode, and the sensitizing dye is photoexcited by irradiating the working electrode with light. A photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the dye to the working electrode is detected. As a sensor unit at that time, a sensor unit using the water-absorbent sheet of the present invention is used.

  According to a preferred aspect of the present invention, when two or more kinds of test substances are individually detected using two or more sensitizing dyes that can be excited at different light wavelengths, a specific wavelength is detected from the light source via the wavelength selection means. It is possible to excite a plurality of dyes individually by irradiating the light. Examples of wavelength selection means include a spectroscope, a colored glass filter, an interference filter, a bandpass filter, and the like. In addition, a plurality of light sources capable of irradiating light of different wavelengths depending on the type of sensitizing dye may be used. Examples of preferable light sources in this case include laser light and LED irradiated with light of a specific wavelength. It may be used. Further, in order to efficiently irradiate the working electrode with light, light may be guided using quartz, glass, or a liquid light guide.

  The photocurrent flowing through the system by light irradiation is measured by an ammeter. Thereby, the test substance can be detected. The current value at that time reflects the amount of sensitizing dye trapped on the working electrode. For example, when the test substance is a nucleic acid, the amount of double strands formed between complementary nucleic acids is reflected as the current value. Therefore, the test substance can be quantified from the obtained current value. Therefore, according to a preferred aspect of the present invention, it is preferable that the ammeter further comprises means for calculating the concentration of the test substance in the sample solution from the obtained amount of current or electricity.

  According to a preferred aspect of the present invention, the step of detecting the photocurrent can measure the current value, and calculate the concentration of the test substance in the sample liquid from the obtained current value or electric quantity. The calculation of the test substance concentration can be performed by comparing a calibration curve between the test substance concentration and the current value or the electric quantity prepared in advance with the obtained current value or the electric quantity. In the method of the present invention, since the current value reflects the amount of the sensitizing dye trapped on the working electrode, an accurate current value corresponding to the test substance concentration can be obtained, which is suitable for quantitative measurement. .

  According to another preferred embodiment of the present invention, a test substance previously labeled with a sensitizing dye is used as a competitor, and a second substance capable of specifically binding to a probe substance that is not labeled with a sensitizing dye. The test substance can be quantified. It is preferable that the second test substance has a property of easily binding to the probe substance more specifically than the labeled test substance. When these two kinds of test substances are made to compete and specifically bind to the probe substance, a correlation is obtained between the detected current value and the concentration of the second test substance. That is, as the number of second analytes that are not dye-labeled increases, the number of competitors that specifically bind to the probe substance decreases, so that the detection current increases as the second analyte concentration increases. A calibration curve with decreasing values can be obtained. Therefore, it is possible to detect and quantify the second test substance that is not labeled with a sensitizing dye.

  According to a more preferred embodiment of the present invention, the test substance and the second test substance are preferably antigens, and the probe substance is preferably an antibody. FIG. 8 shows the step of immobilizing the test substance and the second test substance on the probe substance in this embodiment. As shown in FIG. 8, the antigen 141 labeled with the sensitizing dye and the antigen 142 not labeled with the dye compete with each other and specifically bind to the antibody 143. Therefore, as the amount of the antigen 142 that is not dye-labeled increases, the amount of the dye-labeled antigen 143 that specifically binds to the antibody decreases. Therefore, the detection current value decreases as the second analyte concentration increases. You can get a line.

Example 1: Photocurrent measurement using a water-absorbent sheet (1) Preparation of dye-labeled DNA-immobilized working electrode Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (as a glass substrate for the working electrode) A special glass company, U film, sheet resistance: 12Ω / □, shape: 50 mm × 26 mm) was prepared. This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking for 5 minutes in ultrapure water was performed 3 times with the water changed. After the glass substrate was taken out and air was blown to scatter the remaining water, the glass substrate was immersed in anhydrous methanol for dehydration.

Using 95 wt% methanol 5 wt% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2 vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was immersed in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling solution, and the methanol was changed 3 times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, 5′-end rhodamine-labeled ssDNA (25mer) adjusted to 100 nM and unlabeled ssDNA (24mer) adjusted to 100 nM were held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes. Then, the DNA was denatured. The denatured DNA was filled in 5 μl each in 9 openings of the seal on the glass prepared previously, and kept at 95 ° C. for 10 minutes to evaporate the solvent. Then, 120 mJ ultraviolet light was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the labeled ssDNA on the glass substrate. The seal was peeled off from the glass substrate, and the glass substrate was shaken in a 0.2% SDS solution for 15 minutes × 3 times and rinsed with three changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. Thus, a dye-labeled DNA-immobilized working electrode was obtained. The base sequence of the probe DNA used here is as follows.
5 'terminal rhodamine labeled ssDNA (probe 1): 5'-Rho-GCGGCATGAACCTGAGGCCCATCCT-3'
Unlabeled ssDNA (probe 2): 5'-TTGAGCAAGTTCAGCCTGGTTAAG-3 '

(2) Production of water-absorbing sheet An aqueous solution containing 0.2M, 0.4M, and 0.6M tetrapropylammonium iodide (NPr4I) was prepared. A 0.9 mm-thick blotting filter paper (CB-13A; Ato Co., Ltd.) cut to 26 mm × 20 mm was immersed in this aqueous solution, and lightly drained to obtain a water-absorbent sheet.

(3) Photocurrent measurement using a water-absorbing sheet The dye-labeled DNA-immobilized working electrode prepared in (1) above and a counter electrode obtained by depositing platinum on a glass plate were prepared. The water-absorbent sheet produced in (2) above was sandwiched between both electrodes and brought into close contact. At this time, the working electrode was placed so that the surface on which the ssDNA was immobilized was opposed to the platinum deposition surface of the counter electrode. With both electrodes connected to the electrochemical analyzer, the working electrode was irradiated with a laser light source (output 60 mW, diameter of irradiated region 1 mm, wavelength 530 nm green laser), and the current value observed at that time was recorded.

  The results are shown in FIG. As shown in FIG. 9, an increase in photocurrent was observed depending on the concentration of tetrapropylammonium iodide, but it was revealed that any concentration of tetrapropylammonium iodide can be used. It was.

Example 2: Examination of various electrolytes Photocurrent measurement was performed using various electrolytes. Specifically, filter paper was used as the water-absorbing sheet, and the concentration of each reducing agent was fixed at 0.2 M to produce a water-absorbing sheet. As the electrolyte, use _{NaI, KI, CaI 2, LiI} , NH 4 I, tetrapropylammonium iodide _(NPr 4 I), sodium thiosulfate _{(Na 2} S 2 _{O 3)} and sodium sulfite _(Na 2 SO ₃₎ It was. The preparation method is to prepare electrolytes containing various electrolytes and water, soak a 0.9mm thick blotting filter paper (same as in Example 1) cut into 26mm x 20mm, and drain lightly to obtain a water-absorbent sheet It was. The photocurrent was measured in the same manner as in Example 1. The dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example 1, and working electrodes prepared using solutions each having a rhodamine-labeled ssDNA concentration of 10 nM and a rhodamine-unlabeled ssDNA concentration of 100 nM were also prepared. Current measurement was performed.

  The results are shown in FIG. In any of the reducing agents studied, an increase in photocurrent was observed depending on the amount of immobilized ssDNA, and it was revealed that it can be used.

Example 3: Influence of the thickness of the water-absorbent sheet (1) Preparation of dye-labeled DNA immobilization working electrode Except that the ssDNA concentration to be immobilized was 2 concentrations of 100 nM and 1 μM, the same as in Example 1 (1), A dye-labeled DNA-immobilized working electrode was prepared.
(2) Production of water absorbent sheet
Prepare an electrolyte solution with tetrapropylammonium iodide (NPr ₄ I) and water at a concentration of 0.2M, and stack one, two, and three sheets of 0.9mm-thick blotting filter paper (as in Example 1). Each was immersed in an electrolyte solution to prepare a water absorbent sheet.
(3) Photocurrent measurement In the same manner as in Example 1, using the working electrode having spots with different immobilization concentrations prepared in (1) above and the water-absorbing sheets having different thicknesses prepared in (2) above, The photocurrent was measured.

  The results are shown in FIG. As shown in FIG. 11, almost no influence on the photocurrent value was observed over the entire thickness of the water absorbent sheet of 0.9 mm to 2.7 mm. Therefore, from the viewpoint of obtaining sufficient strength, it is presumed that there is no adverse effect on photocurrent value detection even if the water absorbent sheet thickness is increased.

Example 4: Examination of each water-absorbing sheet As a water-absorbing sheet, 0.9 mm thick blotting filter paper (similar to Example 1), felt (thickness: 1 mm, density: 0.00049 g / mm ³ ), cardboard (thickness: 0.5 mm, density: 0.00071 g / mm ³ ), glass fiber filter paper (GF / D; Whatman: thickness: 0.68 mm), coated paper containing pulp fiber and synthetic fiber (thickness: 0.14 mm, density: 0.00111 g / mm ³ ), and the photocurrent was measured in the same manner as in Example 1 except that a membrane filter (JCWP09025; MILLIPORE: thickness: 0.1 mm) mainly containing a fluorine resin was used. A dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example 1, and a working electrode prepared using solutions with rhodamine-labeled ssDNA concentrations of 100 nM and 1 μM and an unlabeled ssDNA concentration of 100 nM was also prepared. The photocurrent was measured in the same manner as in Example 1. The electrolytic solution used to produce the water-absorbing sheet was an aqueous solution containing 0.2 M tetrapropylammonium iodide (NPr ₄ I), and the other procedures were performed in the same manner as in Example 1. .

  The results are shown in FIG. As shown in FIG. 12, in all the water-absorbent sheets, an increase in photocurrent was recognized depending on the amount of immobilized ssDNA, and it was revealed that the sheets can be used.

Example 5: Examination of water content of water-absorbing sheet (1) Measurement of water content First, 0.4M tetrapropylammonium iodide was used on a blotting filter paper (similar to Example 1) with a thickness of 0.9mm cut into 26mm x 20mm. 500 μl of an aqueous solution containing (NPr4I) was dropped, and the filter paper was completely immersed in the electrolytic solution (production of 6 sheets). Next, it was dried at 50 ° C. for 0 hour, 0.25 hour, 0.5 hour, 1 hour, 1.25 hour, 1.5 hour, respectively, and the weight of the filter paper containing the electrolyte was measured. Tetrapropylammonium iodide The water content around 1 mm ³ was calculated. Thereafter, the ratio of (water content around 1 mm ³ ) / (filter paper density) was calculated and used as the water content of the water-absorbent sheet. The density of the blotting filter paper is 0.00049 g / mm ³ .
(2) Photocurrent measurement Photocurrent was detected in the same manner as in Example 1 using the water-absorbent sheet having each moisture content prepared in (1).

  The results are shown in FIG. As FIG. 13 shows, it turns out that a photocurrent is so high that the moisture content of a water absorbing sheet is high. Photocurrent could not be detected when the water content was 2.2%, but photocurrent could be detected when the water content was 25.3%. From the above, it is desirable that the water absorbent sheet capable of detecting photocurrent has a water content of at least 20%.

Single nucleotide polymorphism (SNPs) detection (1) Example 6: SNPs detection using various electrolytes In this example, a water-absorbent sheet was applied to detect single nucleotide polymorphisms of the p53 gene. A perfect match probe, a single nucleotide variant probe, and a perfect mismatch probe were immobilized on the working electrode side. Each base sequence was as follows.
Exact match (PM) probe: 5'-AGGATGGGCCTC A GGTTCATGCCGC-3 '
Single nucleotide variant (SNP) probe: 5'-AGGATGGGCCTC C GGTTCATGCCGC-3 '
Complete mismatch (MM) probe: 5'-GCGGCATGAACCGGAGGCCCATCCT-3 '

The base sequence of the target DNA to be hybridized with these probes was as follows.
Target DNA: 5'-Rhodamine-GCGGCATGAACC T GAGGCCCATCCT-3 '

Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50 mm × 26 mm) is prepared as a glass substrate for the working electrode did. This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking for 5 minutes in ultrapure water was performed 3 times with the water changed. After the glass substrate was taken out and air was blown to scatter the remaining water, the glass substrate was immersed in anhydrous methanol for dehydration.

  Using 95 wt% methanol 5 wt% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2 vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was immersed in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling solution, and the methanol was changed 3 times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, the probe DNA (25 mer) having a perfectly matched strand, a single nucleotide variant, and a completely mismatched strand prepared to 1 μM was held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes to denature the DNA. . 5 μl of this denatured DNA was filled into 9 openings of the previously prepared seal on the glass and kept at 95 ° C. for 10 minutes to evaporate the solvent. Thereafter, UV light of 120 mJ was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the probe DNA on the glass substrate (three spots were immobilized for each probe). The seal was peeled off from the glass substrate, and the glass substrate was shaken in a 0.2 wt% SDS solution for 15 minutes × 3 times and rinsed with three changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. In this way, a probe DNA fixed working electrode was obtained.

  Thereafter, a 5 × SSC, 0.5 wt% SDS solution containing the target DNA adjusted to a concentration of 100 nM was placed on the electrode on which the probe was fixed and sealed with a cover glass, and kept at 37 ° C. for 10 hours. After that, the cover glass is peeled off in 2 × SSC (room temperature), the electrode is placed on a rack, shaken in a 2 × SSC / 0.2% SDS solution set at 40 ° C. for 30 minutes, and then rinsed with water to dry the electrode. I let you.

The working electrode thus obtained and a water-absorbent sheet produced in the same manner as in Example 1 were incorporated into a measurement cell, and an XY stage for automatic light source movement was attached on the measurement cell. The structure of the cell part is a highly sensitive ammeter through a spring probe in which the working electrode and the platinum counter electrode are opposed to each other to prevent a short circuit due to contact between both electrodes and the working electrode and the counter electrode are in electrical contact with each other. Connected to. As the water absorbent sheet, a filter paper was used as the water absorbent sheet, and the concentration of each reducing agent was fixed at 0.4 M to prepare a water absorbent sheet. Tetrapropylammonium iodide (NPr ₄ I), sodium thiosulfate (Na ₂ S ₂ O ₃ ) and sodium sulfite (Na ₂ SO ₃ ) were used as the electrolyte. The preparation method is to prepare electrolytes containing various electrolytes and water, soak a 0.9mm thick blotting filter paper (same as in Example 1) cut into 26mm x 20mm, and drain lightly to obtain a water-absorbent sheet It was.

  Subsequently, light was irradiated from above the back surface of the working electrode by a light source fixed on the XY stage for automatic movement, and the current flowing between the working electrode and the platinum counter electrode was measured over time. A light-shielding member having the same shape as the spot on the FTO substrate was provided above the working electrode to prevent light irradiation to the adjacent spot, and at the same time, a light non-irradiation spot was provided. In the measurement, the spots were sequentially scanned, and at the same time, the current output at the spots was stored in the personal computer through a high sensitivity ammeter connected to the personal computer.

  The results are shown in FIG. From the results, it was possible to detect single nucleotide polymorphisms (SNPs) as a difference in photocurrent values regardless of which electrolyte was used.

(2) Example 7: Detection of SNPs using a dried water absorbent sheet.
After preparing the water-absorbent sheet, it was dried at 50 ° C. for 2 hours, and after incorporating the dried water-absorbent sheet into the measuring cell, 300 μL of water was dropped on the water-absorbent sheet immediately before photocurrent detection. Photocurrent was detected in the same manner as in Example 6 (dry type). As a comparison, a water-absorbent sheet prepared by immersing a water-absorbent sheet in an electrolyte just before photocurrent detection was used as in Example 6 (immersion type).

  The results are shown in FIG. Even if it was a case where the water-absorbent sheet dried from the result was used, single nucleotide polymorphism (SNPs) was able to be detected as a difference of a photocurrent value.

(3) Example 8: Comparison between aqueous electrolyte and water absorbent sheet.
SNPs in an electrolyte containing 0.4 M tetrapropylammonium iodide (NPr ₄ I) and water, and a water absorbent sheet prepared by immersing a water absorbent sheet in the electrolyte just before photocurrent detection as in Example 6 Detection was performed, and the detection accuracy was compared when using an aqueous electrolyte and when using a water absorbent sheet. SNPs detection of the water-absorbent sheet was performed in the same manner as in Example 6. Detection of SNPs using an aqueous electrolyte was performed in the same manner as in Example 6 in the method for producing the electrode substrate, and a flow cell type measurement cell was used for measurement. 16 and 17 are a cross-sectional view and an exploded view of the flow cell type measurement cell according to the first embodiment.

  The flow cell type measurement cell shown in FIGS. 16 and 17 is provided with a counter electrode 53 on a substrate 57, and an electrolyte solution or cleaning solution supply hole 62 and a discharge hole 63 are formed on the substrate 57. Is provided with an insulating spacer 64 having a space 54 for accommodating an electrolytic solution, a working electrode 52 is provided on the insulating spacer 64, and a plurality of electron-accepting layers 55 are spaced apart from the surface of the working electrode 52 facing the space 54. Is formed.

  A working electrode contact 58 is provided through the substrate 57 so as not to interfere with the counter electrode 53. The photocurrent is taken out by this working electrode contact 58.

  A pressing member 59 is provided on the working electrode 52, and through holes 60 are formed in the pressing member 59 at positions corresponding to the plurality of electron accepting layers 55. The working electrode 52 is irradiated with light from the light source 56 through the through hole 60. An ammeter 61 is connected between the working electrode 52 and the counter electrode 53, and the photocurrent flowing through the system by light irradiation is measured by the ammeter.

  The results are shown in FIG. From the results, even when a water-absorbent sheet was used, single nucleotide polymorphisms (SNPs) could be accurately detected as a difference in photocurrent values as in the case of using an aqueous electrolyte.

(4) Example 9: Comparison of solvents contained in the water absorbent sheet.
In the same manner as in Example 1, SNPs were detected in each of the water-absorbent sheets prepared by immersing the water-absorbent sheet in each electrolyte immediately before photocurrent detection, and the detection accuracy was compared. The composition of each electrolyte is the same as the electrolyte concentration of 0.4M, but 100% water, 10% acetonitrile + 90% water, 25% acetonitrile + 75% water, 50% acetonitrile + 50% water, and 100% acetonitrile are used as solvents. Using. In addition, the density | concentration of a solvent is the mass%.

The results are shown in FIG. From the results, it was possible to accurately detect single nucleotide polymorphisms (SNPs) as a difference in photocurrent values even when any solvent of water, acetonitrile, or a mixed solution of water and acetonitrile was used.

It is a figure which shows an example before and behind the assembly of the sensor unit and measurement apparatus by the 1st aspect of this invention, (a) is sectional drawing after an assembly, (b) shows the exploded view before an assembly. It is a figure which shows an example before and behind the assembly of the sensor unit and measurement apparatus by the 2nd aspect of this invention, (a) is sectional drawing after an assembly, (b) shows the exploded view before an assembly. It is a figure which shows another example before and behind the assembly of the sensor unit and measurement apparatus by the 2nd aspect of this invention, (a) is sectional drawing after an assembly, (b) shows the exploded view before an assembly. It is a figure which shows another example before and behind the assembly of the sensor unit and measurement apparatus by the 2nd aspect of this invention, (a) is sectional drawing after an assembly, (b) shows the exploded view before an assembly. FIG. 4 is a diagram showing a process of immobilizing a test substance on a probe substance when the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid complementary to the nucleic acid. (A) shows the case where the test substance is previously labeled with a sensitizing dye, and (b) shows the case where a sensitizing dye capable of intercalation is added to a double-stranded nucleic acid. It is a figure which shows the immobilization process to the probe substance of a test substance in case a test substance is a ligand, a mediator is a receptor protein molecule, and a probe substance is a double-stranded nucleic acid. It is a figure which shows an example of an electrode unit. It is a figure which shows the fixation process to the probe substance of a test substance in case the test substance and the 2nd test substance which have the specific binding property which mutually compete are antigens, and a probe substance is an antibody. The figure which shows the photocurrent value measured in each tetrapropylammonium iodide (NPr4I) density | concentration in 100 nM ssDNA and 100 nM rhodamine labeled ssDNA when using the water absorbing sheet (filter paper) obtained in Example 1. It is. It is a figure which shows the photocurrent value at the time of using various electrolytes in each rhodamine labeled ssDNA density | concentration of 0 nM, 10 nM, and 100 nM obtained in Example 2. It is a figure which shows the relationship between the thickness of a water absorbing sheet, and a photocurrent value in each rhodamine labeled ssDNA density | concentration of 100 nM and 1 micromol which were obtained in Example 3. It is a figure which shows the photocurrent value at the time of using each water absorbing sheet in each rhodamine labeled ssDNA density | concentration of 100 nM ssDNA obtained in Example 4, 100 nM, and 1 micromol. It is a figure which shows the relationship between the water content of the water absorbing sheet and the photocurrent value obtained in Example 5. It is a figure which shows the photocurrent value of each electrolyte at the time of using the perfect coincidence (PM) probe obtained in Example 6, a single nucleotide variation | mutation chain | strand (SNP) probe, and a perfect mismatch (MM) probe. Prepared by immersing the water-absorbent sheet in the electrolyte just before measurement in the case of using the perfect match (PM) probe, single nucleotide variant (SNP) probe, and perfect mismatch (MM) probe obtained in Example 7 It is a figure which shows a photocurrent with the case where it dries after water immersion sheet | seat is immersed in electrolyte solution and water is dripped just before use. It is sectional drawing of an example of the cell for a flow cell type | mold measurement. FIG. 17 is an exploded perspective view of the measurement cell shown in FIG. 16. In the case of using the aqueous electrolyte solution in the case of using the perfect match (PM) probe, single nucleotide variant (SNP) probe, and perfect mismatch (MM) probe obtained in Example 8, and using the water absorbent sheet It is a figure which shows a photocurrent with the case where it met. The photocurrent in the case of using each solvent for the water-absorbent sheet in the case of using the perfect match (PM) probe, single nucleotide variant (SNP) probe, and perfect mismatch (MM) probe obtained in Example 9 FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Sensor unit 11 ... Working electrode 12 ... Counter electrode 13 ... Water absorbing sheet 14 ... Support base material 15 ... Holding member 16 ... Light source 17 ... Contact probe 20 ... Sensor unit 21 ... Electrode unit 22 ... Opposing member 23 ... Water absorbing sheet DESCRIPTION OF SYMBOLS 24 ... Holding member 26 ... Light source 27 ... Contact probe 30 ... Sensor unit 31 ... Electrode unit 32 ... Opposing member 33 ... Water absorbing sheet 34 ... Support base material 36 ... Light source 37 ... Contact probe 40 ... Sensor unit 41 ... Electrode unit 42 ... Holding member 43 ... Water-absorbing sheet 44 ... Support substrate 46 ... Light source 47 ... Contact probe 121 ... Single-stranded nucleic acid 122 as test substance 122 ... Sensitizing dye 123 ... Working electrode 124 ... Complementary as a probe substance Single-stranded nucleic acid 125 ... conductive substrate 126 ... electron accepting layer 127 ... Conjugate of test substance and probe substance 128 ... Sensitizing dye capable of intercalation 130 ... Ligand as test substance 131 ... Receptor protein molecule 132 ... Sensitizing dye 133 ... Receptor protein to which ligand is bound Molecule 134 ... Double-stranded nucleic acid as probe substance 71 ... Electrode unit 72 ... Insulating substrate 73 ... Working electrode 74 ... Counter electrode 74 '... Lead wire 141 ... Antigen labeled with sensitizing dye 142 ... Not labeled with dye Antigen 143 ... Dye-labeled antigen that specifically binds to antibody 52 ... Working electrode 53 ... Counter electrode 54 ... Space containing electrolyte 55 ... Electron accepting layer 56 ... Light source 57 ... Substrate 58 ... Working electrode contact 59 ... Holding member 60 ... through hole 61 ... ammeter 62 ... supply hole 63 ... discharge hole 64 ... insulating spacer

Claims (27)

A water absorbent sheet used as an electrolyte medium in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A water absorbent substrate,
A water-absorbent sheet comprising an electrolyte contained in the water-absorbent substrate.   The water-absorbent sheet according to claim 1, having a solvent content of 20% by mass or more and 300% by mass or less.   The water absorbing sheet according to claim 2, wherein the solvent is an aprotic solvent, a protic solvent, or a mixture thereof. The water absorbent sheet according to claim 2 or 3, wherein the aprotic solvent is acetonitrile (CH ₃ CN).   The water absorbent sheet according to claim 2 or 3, wherein the protic solvent is water.   The water-absorbent sheet according to any one of claims 1 to 5, having a thickness of 0.01 to 10 mm.   The water absorbent sheet according to any one of claims 1 to 5, having a thickness of 0.1 to 3 mm.   The said water-absorbing base material comprises at least one fiber selected from the group consisting of natural fiber, pulp fiber, regenerated fiber, glass fiber, and synthetic fiber, according to any one of claims 1 to 7. Water absorbent sheet.   The water absorbent sheet according to claim 8, wherein the water absorbent substrate is a filter paper, a membrane filter, a glass filter, or a filter cloth. The electrolyte is sodium iodide (NaI), potassium iodide (KI), calcium iodide (CaI ₂ ), lithium iodide (LiI), ammonium iodide (NH ₄ I), tetrapropylammonium iodide (NPr ₄ I), sodium thiosulfate (Na ₂ S ₂ O ₃ ), sodium sulfite (Na ₂ SO ₃ ), hydroquinone, K ₄ [Fe (CN) ₆ ] · 3H ₂ O, ferrocene-1,1′-dicarboxylic acid, Ferrocene carboxylic acid, sodium borohydride (NaBH ₄ ), triethylamine, ammonium thiocyanate, hydrazine (N ₂ H ₄ ), acetaldehyde (CH ₃ CHO), N, N, N ′, N′-tetramethyl-p-phenylenediamine Dihydrochloride (TMPD), L-ascorbic acid, sodium tellurite (Na ₂ TeO ₃ ), iron (II) chloride tetrahydrate (FeCl ₂ .4H ₂ O), EDTA, cysteine, triethanolamine, tripropylamine, iodine-containing lithium iodide (I / LiI), tris (2- Chloroethyl) phosphate (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoethanolamine, thiourea dioxide, (COOH) ₂ , and HCHO, and at least one selected from the group consisting of The water absorbent sheet according to any one of claims 1 to 9. The _{electrolyte, NaI, KI, CaI 2,} LiI, NH 4 I, tetrapropylammonium iodide _(NPr 4 I), sodium thiosulfate _{(Na 2 S 2 O 3)} , and sodium sulfite _(Na 2 SO ₃₎ The water-absorbent sheet according to any one of claims 1 to 9, which is at least one selected from the group consisting of:
Specific detection of a test substance using the photocurrent,
A probe electrode to which a test substance is directly or indirectly specifically bound is provided on the surface, and a working electrode in which a sensitizing dye is fixed by the binding and a counter electrode are brought into contact with the water absorbent sheet;
Light is applied to the working electrode to excite the sensitizing dye, and a photocurrent flows between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. The water-absorbent sheet according to any one of claims 1 to 11, wherein the water-absorbent sheet is performed by a step including detecting the water content. A specific detection method for a test substance using photocurrent,
A working electrode comprising a probe substance to which a test substance is specifically bound directly or indirectly on the surface and having a sensitizing dye fixed by the binding, and a counter electrode. Contact the water absorbent sheet according to the paragraph,
Light is applied to the working electrode to excite the sensitizing dye, and a photocurrent flows between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. Detecting the method. A sensor unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A working electrode;
A counter electrode provided opposite to the electrode;
A water-absorbent sheet according to any one of claims 1 to 11, which is sandwiched between the working electrode and the counter electrode, and each electrode surface of the working electrode and the counter electrode is the water-absorbing sheet. Sensor unit in contact with the adhesive sheet. A support substrate for supporting the counter electrode;
The sensor unit according to claim 14, further comprising a pressing member that presses the working electrode, the water-absorbing sheet, and the counter electrode so as to closely contact each other toward the support base material. A support substrate for supporting the working electrode;
The sensor unit according to claim 14, further comprising a pressing member that presses the counter electrode, the water absorbent sheet, and the working electrode toward each other toward the supporting base material.   The sensor unit according to claim 15 or 16, wherein the pressing member further includes an opening or a light-transmitting part for allowing light for light excitation to pass through. A sensor unit used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
An electrode unit in which the working electrode and the counter electrode are patterned on the same plane;
A sensor unit comprising the water absorbent sheet according to any one of claims 1 to 11, which is provided in contact with each electrode surface of the working electrode and the counter electrode. Further comprising a facing member provided facing the electrode unit;
The sensor unit according to claim 18, wherein the water absorbent sheet is sandwiched between the electrode unit and the facing member.   The sensor unit according to claim 19, further comprising a pressing member that presses the electrode unit and the water-absorbent sheet toward each other toward the facing member. Further comprising a support substrate for supporting the electrode unit, and
The sensor unit according to claim 18, wherein the facing member functions as a pressing member that presses the water-absorbing sheet and the electrode unit toward each other toward the supporting base material.   The sensor unit according to any one of claims 18 to 21, wherein the pressing member further includes one or more openings or light-transmitting portions for allowing light for light excitation to pass therethrough. A support substrate for supporting the electrode unit;
A pressing member that holds the electrode unit in close contact with the supporting base material,
The sensor unit according to claim 18, wherein the water absorbent sheet is placed on the electrode unit. A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A sensor unit according to any one of claims 14 to 23;
A light source for irradiating the working electrode with light;
A measuring apparatus comprising: an ammeter that measures a current flowing between the working electrode and the counter electrode.   The measurement device according to claim 24, wherein the light source is a light source capable of irradiating light having different wavelengths depending on the type of the sensitizing dye.   26. The measuring apparatus according to claim 24, wherein a plurality of the light sources are provided in advance, and the measuring apparatus further includes a mechanism for switching and irradiating the plurality of light sources.   27. The measuring apparatus according to any one of claims 24 to 26, wherein the light source and / or the sensor unit further includes an XY moving mechanism for relatively moving the light source and the sensor unit in the XY direction.

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