JP2008202995A - Specific detecting method of substance to be inspected using photocurrent, pad used therein, sensor unit, and measuring instrument - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To sharply simplify an apparatus structure and detection procedure in the specific detection of a substance to be inspected using a photocurrent produced by the optical excitation of a sensitizing coloring matter, a sensor chip capable of shortening the time from the spot deposition of the substance to be inspected to the detection thereof, and a measuring instrument. <P>SOLUTION: This pad enables the specific bonding of the substance to be inspected and can be also used as an electrolyte-containing pad in place of an electrolyte. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for 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, a pad used in the method, a sensor unit, and a measuring apparatus. .

  In recent years, attempts have been made to devise a test system that can easily and inexpensively detect hormones and proteins that enable prediction of the disease state and progress of human diseases. In addition, disorders of the reproductive system and nervous system of exogenous endocrine disrupting substances (environmental hormones) such as dioxins have become a social problem, and a method for detecting such problems is desired.

  In addition, using the principle of solar cells that generate electrical energy from light using sensitizing dyes, photocurrents generated by photoexcitation of sensitizing dyes are used to detect test substances (biomolecules such as DNA and proteins). (Patent Literature 1, Patent Literature 2, Patent Literature 3). Furthermore, methods for electrochemically detecting analytical substances such as hormones, proteins, enzymes, etc. using the principle of immunochromatography have been performed (Patent Document 4, Patent Document 5, Patent Document 6).

JP 2002-181777 A JP 2005-251426 A JP 2006-507491 A Japanese Patent Laid-Open No. 2001-337065 JP-A-8-327582 Special Table 2006-524815

  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 addition, for the purpose of simplifying detection and simplifying the apparatus configuration, a method for electrochemically detecting an analyte such as a hormone, protein, enzyme, etc., using the principle of immunochromatography that has been performed, There is a problem that the labeled detection object needs to be separated by an absorption pad, and the structure of the apparatus is complicated, and it takes time from spotting a test substance to detection.

  The present inventors have recently used a water-absorbing pad having a role as a medium for specifically reacting a test substance and an electrolyte medium, and a test using a photocurrent generated by photoexcitation of a sensitizing dye. Specific detection of the substance was performed. By using this detection method, an electrolyte-containing pad could be used instead of the electrolyte solution, and the device structure and detection procedure could be greatly simplified. In addition, it is possible to use a pad with a structure that does not require an absorption pad that is necessary for separation of a labeled detection object, which was necessary in the conventional immunochromatography method. The knowledge that time can be shortened was acquired. Furthermore, when a photocurrent was detected using this technology, a photocurrent derived from a labeled detection substance that was not specifically bound was hardly detected, and it was found that accurate detection can be performed.

  Therefore, an object of the present invention is to greatly simplify the device structure and detection procedure in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye.

That is, the specific detection method of a test substance using photocurrent according to the present invention is as follows:
In the state in which a probe substance to which a test substance is directly or indirectly specifically bound is provided on the surface and a sensitizing dye is fixed by the binding and a counter electrode are in contact with the pad, the electrolyte In the presence, the working electrode is irradiated with light to photoexcite the sensitizing dye, and between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. Detecting a flowing photocurrent. Here, specific binding refers to a sample containing at least a test substance and a solvent after contacting a working electrode having a probe substance on its surface with which the test substance can bind directly or indirectly. It is carried out by impregnating the pad with a liquid. The sensitizing dye and the electrolyte are contained in either the sample solution or the pad, respectively.

The pad of the present invention is a pad used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye in the presence of an electrolyte,
A pad comprising a water-absorbing substance and serving as a medium for a test substance, a sensitizing dye and an electrolyte.

  A single pad or a plurality of pads of the present invention may be used. The pad of the present invention may further contain a sensitizing dye and / or an electrolyte.

Furthermore, according to 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 pad 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 pad.

In addition, a sensor unit according to another aspect of the present invention includes:
An electrode unit in which a working electrode and a counter electrode are patterned;
A pad provided in contact with each electrode surface of the working electrode and the counter electrode, and each electrode surface of the working electrode and the counter electrode is in contact with the pad.

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, in the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye, the structure of the apparatus and the detection procedure can be greatly simplified by using the pad of the present invention, Further, it is possible to shorten the time from the spotting of the sample solution to the detection, and furthermore, it is possible to detect the test substance with high accuracy.

Pad As the pad used in the present invention, an electrolyte-containing pad can be used in place of the electrolytic solution in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. Furthermore, the pad of the present invention is used as a medium for a test substance, a sensitizing dye and an electrolyte. By impregnating the sample solution containing the test substance into the pad, specific binding between the test substance and the probe substance can be performed on the pad. The pad may contain an electrolyte and a sensitizing dye. Further, by using a plurality of pads in the specific detection of the test substance, different substances can be included in each. For example, when two pads are used, an electrolyte can be included in one and a sensitizing dye can be included in the other, and these pads can be used for detection.

In the present invention, the electrolyte is not limited as long as it can freely move in the pad and participate in the exchange of electrons with the sensitizing dye, the working electrode, and the counter electrode, and a wide variety of electrolytes can be used. is there. 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, thiocyanate ammonium, hydrazine _{(N 2 H} 4), acetaldehyde _(CH 3 CH ), N, N, N', N'-tetramethyl--p- phenylenediamine dihydrochloride (TMPD), L-ascorbic acid, sodium tellurite _(Na 2 _TeO 3), iron (II) chloride tetrahydrate sum (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 sulfurous acid Sodium (Na ₂ SO ₃ ), and a mixture thereof, more preferably tetrapropylammonium iodide (NPr ₄ I).

According to a preferred embodiment of the present invention, the pad preferably has a moisture content of 20% or more, more preferably 30% or more, and further preferably 40% or more. When the moisture content is such, a high photocurrent can be detected when the photocurrent is detected, and the detection sensitivity is improved. The water content is obtained from (water content around 1 mm ³ ) / (density of water-absorbing substance). In addition, the moisture content said here is the moisture content of the pad at the time of detecting a photocurrent, Comprising: It does not need to satisfy | fill the said moisture content at the time of storage.

  As a form of the pad of this invention, it is preferable that the contact part with each electrode is made into a smooth plane so that favorable adhesiveness with a working electrode and a counter electrode is ensured. Therefore, when sandwiched between the working electrode and the counter electrode, it is preferable to have a uniform thickness so as not to affect the adhesion. On the other hand, when using an electrode unit in which the working electrode and the counter electrode are patterned in the same plane, it is sufficient that at least one side surface in contact with the electrode unit is a smooth plane. Uniformity is not a particular problem.

  According to a preferred embodiment of the present invention, the pad 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 a strength suitable for handling the pad alone, so it can be easily sandwiched, removed or carried between the working electrode and the counter electrode. 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 pad comprises a water-absorbing substance. Water-absorbing substances include 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; felts and sponges, etc. It is preferable to contain at least one kind of fiber selected from synthetic fibers, and it is not limited as long as it is a water-absorbing substance exhibiting an appropriate strength, water content, and adhesion to an electrode. The fiber processing method is not limited to a specific processing method.

  According to a preferred aspect of the present invention, the pad is in the form of a sheet. Preferable examples include filter paper, membrane filter, glass filter, filter cloth and the like, and filter paper and membrane filter are more preferable.

Specific Detection of Test Substance Using Photocurrent As described above, the pad of the present invention is used for specific detection of a test substance using a 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.

The specific detection method of a test substance using photocurrent according to the present invention is as follows:
A working electrode having a probe substance that can be specifically bound directly or indirectly to the test substance is brought into contact with the pad, the sample liquid is impregnated into the pad, and then the probe is specifically bound to the probe substance. Irradiating light to the working electrode on which a sensitizing dye is fixed to photoexcite the sensitizing dye, and the working electrode and the pad are caused by electron transfer from the photoexcited sensitizing dye to the working electrode. And detecting a photocurrent flowing between the counter electrode and the counter electrode.

In this method, first, a sample solution containing at least a test substance and a solvent and a working electrode are prepared. Here, the solvent is a liquid capable of dissolving or dispersing the test substance, the sensitizing dye, and the electrolyte, and is water and / or an aqueous solvent, preferably water. The working electrode used in the present invention is an electrode provided on the surface with a probe substance capable of specific 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. The sensitizing dye is a substance that can emit electrons to the working electrode in response to photoexcitation. When the specific detection method of the test substance is the sandwich method, the probe substance is the primary antibody and the sensitizing dye is the secondary antibody. When the antibody is labeled and the specific detection method of the test substance is a receptor binding assay, peptide, protein, or DNA is used as a probe substance, and the sensitizing dye is labeled with the receptor protein. When the specific detection method is a competitive method, the sensitizing dye is labeled with a second test substance that can specifically bind to the probe substance.
The substance labeled with the sensitizing dye and the electrolyte may be contained in either the sample solution or the pad.

  Then, after the working electrode is brought into contact with the pad, when the sensitizing dye is photoexcited by irradiating the working electrode with light, electron transfer from the photoexcited sensitizing dye to the electron accepting substance occurs. 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.

Sensor Unit and Measuring Device By using the pad of the present invention, an inexpensive and small sensor unit or measuring device having a greatly simplified structure can be constructed. This is because the use of the pad of the present invention requires a mechanism (for example, a pump, a valve, And their control mechanism), liquid leakage prevention mechanism (for example, packing), and complicated mechanism or process such as waste liquid treatment of the electrolyte are not required, and the specific binding of the test substance is performed on the pad. It becomes a simple reactor mechanism.

  The sensor unit according to the first aspect of the present invention includes a working electrode, a counter electrode provided opposite to the working electrode, and a pad sandwiched between the working electrodes, and the surface of the working electrode is in contact with the pad. It is made. The sensor unit according to the first aspect of the present invention may further include a counter electrode, in which case the pad is further in contact with the counter electrode. 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 pad. 1 and 2 show 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 below the counter electrode 12, and the pad 13 is sandwiched between the working electrode 11 and the counter electrode 12. The sensor unit 10 also includes a pressing member 14 that supports the working electrode 11. On the uppermost part of the sensor unit 10, an opposing member 15 is provided that presses the counter electrode 12, the pad 13, and the working electrode 11 downward toward the pressing member 14 so as to closely contact each other. 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. The counter member that supports the counter electrode and the pressing member that presses the counter electrode, the pad, and the working electrode in close contact with each other toward the counter member may be provided. 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 14 further includes an opening or a light transmitting portion for allowing light for light excitation to pass through. 2 is a sectional view and an exploded view of the sensor unit 10 ′ when two pads 13′a and 13′b formed of a water-absorbing substance are used in the sensor unit of FIG. Indicates. The configuration other than the pad is the same as the configuration of FIG. 1 described above. In addition, it is preferable to make the contact for connecting with the ammeter of a working electrode by the contact probe 16 and the contact probe 16 ', and the same is applied to the counter electrode.

  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 pad provided in contact with each electrode surface of the working electrode and the counter electrode. Become. 3 to 6 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. 3, a facing member 22 is provided to face the electrode unit 21, and a pad 23 is sandwiched between the electrode unit 21 and the facing member 22. The sensor unit 20 includes a pressing member 24 that supports the electrode unit 21. That is, the electrode unit 21 is arranged so that the side on which the test substance is immobilized is on the pressing member 24, and the opposing member 22 contacts the electrode unit 21 and the pad 23 toward each other toward the pressing member 24. Press down on. 4 is a sectional view and an exploded view of the sensor unit when two pads 23'a and 23'b formed of a water-absorbing substance are used in the sensor unit 20 'shown in FIG. Indicates. The configuration other than the pad is the same as the configuration of FIG. 3 described above. On the other hand, in the sensor unit 30 shown in FIG. 5, the electrode unit 31 is further provided with a facing member 34 that supports the electrode unit, and the pressing member 32 faces the facing member 34 with the pad 33 and the electrode unit. Press 31 so that they are in close contact with each other. That is, the electrode unit 31 is placed on the facing member 34 such that the side on which the test substance is immobilized is on the upper side, and the pad 33 is placed on the electrode unit 31. The sensor unit 30 ′ shown in FIG. 6 is a sectional view and an exploded view of the sensor unit when two pads 33′a and 33′b formed of a water-absorbing substance are used in the sensor unit of FIG. Show. 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 reverse 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, 24 ′, 32, and 32 ′ further include one or more openings or light-transmitting portions for allowing light for light excitation to pass through. In addition, in the sensor unit 30 and sensor unit 30 ′ shown in FIGS. 5 and 6, since the irradiated light passes through the pad, attenuation of light intensity is a problem when using an electrolyte having coloring properties. It is preferable to adjust the light source and the electrolyte concentration as appropriate so as not to occur. The contact points for connection to the working electrode and the counter electrode ammeter are preferably provided by the contact probes 25, 25 ', 35, 35', and the same applies to the counter electrode.

  In any of the first aspect and the second aspect, the sensor units 10, 10 ', 20, 20', 30, 30 'include light sources 17, 17', 27 for irradiating light to the working electrode. 27 ', 37, 37' and an ammeter (not shown) for measuring the current flowing between the working electrode and the counter electrode can be further 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, 10 ′, 20 and 20 ′ shown in FIGS. 1 to 4, the light from the light sources 17, 17 ′, 27 and 27 ′ is emitted from the working electrodes 11 and 11 ′ and the electrode units 21 and 21 ′. Irradiated from the back side, it passes through the transparent working electrodes 11, 11 ′ and the electrode units 21, 21 ′ and reaches the surfaces of the working electrodes 11, 11 ′ and the electrode units 21, 21 ′. On the other hand, in the sensor units 30 and 30 ′ shown in FIGS. 5 and 6, the light from the light sources 37 and 37 ′ passes through the openings of the pressing members 32 and 32 ′ and the surfaces of the electrode units 31 and 31 ′. To reach. 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 wires are directly connected, or the contact probes 16, 16 ', 25, 25', 35, 35 'as shown in the illustrated example. It is possible to adopt a means such as connection through the network. 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 another preferred aspect of the present invention, it is also possible to produce a measuring device in which a plurality of sensor units are combined. In that case, the sensor unit may be provided with a plurality of light sources in advance, and the measuring apparatus may further include a mechanism for switching and irradiating the plurality of light sources.

  Furthermore, according to a preferred aspect of the present invention, an XY moving mechanism (not shown) is attached to a measuring apparatus that combines a plurality of sensor units, and the light source and sensor unit are moved relative to each other in the XY direction so that the light source It is preferable that the irradiation can be performed while scanning the working electrode in the X and Y directions. Thereby, light can be irradiated to the test substance fixed spots on the plurality of sensor units. 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. In this case, it is preferable that the XY moving mechanism is configured so that the moving speed, the moving route, and the like can be designated by software incorporated in the computer or apparatus.

  According to a preferred aspect of the present invention, it is preferable that a current value generated by light irradiation is measured by an ammeter, and the result is sent to a memory in a computer or apparatus so that data can be stored sequentially. 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 is read from an appropriate data point, and the concentration of the substance in the sample can be determined using the difference. In addition, it is possible to automatically process from reading to quantification of these data on software.

Test substance and probe substance The test substance in the present invention is not particularly limited as long as it is a substance having specific binding properties with the probe substance. In this method, a probe substance capable of specifically binding directly or indirectly to the test substance is supported on the surface of the working electrode, so that the test substance is directly or indirectly specifically bound to the probe substance. Can be detected.

  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. A preferable example of the combination of the test substance and the probe substance in such a manner that the test substance is directly and specifically bound is a combination of an antigen and an antibody. On the other hand, preferred examples of the combination of the test substance and the probe substance in such a manner that the test substance is bound to the probe substance in such a manner that it can be specifically and indirectly bound to the probe substance include the case where the sandwich method shown below is adopted and receptor binding An example of employing an assay is given.

Measurement Method In the measurement method using the pad of the present invention, first, a probe substance to which a test substance specifically binds directly or indirectly is immobilized on the surface of the working electrode.

  In this way, the working electrode on which the probe substance that specifically binds directly or indirectly to the test substance is immobilized is brought into contact with the pad, and the sample liquid containing the test substance is spotted and then directly or indirectly specific. The sensitizing dye is fixed to the working electrode by the binding.

  According to a preferred aspect of the present invention, when an immunoassay, particularly a sandwich method, is employed using an antigen as a test substance, the following aspects can be taken. A primary antibody is used as a probe substance, and an antigen as a test substance is specifically bound to the primary antibody, and then a secondary antibody labeled with a sensitizing dye is immobilized on the working electrode by specific binding with the antigen. . FIG. 7 shows an immobilization process of the secondary antibody labeled with the antigen as the test substance and the sensitizing dye to the primary antibody as the probe substance in this embodiment. As shown in FIG. 7, the secondary antibody 41 labeled with the sensitizing dye 40 specifically binds to the antibody 43 in the presence of the antigen 42 as the test substance.

  According to a preferred aspect of the present invention, when a competitive immunoassay is further employed using an antigen as a test substance, the following aspects can be taken. Using a probe substance that is an antibody, an antigen that is a test substance, and an antigen that is the second test substance that has the same binding ability as the test substance and is labeled with a sensitizing dye, And the sensitizing dye is immobilized on the working electrode. FIG. 8 shows an immobilization process of the antigen as the test substance and the antigen as the second test substance labeled with a sensitizing dye to the antibody as the probe substance in this embodiment. As shown in FIG. 8, the antigen 141, which is the second test substance labeled with a sensitizing dye, and the antigen 142, which is the test substance not labeled with the dye, compete to specifically bind to the antibody 143. Join.

  In the present invention, the specific binding between the test substance and the probe substance may be indirect. That is, when a receptor binding assay is employed, a sensitizing dye that specifically binds to a ligand as a test substance is labeled on a working electrode to which a nucleic acid (a peptide, protein, etc.) as a probe substance is fixed. A sensitizing dye is immobilized in the presence of a ligand in the presence of a receptor protein. FIG. 9 shows the step of immobilizing the ligand, which is the test substance, on the working electrode in this embodiment. As shown in FIG. 9, the ligand 341 that is the test substance first specifically binds to the receptor protein 343 labeled with the sensitizing dye 342. Then, the receptor protein 344 to which the ligand is bound specifically binds to the double-stranded nucleic acid 345 as the probe substance.

  Furthermore, an immunoassay by the open sandwich method is also applicable to this method. In this case, the antibody L chain or H chain labeled with a sensitizing dye that specifically binds to the test substance antigen is attached to the working electrode to which the antibody H chain or L chain is immobilized. The sensitizing dye is immobilized in the presence of the test substance. FIG. 10 shows the step of immobilizing the antigen as the test substance in this embodiment on the working electrode. As shown in FIG. 10, the test substance antigen 441 is transferred to the antibody H chain 444 (or the probe substance) in the presence of the L chain 443 (or H chain) of the antibody labeled with the sensitizing dye 442 (or L chain) specifically.

  In this way, the working electrode on which the test substance is fixed together with the sensitizing dye is irradiated with light in the presence of an electrolyte to photoexcite the sensitizing dye and act due to electron transfer from the photoexcited sensitizing dye to the working electrode. A photocurrent flowing between the electrode and the counter electrode in contact with the pad is detected by an ammeter.

  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 an antigen, the amount of antibody specifically bound to the antibody 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 a preferred embodiment of the present invention, when the competition method is adopted, the antigen or single-stranded nucleic acid that is the test substance and the antigen or single-stranded nucleic acid labeled with the sensitizing dye are caused to compete with each other to be specific to the probe substance. When the binding is performed, a correlation is obtained between the detected current value and the concentration of the antigen or single-stranded nucleic acid labeled with the sensitizing dye. That is, as the number of analytes that are not labeled with a sensitizing dye increases, the number of competitors that specifically bind to the probe substance decreases, so as the concentration of the analyte that is not labeled with the dye increases, A calibration curve in which the detected current value decreases can be obtained. Therefore, it is possible to detect and quantify a test substance that is not labeled with a sensitizing dye.

Sensitizing dye In the present invention, in order to detect the presence of the test substance by photocurrent, in the presence of the sensitizing dye, the test substance is directly or indirectly specifically bound to the probe substance, The sensitizing dye is fixed to the working electrode by the binding. Therefore, in the present invention, the sensitizing dye is a primary antibody, antigen, ligand, receptor protein, single-stranded nucleic acid, antibody H chain, antibody L chain as shown in FIGS. It is preferable that the affinity substance such as is labeled.

  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.

Working electrode and production thereof The working electrode used in the present invention is an electrode having the probe substance on its surface, and is an electrode that can accept electrons emitted by a sensitizing dye immobilized via the probe substance in response to photoexcitation. is there. 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 in response to photoexcitation of the sensitizing dye, 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. 7 to 10 includes a conductive substrate 125 and an electron-accepting layer 126 formed on the conductive substrate and containing an electron-accepting substance. 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) at which electrons can be injected 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, this A level 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. Further, for the purpose of improving the light capture rate by scattering incident light, an electron-accepting layer may be formed using a combination of 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. Examples thereof include a method (sol-gel method) that is applied to the substrate and hydrolyzed with moisture in the air to obtain a fine particle film, 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. Moreover, it is preferable that the application quantity of the semiconductor fine particle per 1 m < ² > of electroconductive base materials is 0.5-400g, More preferably, it is 5-100g.

  According to a preferred embodiment of the present invention, when the electron-accepting material comprises indium-tin composite oxide (ITO) or metal oxide (FTO) in which tin oxide is doped with fluorine (FTO), 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 degrees, and more preferably 100 to 600 degrees. 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, thiol group, hydroxyl group, phosphoric acid and the like can be suitably used as a functional group for immobilizing the probe substance to the working electrode. Further, according to a preferred embodiment of the present invention, in order to firmly fix the probe substance to the working electrode, it is possible to use a material that crosslinks between the working electrode and the probe substance. 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 probe substance can be immobilized efficiently by a simple operation called physical adsorption. The physical adsorption of the 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 probe substance, and the probe substance is adsorbed on the surface of the carrier.

  Moreover, nonspecific adsorption | suction can be suppressed by adding a blocking agent after carrying | supporting a probe substance. 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 probe substance is not supported and can be adsorbed to the electron-accepting substance by chemical adsorption or physical adsorption. However, it is preferably a substance having a functional group that can be adsorbed via a chemical bond. Examples of the blocking agent include substances having a functional group such as a carboxylic acid group, a phosphoric acid group, a sulfonic acid group, a hydroxyl group, an amino group, a pyridyl group, and an amide.

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 is a glass on which a metal or a conductive oxide is deposited. 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.

Electrode Unit According to a preferred embodiment of the present invention, an electrode unit in which the working electrode and the counter electrode are patterned on the same plane may be used. 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. 11 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 73 ′ and 74 ′ are provided so as to extend from the working electrode 73 and the counter electrode 74, respectively.

  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.

Example 1. Protein detection (1) Preparation of biotin-labeled DNA-immobilized working electrode Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U membrane) 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% methanol and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2vol% 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) in which an opening having a diameter of 3 mm was formed was placed and adhered. Subsequently, biotin-labeled ssDNA (25 mer) adjusted to a concentration of 1 μM was held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes to denature the DNA. The denatured DNA was filled in 5 μl each in the opening of the previously prepared seal on the glass 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 biotin-labeled ssDNA on the glass substrate. The seal was peeled off from each glass substrate, and each glass substrate was shaken in a 0.2% SDS solution for 15 minutes × 3 times, and rinsed by changing the ultrapure water three times. These glass substrates were immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, each 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 biotin-labeled DNA-immobilized working electrode was obtained.

(2) Example 1A . Photocurrent detection using a pad containing a water-absorbing substance and an electrolyte (competitive method).
i. Preparation of Pad An aqueous solution containing 0.4 M tetrapropylammonium iodide (NPr ₄ I) was prepared. A 0.9 mm-thick blotting filter paper (manufactured by Ato Co., Ltd.) cut into 43 mm × 16 mm was immersed in this aqueous solution and dried at 50 ° C. for 2 hours to obtain a pad.

ii. Photocurrent measurement FIG. 12 shows a sectional view and an exploded view of the sensor unit used in this experiment. In the embodiment, a biotin-labeled DNA immobilization working electrode 51 prepared in the above (1) and a counter electrode 52 formed by depositing platinum on a glass plate were prepared. The pad 53 produced in i above was sandwiched between both electrodes and adhered. Thereafter, a rhodamine-labeled streptavidin solution (solvent composition: 10 mM HEPES PH7.4, 150 mM NaCl, 0.05% Tween solution) prepared to 10 μg / ml assumed to be a competitive substance and 10 μg / ml, 1 μg / ml assumed to be a test substance. 500 μl each of the mixed solutions 54 prepared by mixing 1: 1 the streptavidin solution (solvent composition: 10 mM HEPES PH7.4, 150 mM NaCl, 0.05% Tween solution) prepared to 0 μg / ml was added to the end of the pad. I let it soak. At this time, the working electrode was fixed so that the surface on which the DNA was immobilized and the platinum deposition surface of the counter electrode were opposed to each other. With both electrodes connected to an electrochemical analyzer, the working electrode was irradiated with a laser light source 55 (output 60 mW, diameter of irradiated region 1 mm, wavelength 530 nm green laser), and the current value observed at that time was recorded. In FIG. 12, 56 is a pressing member, 57 is a counter member, 58 is an electrolyte, 59 is biotin-labeled DNA, 60 is rhodamine-labeled streptavidin, and 61 is streptavidin.

  The results are shown in FIG. As shown in FIG. 13, when the concentration of the streptavidin solution in the dropped mixed liquid 54 increased, a decrease in photocurrent was observed.

(3) Example 1B. Photocurrent detection using a pad containing an affinity substance labeled with a water-absorbing substance, electrolyte, or sensitizing dye (competitive method)
i. Preparation of Pad An aqueous solution containing 0.4 M tetrapropylammonium iodide (NPr ₄ I) was prepared. A 0.9 mm thick blotting filter paper (Ato Co., Ltd.) cut into 43 mm × 16 mm was immersed in this aqueous solution and dried at 50 ° C. for 2 hours to obtain a pad (1). Thereafter, rhodamine-labeled streptavidin solution (solvent composition: 10 mM HEPES PH7.4, 150 mM NaCl, 0.05% Tween solution) 500 μl prepared to 10 μg / ml was cut into 31 mm × 16 mm 0.9 mm thick blotting filter paper (Ato shares) Was added dropwise to the company) and dried at 50 ° C. for 2 hours to obtain a pad (2).

ii. Photocurrent measurement FIG. 14 shows a sectional view and an exploded view of the sensor unit used in this experiment. In the embodiment, a biotin-labeled DNA-immobilizing working electrode 51 ′ prepared in the above (1) and a counter electrode 52 ′ formed by depositing platinum on a glass plate were prepared. The pad (1) 53'-a was sandwiched between the counter electrode side and the pad (2) 53'-b was sandwiched between the working electrode side, thereby being brought into close contact. Thereafter, a streptavidin solution (solvent composition: 10 mM HEPES PH7.4, 150 mM NaCl, 0.05% Tween solution) 54 ′ prepared to 10 μg / ml, 1 μg / ml, and 0 μg / ml assumed to be a test substance is put in a 700 μl pad ( It dripped at the edge part of 2), and the pad (1) and the pad (2) were made to soak with water. At this time, the working electrode was fixed so that the surface on which the DNA was immobilized and the platinum deposition surface of the counter electrode were opposed to each other. With both electrodes connected to an electrochemical analyzer, the working electrode was irradiated with a laser light source 55 '(output 60 mW, diameter of irradiated region 1 mm, wavelength 530 nm green laser), and the current value observed at that time was recorded. In FIG. 14, 56 ′ represents a pressing member, 57 ′ represents an opposing member, 58 ′ represents an electrolyte, 59 ′ represents biotin-labeled DNA, 60 ′ represents rhodamine-labeled streptavidin, and 61 ′ represents streptavidin.

  The results are shown in FIG. As shown in FIG. 15, the photocurrent decreased as the streptavidin concentration in the dropped solution increased. Furthermore, it has been found that detection is possible even using a plurality of pads.

Example 2. Examination of conditions for electrolyte-containing pads by DNA detection (reference example)
(1) Production of dye-labeled DNA-immobilized working electrode As a glass substrate for the working electrode, 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) 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% methanol and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2vol% 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 degrees 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) in which an opening having a diameter of 3 mm was formed was placed and adhered. 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 the opening of the previously prepared seal on the glass 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 and unlabeled ssDNA on each glass substrate. The seal was peeled off from each glass substrate, and each glass substrate was shaken in a 0.2% SDS solution for 15 minutes × 3 times, and rinsed by changing the ultrapure water three times. These glass substrates were immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, each glass substrate was immersed in 4 degrees of absolute ethanol for 1 minute to dehydrate, and air was blown to disperse residual ethanol. Thus, a dye-labeled DNA immobilization working electrode and an ssDNA immobilization working electrode were obtained. The base sequence of the probe DNA used here is as follows.
5'-end rhodamine labeled ssDNA (probe 1): 5'-Rho-GCGGCATGAACCTGAGGCCCATCCT-3 '
Unlabeled ssDNA (probe 2): 5´-TTGAGCAAGTTCAGCCTGGTTAAG-3´

(2) Production of Electrolyte-Containing Pad Aqueous solutions containing 0.2M, 0.4M, and 0.6M tetrapropylammonium iodide (NPr ₄ I) were prepared. A 0.9 mm-thick blotting filter paper (CB-13A; Ato Co., Ltd.) cut to 26 mm × 20 mm was dipped in this aqueous solution, and lightly drained to obtain an electrolyte-containing pad.

(3) Example 2A . Examination of electrolyte concentration A dye-labeled DNA-immobilized working electrode prepared in the above (1) and a counter electrode obtained by depositing platinum on a glass plate were prepared. The electrolyte-containing pad produced in the above (2) was sandwiched between the electrodes and adhered. 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. 16, an increase in photocurrent was observed depending on the concentration of tetrapropylammonium iodide, but it is clear that any concentration of tetrapropylammonium iodide can be used for measurement. Became.

(4) Example 2B . Examination of various electrolytes Photocurrent measurement was performed using various electrolytes. Specifically, a filter paper was used as the water-absorbing substance, and the concentration of each reducing agent was fixed at 0.2 M to prepare an electrolyte-containing pad. 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. Electrolytes containing these various electrolytes and water were prepared, and a blotting filter paper (CB-13A; Ato Co., Ltd.) with a thickness of 26 mm x 20 mm was soaked, and then drained lightly. Got. The dye-labeled DNA-immobilized working electrode was prepared in the same manner as in (1), and working electrodes prepared using solutions having a rhodamine-labeled ssDNA concentration of 10 nM and a rhodamine-unlabeled ssDNA concentration of 100 nM were also prepared (3). The photocurrent was measured in the same manner as above.

  The results are shown in FIG. As shown in FIG. 17, in any of the studied electrolytes, an increase in photocurrent was recognized depending on the amount of immobilized ssDNA, and it was revealed that it can be used for measurement.

(5) Example 2C . Examination of the thickness of the electrolyte-containing pad After preparing the dye-labeled DNA-immobilizing working electrode in the same manner as (1) except that the ssDNA concentration to be immobilized was changed to two concentrations of 100 nM and 1 μM, the concentration of 0.2 M Prepare an electrolyte with tetrapropylammonium iodide (NPr ₄ I) and water, and stack one, two or three sheets of 0.9 mm thick blotting filter paper (CB-13A; Ato Co., Ltd.) An electrolyte-containing pad was prepared by dipping in an electrolytic solution. Thereafter, the photocurrent was measured in the same manner as in (3) using the prepared working electrodes having different immobilization concentrations and respective electrolyte-containing pads having different thicknesses.

  The results are shown in FIG. As shown in FIG. 18, almost no influence on the photocurrent value was observed over the entire area of the electrolyte-containing pad thickness of 0.9 mm to 2.7 mm. Therefore, it is presumed that there is no adverse effect on the detection of the photocurrent value even if the electrolyte-containing pad thickness is increased from the viewpoint of obtaining sufficient strength.

(6) Example 2D . Examination of various pads Blotting filter paper (CB-13A; ATTO Co., Ltd.) having a thickness of 0.9 mm, 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 ), fluorine resin The photocurrent was measured in the same manner as (3) except that a membrane filter (JCWP09025; MILLIPORE: thickness: 0.1 mm) mainly containing The dye-labeled DNA-immobilized working electrode was prepared in the same manner as (1), and a working electrode prepared using each solution with rhodamine-labeled ssDNA concentrations of 100 nM and 1 μM and an unlabeled ssDNA concentration of 100 nM was also prepared. (1) Photocurrent measurement was performed in the same manner. As the electrolytic solution, an aqueous solution containing 0.2 M tetrapropylammonium iodide (NPr ₄ I) was used.

  The results are shown in FIG. As shown in FIG. 19, in all the pads, an increase in photocurrent was observed depending on the amount of immobilized ssDNA, and it was revealed that the pads can be used as electrolyte-containing pads.

(7) Example 2E . Examination of moisture content of electrolyte-containing pad i. Measurement of water content First, 500 μl of an aqueous solution containing 0.4 M tetrapropylammonium iodide (NPr ₄ I) was dropped on a 0.9 mm thick filter paper (CB-13A; Atto Co., Ltd.) cut to 26 mm × 20 mm. The filter paper was completely immersed in the electrolyte. Thus, a filter paper impregnated with the electrolytic solution was produced. Next, each impregnated filter paper was dried at 50 degrees for 0 hour, 0.25 hour, 0.5 hour, 1 hour, 1.25 hour and 1.5 hour, respectively. The weight of the filter paper after drying was measured, and after removing the weight of 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 electrolyte-containing pad. The density of the blotting filter paper is 0.00049 g / mm ³ .
ii. Photocurrent measurement i. The photocurrent was detected in the same manner as in Example 1 by using the electrolyte-containing pad having each moisture content prepared in (1).

The results are shown in FIG. As shown in FIG. 20, it can be seen that the higher the moisture content of the electrolyte-containing pad, the higher the photocurrent. 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 electrolyte-containing pad capable of detecting photocurrent has a water content of at least 20% or more.

It is a figure which shows an example of the sensor unit by the 1st aspect of this invention using the pad comprised by the sheet of 1 sheet | seat, and an example before and behind a measurement, a) is sectional drawing after an assembly, b) is before an assembly An exploded view of is shown. It is a figure which shows an example of the sensor unit by the 1st aspect of this invention using the pad comprised with two water-absorbing substances, and an example before and behind a measurement, a) is sectional drawing after an assembly, b) is before an assembly An exploded view of is shown. It is a figure which shows an example before and after the sensor unit by the 2nd aspect of this invention which irradiated light from the electrode unit back side using the pad comprised with the sheet | seat of one water absorbing material, and a measurement. B) shows an exploded view before assembly. It is a figure which shows an example before and after a sensor unit by the 2nd aspect of this invention which irradiated light from the electrode unit back side using the pad comprised with two water absorbing materials, and a measurement, B) shows an exploded view before assembly. It is a figure which shows an example before and after the sensor unit by the 2nd aspect of this invention which irradiated the light from the electrode unit front side using the pad comprised with the sheet | seat of one water absorbing material, and a measurement. B) shows an exploded view before assembly. It is a figure which shows an example before and after the sensor unit by the 2nd aspect of this invention which irradiated the light from the electrode unit front side using the pad comprised with two water absorbing substances, and a measurement. B) shows an exploded view before assembly. When the test substance is an antigen, the probe substance is a primary antibody that specifically binds to the antigen, and the sensitizing dye is labeled on the secondary antibody, the test substance and the sensitizing dye probe substance The immobilization process is shown in FIG. 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. 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. In the case where the test substance is an antigen and the probe substance is the H chain (or L chain) of the antibody, the immobilization process of the test substance to the probe substance in the presence of the L chain (or H chain) of the antibody is shown. FIG. It is a figure which shows an example of an electrode unit. It is a figure which shows sectional drawing and exploded view of a sensor unit in the case of using the pad containing a water absorbing material and an electrolyte used in the experiment of Example 1A. It is a figure which shows the photocurrent value measured in each streptavidin density | concentration at the time of using the pad containing a water absorbing material and electrolyte obtained in Example 1A. It is a figure which shows the cross-sectional view and exploded view of a sensor unit at the time of using the pad containing the affinity substance labeled with the water absorbing substance, electrolyte, and sensitizing dye used in the experiment of Example 1B. It is a figure which shows the photocurrent value measured in each streptavidin density | concentration at the time of using the pad containing the water absorbing substance obtained in Example 1B, an electrolyte, and the affinity substance labeled with the sensitizing dye. Obtained in Example 2A, when using an electrolyte containing pad, the rhodamine-labeled ssDNA of ssDNA and 100nM of 100nM, a diagram showing the measured photocurrent values at each tetrapropylammonium iodide _(NPr 4 I) concentrations is there. It is a figure which shows the photocurrent value at the time of using each electrolyte in each rhodamine labeled ssDNA density | concentration of 0 nM, 10 nM, and 100 nM obtained in Example 2B. It is a figure which shows the relationship between the thickness of an electrolyte containing pad, and a photocurrent value in each rhodamine labeled ssDNA density | concentration of 100 nM and 1 micromol which were obtained in Example 2C. It is a figure which shows the photocurrent value at the time of using each electrolyte containing pad in each rhodamine labeled ssDNA density | concentration of 100 nM ssDNA obtained in Example 2D, 100 nM, and 1 micromol. It is a figure which shows the relationship between the moisture content of an electrolyte containing pad obtained in Example 2E, and a photocurrent value.

Claims (19)

A working electrode comprising a probe substance specifically bound to the test substance directly or indirectly on the surface, and a sensitizing dye fixed by the binding, and a counter electrode were brought into contact with a water-absorbing pad. In state,
In the presence of an electrolyte, the working electrode is irradiated with light to photoexcite the sensitizing dye, and between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. A method for specifically detecting a test substance by detecting a photocurrent flowing through
The bond is
A working electrode provided on the surface with a probe substance to which a test substance can be directly or indirectly bound is brought into contact with the pad;
It is carried out by impregnating the pad with a sample solution containing at least a test substance and a solvent,
The sensitizing dye and the electrolyte are each contained in either the sample solution or the pad. The method according to claim 1, wherein the sensitizing dye is labeled with an affinity substance capable of specific binding directly or indirectly with a probe substance.
  The method according to claim 1, wherein the pad has a moisture content of 20% or more when detecting a photocurrent.   The method according to claim 1, wherein a plurality of the pads are used. It is a pad used for a method given in any 1 paragraph of Claims 1-4,
A pad comprising at least a water-absorbing substance.   The pad according to claim 5, further comprising a sensitizing dye and / or an electrolyte.   The pad according to claim 6, wherein the sensitizing dye is labeled with an affinity substance capable of specific binding directly or indirectly with a probe substance.   The pad according to any one of claims 5 to 7, which has a thickness of 0.01 to 10 mm.   The pad according to any one of claims 5 to 8, wherein the water-absorbing substance is at least one fiber selected from the group consisting of natural fiber, pulp fiber, regenerated fiber, glass fiber, and synthetic fiber.   The pad according to any one of claims 5 to 9, wherein the pad is at least one sheet selected from the group consisting of a filter paper, a membrane filter, a glass filter, and a filter cloth. 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 sensor unit comprising the pad according to claim 5 sandwiched between the working electrode and a counter electrode, wherein the surface of the working electrode is in contact with the pad.   The sensor unit according to claim 11, further comprising a counter electrode, wherein the counter electrode is in contact with the pad. A support substrate for supporting the counter electrode;
The sensor unit according to claim 12, further comprising a pressing member that presses the working electrode, the pad, and the counter electrode in close contact with each other toward the support base. 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 pad according to claim 5, 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 14, wherein the pad is sandwiched between the electrode unit and the facing member.   The sensor unit according to claim 15, further comprising a pressing member that presses the electrode unit and the pad toward each other toward the facing member. Further comprising a support substrate for supporting the electrode unit, and
The sensor unit according to claim 15, wherein the facing member functions as a pressing member that presses the pad and the electrode unit toward each other toward the support base material. 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 14, wherein the pad 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 11 to 18,
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.

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