JP2012098252A - Measuring device used for specific detection of specimen using photocurrent and measuring method employing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring device which is used for specific detection of a specimen using a photocurrent, having a light radiation mechanism and a light radiation method, capable of solving the problems that the number of light sources is decreased as further as possible, an integrated spot can be detected, an overall shape of the apparatus is reduced as further as possible, and a plurality of spots can be accurately detected in a short time as further as possible.SOLUTION: A measuring device is used for the specific detection of a specimen using a photocurrent generated by optical excitation of sensitized pigments by mounting an active electrode which is obtained by carrying a probe material that can be coupled with a specimen, while being divided for each of a plurality of spots separated from one another. The measuring device includes a plurality of light sources which irradiate the spots with light, and an ammeter which measures a photocurrent generated by the optical excitation of the sensitized pigments. The light sources are provided on an extension of a centerline corresponding to the spots at least as many as the number of columns or rows, and a light source moving mechanism is provided for moving the light sources toward the extension of the centerline.

Description

  The present invention relates to a measuring apparatus for detecting DNA with high accuracy and simplicity using a photocurrent, and a measuring method using the measuring apparatus.

  Genetic diagnostic methods for detecting and analyzing DNA in biological samples are promising as new preventive and diagnostic methods for various diseases. The following have been proposed as techniques for easily and accurately detecting such DNA.

  There is known a DNA analysis method in which a sample DNA is hybridized with a DNA probe having a base sequence complementary thereto and a fluorescent substance labeled, and a fluorescent signal at that time is detected (for example, JP, 7-107999, A (patent documents 1) and JP, 11-315095, A (patent documents 2)). In this method, the formation of double-stranded DNA by hybridization is detected by fluorescence of the dye. In fluorescence detection, since a plurality of detection spots on which probe DNA is immobilized can be formed on the same substrate, the specificity to a plurality of probe DNAs can be elucidated by one detection. However, since a detection apparatus including a light receiver necessary for fluorescence detection is large and expensive, DNA cannot be easily detected.

  In addition, the analyte DNA has a base sequence complementary to this, and the enzyme is hybridized with a labeled DNA probe, and an optically detectable change amount generated in the temperament due to the activity of the enzyme is obtained. An automatic measuring device for measuring and a detecting method thereof are known (see Japanese Patent Application Laid-Open No. 6-225752 (Patent Document 3)). By using the apparatus described in JP-A-6-225752, a photometric process for single-stranded double-stranded DNA, hybridization, washing, and specific detection of DNA can be performed automatically. Optical equipment for photometry is required, and the detection device becomes large and expensive.

  Furthermore, after hybridization to a single-stranded nucleic acid probe complementary to a single-stranded denatured gene sample, an intercalator that is a double-stranded recognizer is added to perform electrochemical detection. There is a known method (see JP-A-5-199898 (Patent Document 4)). When the electrochemical detection method described in this method is used, an optical instrument such as fluorescence detection or photometry is not required, so that the detection device is small and inexpensive. However, in order to confirm the specificity of the detection DNA with respect to a plurality of types of probe DNA, it is necessary to immobilize one type of probe DNA on one electrode, so it is necessary to provide a plurality of electrodes. Even if a plurality of electrodes are provided on the same substrate, a wiring for connecting each electrode is necessary to apply a voltage to each electrode, and it is not possible to easily detect double-stranded DNA. .

  By the way, a solar cell that generates electric energy from light using a sensitizing dye is known (for example, see JP-A-1-220380 (Patent Document 5)). This solar cell has a polycrystalline metal oxide semiconductor and has a sensitizing dye layer formed over its surface area over a wide range.

  As an attempt to apply the characteristics of such a solar cell to biochemical analysis, there is a proposal to use a photocurrent generated by photoexcitation of a dye for detection of a test substance (biomolecules such as DNA and protein). (For example, Nakamura et al. “New DNA Double-Strand Detection Method by Photoelectric Conversion” (The Chemical Society of Japan, Proceedings Vol.81ST NO.2 (2002), page 947 (Non-patent Document 1)) and JP-A-2006- See 119128 (Patent Document 6) However, these proposals mainly relate to a method for detecting a chip after hybridization of DNA, and do not describe a detailed apparatus configuration at the time of detection.

  When using the photocurrent generated by photoexcitation of the dye for specific detection of the test substance, the entire semiconductor electrode substrate and the counter electrode may be directly connected to an ammeter (no wiring for each detection spot is required), and each spot The proposal which detects an electric current by light irradiation to is made (refer to WO07 / 037341 (patent document 7)). In this proposal, in the light irradiation to each spot, a plurality of detection spots formed on the semiconductor electrode are sequentially irradiated. In this detection method, the entire semiconductor electrode substrate and an ammeter are connected. Since they are connected, in order to accurately detect the current value generated at each detection spot, it is necessary to design a light irradiation mechanism so that light is not irradiated to adjacent detection spots. Therefore, when using a laser with high directivity or an LED with low directivity as the light source used for the light irradiation mechanism, a condensing lens or the like is used. When designing a device capable of this detection method, the light irradiation mechanism installs a light source for each detection spot, connects the light source to an XY movement mechanism that can move the light source, and sequentially moves the light source to the detection spot. , Etc. can be considered. However, in the former method, it is necessary to prepare a plurality of expensive lasers and condenser lenses, and it is difficult to cope with detection of an integrated electrode with a narrow detection spot interval. In the latter method, the XY moving mechanism increases the overall shape of the apparatus, and is not suitable for downsizing the apparatus. Furthermore, in the former method, the light source is turned on for each spot, and it is necessary to wait until the light intensity of the light source becomes stable in each spot in order to perform accurate current detection of each detection spot. There is a problem that it takes time to complete the detection.

JP-A-7-107999 Japanese Patent Laid-Open No. 11-315095 JP-A-6-225752 JP-A-5-199898 Japanese Patent Laid-Open No. 1-220380 JP 2006-119128 A WO07 / 037341

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)

In the detection using the photocurrent generated by photoexcitation of the sensitizing dye, the present inventors have now accurately performed detection of double-stranded DNA using the photocurrent generated by photoexcitation of the sensitizing dye with a plurality of spots. We have gained knowledge to do well in a short time.

  Therefore, the present invention can solve the problem of accurately detecting a plurality of spots in a short time without using a large apparatus, and is used for specific detection of a test substance using photocurrent. Its purpose is to provide a device.

That is, according to the present invention, a working electrode in which a probe substance capable of binding to a test substance is separated and supported by a plurality of spots separated from each other is attached, and a photocurrent generated by photoexcitation of a sensitizing dye is detected. A measuring device used for specific detection of a test substance used,
A plurality of light sources for irradiating the spot with light;
An ammeter for measuring a photocurrent generated by photoexcitation of a sensitizing dye, and
The light source is provided on at least the number of columns or rows on an extension of the center line corresponding to the spot,
There is provided a measuring apparatus comprising a light source moving mechanism for moving the light source in an extension line direction of the center line.
Thereby, it becomes possible to irradiate each spot arranged two-dimensionally by moving the light source only in one axis direction, and the apparatus can be simplified and miniaturized. In addition, by moving the light source while it is lit, accurate detection can be performed in a short time.

In addition, according to the present invention, there is provided a measuring apparatus in which the light source can be moved to a position where the light from the light source is not irradiated to the spot by the light source moving mechanism.
Since the light source can be moved to a position where no light is irradiated to the spot, the light source can be turned on at this position to stabilize the light intensity.

In addition, according to the present invention, there is provided a measuring apparatus in which the light source is a pigtail laser.
By using a pigtail laser as a light source, it becomes possible to irradiate light with high directivity, and unintentional light irradiation to neighboring spots can be reduced. Thereby, the noise current can be reduced.

Further, according to the present invention, a pressing member is provided on the side of the working electrode opposite to the surface on which the spot is formed to hold the sensor cells in close contact with each other, and the pressing member emits light for photoexcitation. There is provided a measuring apparatus including a plurality of openings or light-transmitting portions for passing through.
Thereby, it is possible to irradiate each spot with light from the light source while keeping the sensor cell in close contact. By stabilizing the light applied to each spot, the generated photocurrent can be detected stably.

In addition, according to the present invention, there is provided a measuring apparatus in which the pressing member has light shielding properties except for the opening and the light transmitting portion.
Thereby, when irradiating light to a spot, it can suppress that light leaks to a nearby spot and an unexpected photocurrent is generated. In addition, it is possible to prevent unnecessary light current from being generated by irradiating a plurality of spots with light while the light source is moving.

Further, according to the present invention, there is provided a measurement method for performing specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A first light irradiation step of turning on the first light source among the light sources and irradiating light to the spot at the corresponding position while moving in the first direction on an extension of the center line,
A second light irradiation step of illuminating the spot at the corresponding position while turning on the second light source among the light sources and moving in the second direction on the extension of the center line;
Is provided.
This makes it possible to irradiate a spot on the extension line of two different center lines by reciprocating once by irradiating light while moving in the second direction opposite to the first direction. Thus, the measurement time can be shortened.

Further, according to the present invention, there is provided a measurement method in which, in the light irradiation step, the light source is turned on at a position where the spot is not irradiated with light.
This makes it possible to stabilize the light intensity by illuminating the light source at a position where no light is irradiated to the spot, and to irradiate each spot with stable light, and to stably detect the generated photocurrent. Become.

According to the present invention, light irradiation is performed on each working electrode in which a plurality of probe substances capable of specifically binding directly or indirectly to a test substance on the same surface are fixed. Since the number of times of stabilizing the light intensity of each light source is continuously reduced, the detection time can be greatly shortened.
In addition, the number of light sources can be reduced to the number of columns or rows, and light can be irradiated to all detection spots with a single-axis moving mechanism, so that the overall shape of the apparatus can be reduced.

It is a top view which shows an example of the measuring apparatus of this invention. It is a figure which shows the piping diagram used for this invention, (a) injects the test substance directly into the sensor cell 1, (b) injects the sample containing the test substance in the upstream of the sensor cell 1. It is a figure which shows a case. It is a schematic diagram which shows the movable light source 3 and the sensor cell 1 in this invention. It is a time chart of each member of the movable light source 3 in this invention. It is the figure which showed the mode of the movement of the movable light source 3 in this invention typically, (a) is before the light irradiation start to the a-axis spot, (b) is the light irradiation to the a-axis spot. It is a figure which shows a state, respectively after (c) light irradiation completion | finish to the spot of an a axis | shaft. It is a figure which shows an example of the sensor cell 1 used for this invention, Fig.6 (a) is a block diagram of a working electrode, FIG.6 (b) is before a working electrode attachment, FIG.6 (c) is when a working electrode is attached. FIG. 6D is a state diagram after the working electrode is attached, FIG. 6E is a configuration diagram of a sensor cell in which the working electrode and the counter electrode are fixed and a flow path is formed, and FIG. The side views of FIG. It is a figure which shows how to flow the fluid in the sensor cell 1 used for this invention, Fig.7 (a) is sectional drawing of the sensor cell 1 seen from the light source side, FIG.7 (b) is a side view. It is the top view and sectional drawing which show an example of another aspect of the temperature control mechanism used for this invention. It is sectional drawing which shows an example of the spacer which is a sensor cell base material which has a fluid guidance | induction function, (a) is a case where a test substance is directly inject | poured into the sensor cell 1, (b) is a test in the upstream of a sensor cell. It is a figure which shows the case where the sample containing a substance is inject | poured. It is a figure which shows an example of the 2nd aspect of the sensor cell 1 used for this invention. 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.

Measuring Device The basic features of the device and detection method according to the present invention will first be described with reference to the drawings. As shown in FIG. 1, a measurement device that uses a photocurrent generated by photoexcitation of a dye for specific detection of a test substance has a basic configuration, as shown in FIG. 1, and a fluid supply means for supplying fluid to the sensor cell 1. 2, a movable light source 3 for irradiating light to a spot 323 carrying a probe substance provided on an electron accepting layer of a working electrode provided in the sensor cell 1, and a current generated by photoexcitation are detected Used to detect a photocurrent generated by photoexcitation after a hybridization reaction or a binding reaction for reacting a test substance with a probe substance that specifically binds on the working electrode. A solution holding unit 5 for holding a liquid, a temperature adjustment control unit 6 for controlling a temperature adjustment function at the time of reaction and detection provided in the sensor cell 1, a power source 7, and a temperature rise in the apparatus Comprising a fan 8 to prevent. In the illustrated apparatus, a syringe pump 20 is used as the fluid supply means 2 and a switching valve 21 is connected. The sensor cell 1, fluid supply means 2, moving light source 3, ammeter 4, temperature adjustment control unit 6, power source 7, etc. are connected to a terminal 41. As will be described later, the sensor cell 1 is supported by being divided into a plurality of spots 323 in which probe substances capable of binding to the test substance are separated from each other on the electron accepting layer. . By forming a plurality of spots 323, a plurality of measurements can be performed simultaneously.

  FIG. 2 is a diagram showing a piping diagram of the flow path configuration based on the fluid supply means. 2A shows a case where a sample containing a test substance is directly injected into the sensor cell 1, and FIG. 2B shows a case where a sample containing the test substance is injected upstream of the sensor cell 1. FIG.

  In FIG. 2A, as a fluid supply means, a switching valve 21 communicated with the syringe pump 20 includes a hybridization reaction solution tank 51, a post-reaction washing solution tank 52 used for subsequent washing, and a current detection. An electrolyte tank 53 for use, a post-measurement cleaning solution tank 54 for cleaning the sensor cell 1 and each pipe after current measurement, and air connected to an opening to the atmosphere when air is used as a fluid The supply port 22, the sensor cell 1 provided with the injection port 101 for injecting the sample containing the test substance, and the waste liquid tank 56 are connected by piping. The waste liquid tank 56 is also connected to the fluid discharge port 14 provided in the sensor cell 1 by piping.

  FIG. 2B shows piping in a mode in which a sample containing a test substance is injected on the upstream side of the sensor cell. In this configuration, the switching valve 21 communicated with the syringe pump 20 as a fluid supply means includes a post-reaction cleaning solution tank 52 used for cleaning after hybridization, and an electrolyte solution tank used for current detection. 53, a post-measurement cleaning solution tank 54 for cleaning the sensor cell 1 and each pipe after current measurement, an inlet 23 for injecting a sample containing a test substance, and the atmosphere when air is used as a fluid Are connected to the air supply port 22 connected to the open port, the fluid supply port 15 provided in the sensor cell 1, and the waste liquid tank 56 by piping. The waste liquid tank 56 is also connected to the fluid discharge port 14 provided in the sensor cell 1 by piping.

  2A and 2B, the fluid is supplied to and discharged from the sensor cell by switching the switching valve 21 to each pipe. Further, it is more preferable that the hybridization reaction solution tank 51, the post-reaction washing solution tank 52 used for the subsequent washing, and the electrolytic solution tank 53 for detection have a temperature adjusting mechanism so as to be maintained at a set temperature. . In the post-measurement cleaning solution tank 54 for cleaning the sensor cell 1 after the current measurement, alcohol, water, or the like is prepared as a cleaning solution. In consideration of cleaning performance and waste liquid treatment, it is preferable to provide a plurality of tanks of ethanol and water.

  In the apparatus configuration of FIGS. 1 and 2A, the hybridization reaction and detection of the test substance will be described. First, a reaction solution is supplied from the hybridization reaction solution tank 51 into the sensor cell 1. Next, a test substance for hybridization reaction is supplied from the sample supply port 101 provided in the sensor cell 1 and directly with the test substance provided on the working electrode in the sensor cell 1 adjusted to the set temperature. Alternatively, a hybridization reaction is performed with a probe substance that can specifically bind indirectly. Thereafter, a cleaning solution for cleaning unbound substances is supplied from the cleaning solution tank 52.

  In this washing, it is necessary to remove only the unbound substance while the test substance bound to the probe substance remains bound. Therefore, it is preferable to add a surfactant to the cleaning solution. However, reaction bubbles on the surface of the working electrode are generated when bubbles generated by supplying and discharging the cleaning fluid remain on the probe provided on the working electrode in order to perform reaction and detection on the working electrode in the sensor cell 1. Therefore, attention must be paid to the generation and remaining of bubbles. In consideration of the washing efficiency, it is preferable to use a washing solution containing 0.001% or more and 10% or less of a surfactant as a washing solution used after hybridization.

  After cleaning, the cleaning solution is discharged from the sensor cell 1 through the discharge port 102, and then the electrolyte is supplied from the electrolyte tank 53, and the light from the movable light source 3 enters the sensor cell 1 adjusted at a temperature lower than that of the hybridization solution. Is measured, and the current value accompanying photoexcitation is measured.

FIG. 3 shows a schematic diagram of the movable light source 3. The movable light source 3 includes a laser mounting arm 810 having light sources 801, 802, and 803 for emitting a pigtail laser at the tip, and a light source moving mechanism 820 for moving the laser mounting arm 810 in one axial direction. Yes. At this time, the light source 801 is located on the extension of the center line corresponding to the spot 323 shown as the a-axis in the drawing. Similarly, the light sources 802 and 803 are located on the b-axis and the c-axis corresponding to the spot 323, respectively.
The light source may be any light source that can irradiate light having a wavelength that can be photoexcited, and various light sources other than the pigtail laser can be used. In addition, it is preferable that what is used as a light source has the directivity of the light to irradiate. Moreover, it is preferable that only a specific wavelength used for photoexcitation can be irradiated.

  When the laser mounting arm 810 moves in the uniaxial direction, the spot 323 provided on the working electrode 320 is sequentially irradiated with light, and the current value can be detected. At this time, the spots 323 are preferably provided on the working electrode 320 in a substantially rectangular shape so as to form rows and columns. By providing the spot 323 in this manner, the movable light source 3 can be moved efficiently.

The light irradiation will be described in detail below. FIG. 4 shows a time chart of each member, and FIG. 5 shows a schematic diagram of the movable light source 3 and the sensor cell 1 during each operation.
First, as shown in FIG. 5 (a), the light source moving mechanism moves to a position where the laser irradiated from the light sources 801, 802, and 803 is irradiated onto the position A on the working electrode 320 where no spot is provided. The laser mounting arm 810 is moved by 820. This state is the timing indicated by a in the time chart of FIG. At this timing, the light source 801 is turned on to stabilize the light intensity.
After the light intensity is stabilized, as shown in FIG. 5B, the light source moving mechanism 820 is operated for a certain period of time so that the laser emitted from the light source 801 reaches the spot position Ba on the working electrode 320. The laser mounting arm 810 is moved. Thereby, the laser beam from the light source 801 is irradiated to the spot Ba. Similarly, the light source moving mechanism 820 is operated for a certain period of time, and the laser mounting arm 810 is sequentially moved in the right direction as indicated by the arrow in the drawing from Ba to Ea, and exists on the a axis. Irradiate each spot with laser light.

  After that, as shown in FIG. 5C, the laser mounting arm 810 is moved to the position where the laser irradiated from the light sources 801, 802, 803 is irradiated to the position F on the working electrode 320 where no spot is provided. Moving. Here, as indicated by b in the time chart of FIG. 4, the light source 801 is turned off and the light source 802 is turned on to stabilize the light intensity.

  After the light intensity is stabilized, the light source moving mechanism 820 is operated in the reverse direction, and the laser mounting arm 810 is moved in the left direction in the figure, whereby the laser light from the light source 802 is sequentially irradiated from the spots Fb to Bb. To do. Thereafter, the laser mounting arm 810 is moved to a position where the laser irradiated from the light sources 801, 802, 803 is irradiated on the working electrode 320 to the position A where no spot is provided. Here, as indicated by c in the time chart of FIG. 4, the light source 802 is turned off and the light source 803 is turned on to stabilize the light intensity. After the light intensity is stabilized, by moving the light source moving mechanism 820 in the forward direction to move the laser mounting arm 810 in the right direction in the figure, the laser light from the light source 803 is changed from cB to cE. Irradiate the spot.

By moving the light source only in one axial direction by the above procedure, it is possible to irradiate light to a plurality of spots provided two-dimensionally on the working electrode. In addition, since current measurement can be performed while the laser at the time of light irradiation at each detection spot is continuously lit, stable detection is possible as compared with a device that is lit at each detection spot.
Although an example in which spots are arranged in 3 rows and 4 columns is shown here, the number of spots is not limited to this. For example, the number of spots such as 3 rows and 5 columns and 3 rows and 6 columns can be increased. In this case, it is only necessary to prepare as many light sources as the number corresponding to each column or row.

  After completion of the measurement, the electrolytic solution in the sensor cell 1 is discharged to the waste liquid tank 56 by a syringe pump. In order to clean the electrolyte remaining in the sensor cell 1, the cleaning solution is supplied from the cleaning solution tank 54 a plurality of times to clean the inside of the sensor cell 1 including the counter electrode surface. By washing the inside of the sensor cell 1 after the measurement is completed, the next reaction and detection can be performed continuously by replacing the working electrode without replacing the counter electrode. In this configuration, since the sample does not come into contact with other piping by directly injecting the sample containing the test substance into the sensor cell 1, the occurrence of contamination in the device configuration in which the reaction and detection are performed multiple times. Can be prevented and stable detection can be performed.

  Next, hybridization reaction and detection of a test substance in the configuration of FIG. First, a test substance is mixed with a hybridization solution in a sample tube before injection of the apparatus. Thereafter, a hybridization solution containing a test substance is injected into the injection port 23 and flows into the sensor cell 1 by a syringe pump. Thereafter, the same processing as in FIG. 2A can be performed. In this configuration, the apparatus does not have to be provided with the hybridization reaction solution tank 51, so that the apparatus becomes compact.

  The structure of the sensor cell 1 in the present invention is shown in FIGS. 6A, B, C, D, E, and F. FIG. 6A is a configuration diagram of the working electrode case, FIG. 6B is a state diagram before attaching the working electrode case, FIG. 6C is a state diagram when the working electrode case is attached, FIG. 6D is a state diagram after the working electrode case is attached, and FIG. And FIG. 6F shows a top view of FIG. 6D, respectively.

  As shown in FIG. 6A, the working electrode 320 used in the present invention includes a conductive substrate 321 and an electron accepting layer 322 that can accept electrons emitted from the sensitizing dye in response to photoexcitation thereon. It is an electrode and is arranged so that the electron-accepting layer side is exposed to the flow path. The electron accepting layer 322 includes a spot 323 carrying a probe substance capable of specifically binding directly or indirectly to a test substance. The working electrode preferably has translucency so that the electron-accepting layer can be easily irradiated with light for photoexcitation. A spacer 330 is disposed as a sensor cell base material on the flow path side of the working electrode, and is accommodated in a working electrode case 324 including a working electrode receiving member 324a and a working electrode lid member 324b. The spacer 330 has contact probe openings 331 at both ends for taking out a current generated on the working electrode. The working electrode receiving member 324a has a space 325 for transmitting light and a working electrode case positioning pin hole 326. The working electrode lid member 324b has a space 327 for bringing the counter electrode and the spacer into contact with each other and an opening 305 for a contact probe. Then, both the working electrode receiving member 324a and the working electrode lid member 324b are fixed by a fixing screw 328 at a position of a screw receiving 329 provided on the working electrode receiving member 324a to form a working electrode case 324.

  The formation of the sensor cell 1 will be described below with reference to FIGS. 6B, C, D and F. First, a pressing member for bringing the working electrode 320, the spacer 330, and the counter electrode 350 into close contact with each other is provided on the side opposite to the surface on which the electron accepting layer is formed. The pressing member preferably includes a pressing member cover 310 provided in contact with the working electrode and a working electrode heater 312 provided as a temperature adjustment mechanism. The holding member cover 310 includes a plurality of light-transmitting portions 311 for allowing light for light excitation to pass therethrough, and is thermally conductive so that the heat of the working electrode heater 312 provided on both side surfaces is uniform over the entire surface. It is preferable to consist of a high member. An opening portion may be provided instead of the light transmitting portion 311. Specifically, aluminum, S45C or the like can be used, and aluminum is preferable in consideration of strength, durability, and the like. The pressing member is fixed to the apparatus by a pressing member stage 302 that is fixed to the sensor cell base 301 provided in the apparatus main body. The pressing member stage 302 is preferably made of a material that does not allow the heat of the pressing member cover to escape to the outside. By adopting such a configuration, heat is uniformly transmitted to the entire surface of the pressing member cover, the reaction and detection on the working electrode are maintained at the set temperature, and accurate measurement is possible.

  Next, the state which attaches a working electrode to an apparatus is shown to FIG. 6C. The working electrode 320 moves to the holding member fixing stage 302 side along the working electrode guide member 306 while being included in the working electrode case 324 together with the spacer 330. And it positions according to the working electrode case positioning pin 303, and is temporarily fixed. The state after temporary fixing is shown in FIG. 6D.

  Next, as shown in FIG. 6E, when forming the sensor cell, the counter electrode side stage 307 is moved along the shaft 313, joined to the working electrode case, and the snap lock 308 and the pressing member stage 302 provided on the counter electrode stage 307 are connected. The sensor cell 1 is completed by fixing the snap lock receiver 309.

  The sensor cell 1 is provided with a working electrode 320 and a counter electrode 350 facing each other through a spacer 330 having an opening, and as a result, a flow path is formed. Then, at least one of the working electrode 320, the counter electrode 350, and the spacer 330 is provided with a fluid supply port 103 communicating with the flow path, and the discharge port 104 is generated at a position different from the supply port 103 and generated by photoexcitation. A contact probe 304 for taking out current is provided. Here, the flow path is a solution or cleaning liquid for performing a hybridization in which a test substance specifically binds to a probe substance provided on the working electrode in the sensor cell 1, a temperature adjusting fluid, and a photocurrent. It is a space for allowing fluid such as an electrolyte-containing solution to be used and flowing.

  The counter electrode 350 is provided with a counter electrode heater 351 as a second temperature adjusting mechanism so as to be in contact with the surface opposite to the surface forming the flow path. In order to adjust the temperature of the fluid in the flow channel in a short time, it is preferable to provide a temperature adjusting mechanism for both the counter electrode 350 and the working electrode 320. By adjusting the temperature in the sensor cell 1 from both the working electrode 320 and the counter electrode 350, the temperature in the sensor cell 1 can be quickly and uniformly adjusted, and is specific to the probe provided on the working electrode 320 side. The photoelectric efficiency of the sensitizing dye bonded to is improved.

  As shown in FIG. 6F, the working electrode heater 312 is provided at both ends of the pressing member so as not to block the light irradiated from the light source to the spot 323, and is arranged to have an opening at the center. Here, the opening is a region through which light passes, and a part of the working electrode heater 312 may be hollowed out, and includes a region where a plurality of working electrode heaters are separated as shown in FIG. 6F. Use for meaning. The working electrode heater 312 may be disposed at any other location as long as it does not block the light irradiated from the light source to the spot 323. In addition to providing the opening, a light transmitting portion may be provided so that light irradiation can be performed. The term “translucent portion” as used herein refers to a portion made of a member that transmits light. Furthermore, the working electrode temperature sensor 314 is disposed between the spots so as not to block the light emitted from the light source to the spots 323.

  This temperature adjustment mechanism adjusts the temperature in both hybridization and detection. When the temperature is set higher than room temperature, a mechanism capable of at least heating is required. Specifically, a ceramic heater or the like can be used as a heater for heating, and the temperature is adjusted using a resistance temperature detector, a temperature controller, or the like. In addition, according to a preferred aspect of the present invention, in consideration of shortening of the rise time to the set temperature and temperature control accuracy, both the heater for heating and the resistance temperature detector capable of detecting temperature are provided with the counter electrode. In addition, it is preferable to directly contact with the working electrode or through a member having high thermal conductivity. Further, the temperature inside the sensor cell 1, that is, the surface of the working electrode on which the probe DNA is immobilized, and the temperature indicated by the resistance temperature detector are checked in advance, and the temperature data is corrected so that the probe cell is immobilized in the sensor cell 1. It is preferable to further improve the temperature control accuracy of the working electrode surface.

  In the present invention, the sample containing the test substance is directly supplied into the sensor cell 1 from the supply port 101. After the sample is supplied, the sample supply port 101 is closed using the stopper 102.

  FIG. 7 shows how the fluid flows in the sensor cell 1 of the present invention. FIG. 7A is a cross-sectional view of the sensor cell viewed from the light source side, and FIG. 7B is a side view. A hybridization solution, a subsequent cleaning fluid, and a fluid such as an electrolyte for current detection are introduced from a fluid supply port 103 provided at the bottom of the sensor cell channel from a pipe communicating with each fluid. Thereafter, the fluid is filled into the sensor cell 1 from the bottom, and after completion of each reaction, washing, and detection, the fluid is led out to the pipe from the upper fluid discharge port 104 and discharged. In this configuration, since the fluid is introduced from the bottom and discharged to the top, the probe material provided on the working electrode 320 and capable of specifically binding directly or indirectly to the test substance is supported. Contact with the spot 323 surely. As a result, it is possible to prevent current detection errors and the like due to uneven reaction and poor electrolyte contact at the time of detection.

  FIG. 8 shows a configuration of a temperature adjustment mechanism according to another aspect of the present invention. The temperature adjustment mechanism of this aspect has a configuration in which when a counter electrode is formed, a thermocouple 502 is formed on the counter electrode substrate 501 and a conductive thin film 503 is formed thereon by sputtering or the like. . The thermocouple is connected to a conducting wire 505 connected at a contact 504 between the thermocouple and the counter electrode base material, and is controlled by a temperature adjuster so as to reach a set temperature. At this time, the counter electrode substrate 501 needs to form a flow path as a sensor cell by a working electrode (not shown), a spacer 506, a fluid supply port 507, a fluid discharge port 508, and a pressing member (not shown). . For this reason, the counter electrode base material 501 needs to be made of a material that is not stress-deformed by the pressing member, and for example, glass, SUS, resin having excellent heat resistance (PEEK, polycarbonate), or the like can be used. Furthermore, it is more desirable to select a material having high thermal conductivity so that the temperature of the entire counter electrode is not uneven. For example, the use of SUS is preferable. As the conductive thin film, Pt, carbon or the like is used, and Pt is preferable for the same reason as the counter electrode base material.

  FIG. 9 shows a configuration of another aspect of the sensor cell according to the present invention. The sensor cell includes a pressing member, a working electrode 701, a spacer 702, and a counter electrode 703, and a fluid supply port 705 and a fluid discharge port 706 are provided on the counter electrode. As the pressing member, a metal plate 709, a lid member 712, and a metal plate 709 each having an opening corresponding to the spot 323 so that light can be emitted to each spot 323 corresponding to the probe substance provided on the working electrode. And a heater 707 as a temperature adjusting member. A spring damper 710 is installed between the metal plate 709 and the lid member 712, and a space 708 for heat insulation is formed between the metal plate 709 and the lid member 712. By fixing the pressing member to the base 715 on the counter electrode side by the fixing member 713, the working electrode 701, the spacer 702, and the counter electrode 703 are connected to form a flow path. A heater 707 serving as a temperature adjusting member is provided in contact with a portion corresponding to the projection surface of the probe substance immobilization portion on the surface opposite to the flow path side of the counter electrode. The heater 707 is included in a base 715 provided with a flow path for supplying and discharging fluid, and a space 708 for insulating heat from the outside is formed in the same manner as the heater provided in the pressing member. ing. The contact probe 704 that penetrates the lid member is connected to the working electrode by fixing the pressing member, and is configured to take out a photocurrent. By contacting a metal plate with high heat conduction efficiency and a heater, and setting the working plate with a metal plate that has a uniform set temperature across the entire surface, the set temperature at the time of hybridization differs from that at the time of detection. In addition, the temperature adjustment in the vicinity of the probe-immobilized portion on the working electrode can be performed more accurately in a short time.

  According to a preferred aspect of the present invention, when detecting the photocurrent after hybridization, it is preferable to supply a temperature adjusting fluid before feeding a detection solution containing an electrolyte for detection. In the photocurrent detection method using a sensitizing dye bonded to a probe substance provided on the working electrode in the sensor cell 1, the photoelectric efficiency and the conductive base material are obtained by making the temperature in the sensor cell 1 uniform during detection. The influence of the resistance of the current on the current value can be suppressed. Further, when the photocurrent is detected, the inside of the sensor cell 1 is filled with the adjusted temperature adjusting fluid, so that the temperature inside the sensor cell 1 can be uniformly adjusted and stable photocurrent detection can be performed. It becomes.

  In the above aspect, when air is used as the temperature adjusting fluid, air is supplied into the sensor cell 1 set to a temperature higher than room temperature using a fluid supply pump. Further, as the temperature adjusting fluid, a washing reagent for washing unbound test substance after hybridization or an electrolytic solution used for detecting photocurrent may be used.

  According to a preferred embodiment of the present invention, when detecting the photocurrent after hybridization, it is preferable to detect the photocurrent at a temperature lower than that in the hybridization step. This increases the photoelectric efficiency of the sensitizing dye and the electronic efficiency of the conductive substrate of the working electrode. Usually, hybridization is mainly performed at a temperature higher than room temperature. When the temperature in the sensor cell 1 is lowered after hybridization, the heating function is stopped so as to naturally cool, the temperature adjusting fluid is supplied into the sensor cell 1, or the function is forcibly cooled. A mechanism having the above may be provided, or a combination thereof may be provided. When the heating function is stopped so as to naturally cool, it is preferable to supply the temperature adjusting fluid to the sensor cell 1 in addition to that because the temperature in the sensor cell 1 becomes constant in a short time.

  According to a preferred embodiment of the present invention, it is preferable to supply a temperature adjusting fluid before the hybridization step. Thereby, the efficiency of a hybridization process can be raised. At this time, as the temperature adjusting fluid, air, a sample solution containing a test substance, a hybridization reaction solution, or the like can be used.

  According to a preferred aspect of the present invention, it is preferable to keep the temperature in the sensor cell 1 constant in the photocurrent detection step. Thereby, the influence which photoelectric efficiency and the resistance of an electroconductive base material have on an electric current value can be suppressed, and the stable electric current measurement can be performed.

  According to a preferred aspect of the present invention, the sensor cell substrate has a configuration in which a fluid guiding function is provided for reliably discharging the fluid from the sensor cell 1. FIG. 8 shows a cross-sectional view of a spacer 330 which is a sensor cell substrate having a fluid guiding function according to the present invention. FIG. 8A shows a case where a test substance is directly injected into the sensor cell 1 and the injection port 101 (B) is a diagram showing a spacer 330 used when injecting a sample containing a test substance upstream of a sensor cell. By setting it as such a structure, the forced flow path with respect to the fluid supplied in the sensor cell 1 can be made, and replacement | exchange of the fluid in the sensor cell 1 can be performed more efficiently and reliably. Specifically, it is preferable that the cross-sectional area of the flow path is narrowed toward the downstream side of the opening of the spacer 330 forming the flow path. More preferably, the cross-sectional area of the flow path is increased from the fluid supply port 103 provided in the sensor cell 1 to the spot 323 portion where the probe provided on the working electrode is fixed, and the flow passage is cut downstream from the spot 323 portion. Reduce the area. With this configuration, when air and a solution are used as fluids to be supplied into the sensor cell 1, even if air remains in the sensor cell 1 at the time of fluid exchange in the sensor cell 1, the next fluid is surely placed in the spot 323 portion. Is satisfied and replaced. For this reason, temperature unevenness is eliminated, and the influence of variations in measured values at the time of current detection is reduced.

  According to a preferred aspect of the present invention, it is preferable that a pressing member that presses the working electrode, the counter electrode, and / or the sensor cell base material is brought into close contact with each other, and the temperature adjusting mechanism is provided on the pressing member. In such a configuration, since the constituent members of the sensor cell are pressed and brought into close contact with each other, leakage of a liquid such as an electrolytic solution can be effectively prevented. At the same time, the temperature adjustment mechanism and the sensor cell can be brought into close contact with each other, and temperature adjustment can be performed efficiently.

  According to a preferred aspect of the present invention, the pressing member is made of a material having a higher thermal conductivity than the working electrode. With this configuration, heat transfer to the working electrode is accelerated, and temperature adjustment by the temperature adjustment mechanism can be performed quickly and uniformly.

  According to a preferred aspect of the present invention, the pressing member further includes at least one fixing means selected from the group consisting of a screw, a spring, and a magnet, and the pressing member, the working electrode, and the counter electrode via the fixing means. And / or detachably fixed to the sensor cell substrate. By using this coupling means, the constituent members of the cell can be more closely attached, and the solution in the sensor cell 1 can be reliably held. In addition, since the coupling means can easily remove each member, the working electrode and the like can be easily replaced and maintained.

  According to a preferable aspect of the present invention, it is preferable that the pressing member shields a portion other than the opening and the light transmitting portion. With this configuration, when light from the light source is irradiated to the opening and the light transmitting part, generation of a photocurrent due to a test substance other than the irradiation target due to light leakage to the other opening and light transmitting part is prevented. It can be surely prevented, and an accurate photocurrent value specific to the test substance can be detected.

  According to a preferred aspect of the present invention, it is preferable that a light source is provided outside the opening of the pressing member or the translucent part. With this configuration, the photocurrent can be detected without moving the sensor cell after the hybridization reaction for detection.

  According to a preferred aspect of the present invention, the pressing member has a plurality of the opening portions or the light transmitting portions, and has a light shielding property except for the opening portions and the light transmitting portions. The plurality of light sources are individually provided for each of the opening and the light transmitting portion. According to this configuration, since the plurality of light sources are provided at positions corresponding to the plurality of probe immobilization units to which the test substance is provided provided on the working electrode, as compared with the aspect in which the light source is moved earlier. Without the driving means, detection can be performed while the sensor cell including the connection part and the fluid holding part for supplying a plurality of fluids such as the electrolyte-containing solution is fixed.

  According to a preferred aspect of the present invention, the distance between the working electrode and the counter electrode is preferably 0.1 to 3 mm. The volume of the sensor cell is preferably a small amount of the test substance when performing the hybridization reaction, but the amount of the solution is constant in consideration of the diffusion efficiency of the solution with respect to the solid surface to the probe substance provided on the working electrode. This is necessary. According to this configuration, the hybridization reaction can be completed in the same sensor cell 1 in a short time, and the photocurrent can be reliably detected.

  According to a preferred embodiment of the present invention, the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye is performed on the surface of a probe substance capable of specifically binding directly or indirectly to the test substance. Supplying a sample solution containing a test substance combined with a sensitizing dye into a sensor cell 1 in which a working electrode and a counter electrode fixed to each other form a flow path via an insulating spacer; and supplying the sample solution to the working electrode A hybridization step in which the test substance is specifically bound to the probe substance directly or indirectly under temperature adjustment, and the sensitizing dye is fixed to the working electrode by the binding; and the sensor cell 1 Photoexcitation of the sensitizing dye at a temperature different from the step of cleaning the inside, the step of supplying an electrolyte solution into the sensor cell 1, and the step of irradiating the working electrode with light to specifically bind to the former. And a step of detecting a photocurrent flowing between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. . By using this measurement method, the temperature in the sensor cell 1 controlled at a temperature suitable for the hybridization step can be changed to a temperature suitable for detection, so that the detection accuracy in the specific detection of the test substance can be improved. It becomes possible to improve. Moreover, since the hybridization step and the photocurrent detection step can be performed in the same sensor cell 1, workability can be greatly improved.

  According to a preferred embodiment of the present invention, the photocurrent detection step is performed at a lower temperature than the hybridization step. More preferably, the photocurrent detection step is preferably performed at a temperature lower by 3 ° C. or more than the hybridization step, and is preferably performed at 20 ° C. or more and 70 ° C. or so. This temperature setting is determined from the optimum temperature in consideration of the photoelectric efficiency necessary for photocurrent detection and the resistance value related to the electron transfer of the conductive substrate. By setting such a temperature range, detection with high measurement accuracy can be performed.

Electrolyte-containing solution The electrolyte-containing solution used in the present invention preferably comprises an electrolyte, at least one solvent selected from an aprotic solvent and a protic solvent, and optionally an additive. The electrolyte is not limited as long as it can move freely in the water-containing substrate 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. 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, 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 sodium sulfite Thorium (Na ₂ SO ₃ ), and a mixture thereof, more preferably tetrapropylammonium iodide (NPr ₄ I). The solvent is an aprotic solvent, a protic solvent, or a mixture thereof. That is, a polar solvent system in which buffer components are mainly mixed with water and 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.

Washing Solution According to a preferred embodiment of the present invention, it is preferred that after the hybridization step, the spot 323 is washed by supplying a washing solution a plurality of times in order to wash unbound substances. As a result, the unbound substance can be removed, and only the test substance bound to the probe substance on the spot 323 can be left.

  According to a preferred embodiment of the present invention, a surfactant is preferably added to the washing solution used after hybridization, and more preferably 0.001% to 10% of the surfactant is added. By incorporating the surfactant in an amount of 0.001% or more, the cleaning efficiency can be increased. By setting the surfactant to 10% or less, the remaining of bubbles generated by supplying and discharging the cleaning fluid on the probe provided on the working electrode for performing the reaction and detection on the working electrode in the sensor cell 1 Can be suppressed, and uneven reaction on the surface of the working electrode can be suppressed.

  According to a preferred aspect of the present invention, it is preferable to wash the inside of the sensor cell 1 including the counter electrode surface by supplying the washing solution a plurality of times even after the photocurrent detection step. Since the inside of the sensor cell 1 can be cleaned after the measurement is completed, the next reaction and detection can be performed continuously only by replacing the working electrode without replacing the counter electrode.

Light source In the present invention, the light source used is not limited as long as it can irradiate light having a wavelength capable of photoexciting the labeling dye. Preferred examples include fluorescent lamps, black lights, sterilizing lamps, and incandescent lamps. , Low-pressure mercury lamp, high-pressure mercury lamp, xenon lamp, mercury-xenon lamp, halogen lamp, metal halide lamp, LED (white, blue, green, red), laser (CO2 laser, dye laser, semiconductor laser), using sunlight More preferable examples include fluorescent lamps, incandescent lamps, xenon lamps, halogen lamps, metal halide lamps, LEDs (white, blue, green, red), and sunlight. Moreover, you may irradiate only the light of a specific wavelength range using a spectroscope and a band pass filter as needed.

  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.

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. The specific binding process of the test substance to the working electrode in this embodiment is shown in FIGS. 8 (a) and 11 (b). As shown in these figures, a single-stranded nucleic acid 1 as a test substance is hybridized with a single-stranded nucleic acid 64 having complementarity as a probe substance carried on the working electrode 63, and A double-stranded nucleic acid 67 is formed.

  When a single-stranded nucleic acid is used as a test substance, it only needs to have a portion complementary to the nucleic acid that is the probe substance, and the length of the base pairs constituting the test substance is not limited. It preferably has a complementary portion of 10 bp or more. According to the method of the present invention, even when a nucleic acid having a base length of 200 bp, 500 bp, and 1000 bp is relatively long, a specific binding formation between the nucleic acid of the probe substance and the test substance can be performed with high sensitivity. Can be detected as

  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. In general, a test substance labeled with a sensitizing dye is obtained by gene amplification using a PCR primer labeled with a sensitizing dye at the 5 'end. In addition, a nucleic acid can be labeled with a sensitizing dye by incorporating dUTP labeled with a sensitizing dye or aminoallyl-modified dUTP. When aminoallyl-modified dUTP is incorporated, the sensitizing dye activated by N-hydroxysuccinimide reacts specifically with the modified dUTP in the next coupling step, and is uniformly sensitized dye. A labeled test substance 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. Moreover, alkali denaturation can be performed to prepare single-stranded nucleic acids.

  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 process of specific binding of the test substance to the working electrode in this embodiment is shown in FIG. As shown in FIG. 12, a ligand 100 as a test substance first specifically binds to a receptor protein molecule 101 that is a mediator. The receptor protein molecule 103 to which the ligand is bound specifically binds to the double-stranded nucleic acid 104 as the probe substance.

  According to the present invention, a target substance can be obtained by simultaneously reacting a single probe substance with a plurality of identical test substances derived from different acquisition routes and determining the difference in the amount of the test substance depending on the origin of the sample. It is also possible to quantify the test substance derived from the route. Specific application examples include expression profile analysis by competitive hybridization on a microarray. In this method, in order to analyze the difference in expression pattern of a specific gene among cells, a test substance labeled with different fluorescent dyes is competitively hybridized with the same probe substance. In the present invention, by using such a technique, an advantage that has not been obtained in the past can be obtained, in which an expression difference analysis between cells can be performed electrochemically.

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 specific to the probe substance in the presence of the sensitizing dye. The sensitizing dye is fixed to the working electrode by the binding. Therefore, in the present invention, the test substance 91 or the mediating substance 101 can be labeled with a sensitizing dye 92 in advance as shown in FIGS. In addition, as shown in FIG. 11B, when using a sensitizing dye 98 that can be intercalated with a conjugate 96 (for example, a double-stranded nucleic acid after hybridization) of a test substance and a probe substance, By adding a sensitizing dye to the solution, the sensitizing dye can be fixed to the probe substance.

  According to a preferred embodiment of the present invention, when the test substance is a single-stranded nucleic acid, it is preferable to label one sensitizing dye per molecule of the test substance. The labeling position in the single-stranded nucleic acid is either the 5 ′ end or the 3 ′ end of the single-stranded nucleic acid from the viewpoint of easily forming a specific bond between the test substance and the probe substance. From the viewpoint of further simplifying the labeling step, the 5 ′ end of the test substance is more preferable.

  According to another preferred embodiment of the present invention, it is preferable to label two or more sensitizing dyes per molecule of the test substance in order to increase the carrying amount of the sensitizing dye per molecule of the test substance. As a result, the amount of dye supported per unit specific surface area of the working electrode on which the electron accepting material is formed can be increased, and the photocurrent response can be observed with higher sensitivity.

  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.

  In the embodiment in which individual detection of a plurality of test substances is performed, the sensitizing dye that labels each test substance may be any one that can be excited by different wavelengths of light. It is only necessary that each test substance can be excited individually by selecting. For example, when a plurality of sensitizing dyes corresponding to a plurality of test substances are used and light of different excitation wavelengths is irradiated for each sensitizing dye, signals are detected individually even if the plurality of probes are on the same spot 323. It becomes possible to do. In the present invention, the number of test substances is not limited, but considering the wavelength of light emitted from the light source and the absorption characteristics of the sensitizing dye, 1 to 5 types may be appropriate. The sensitizing dye that can be used in this embodiment only needs to be photoexcited in the wavelength region of the irradiation light, and the absorption maximum is not necessarily in the wavelength region. In addition, the presence or absence of the light absorption reaction of the sensitizing dye at a specific wavelength can be measured using an ultraviolet-visible spectrophotometer (for example, UV-3150 manufactured by Shimadzu Corporation).

  Specific examples of the sensitizing dye include metal complexes and organic dyes. Preferred examples of the metal complex 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 (for example, cis-dicyanate-bis (2,2′-bipyridyl-4,4′-dicarboxylate) ruthenium (II)) and europium complexes are exemplified. Preferred examples of organic dyes include metal-free phthalocyanine, 9-phenylxanthene dyes, cyanine dyes, metalocyanine dyes, carbocyanine dyes, xanthene dyes, triphenylmethane dyes, acridine dyes, and oxazine dyes. , Coumarin dyes, merocyanine dyes, rhodacyanine dyes, polymethine dyes, indigo dyes, and the like. As another preferred example of the sensitizing dye, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, Cy9 manufactured by GE Healthcare Life Sciences; AlexaFluor 355, AlexaFluor 405 manufactured by Molecular Probes, Inc. , AlexaFluor430, AlexaFluor488, AlexaFluor532, AlexaFluor546, AlexaFluor555, AlexaFluor568, AlexaFluor594, AlexaFluor633, AlexaFluor647, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750; Dyomics manufactured by DY-610, DY-615, DY-630, DY-631, Y-633, DY-635, DY-636, EVOblue10, EVOblue30, DY-647, DY-650, DY-651, DYQ-660, DYQ-661 and the like.

  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.

Working electrode and production thereof The working electrode used in the present invention is an electrode having the probe substance on its surface, and the sensitizing dye immobilized via the probe substance emits electrons emitted in response to photoexcitation. It is an acceptable 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 93 shown in FIGS. 11 and 12 includes a conductive base material 95 and an electron accepting layer 96 formed on the conductive base material 95 and containing an electron accepting substance. A probe substance 94 is carried on the surface of the electron accepting layer 96.

  The electron-accepting layer 96 in the present invention includes an electron-accepting material that can accept electrons emitted by the sensitizing dye fixed through the probe material 94 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) in 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. . 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.

  According to a preferred embodiment of the present invention, the electron accepting material is titanium oxide, zinc oxide, tin oxide, niobium oxide, strontium titanate, indium oxide, indium-tin composite oxide (ITO), fluorine-doped tin oxide. It is preferably at least one selected from the group consisting of (FTO). Most preferably, TiO2, indium-tin composite oxide (ITO) or fluorine-doped tin oxide (FTO) can be used. 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 is used as the electron-accepting substance, the semiconductor may be either single crystal or polycrystalline, but a polycrystalline body is preferable, and a porous body is more preferable than a dense body. 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. 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.

  With the concavo-convex structure, the detection sensitivity can be increased by increasing the surface area of the electron-accepting layer and immobilizing many probe molecules. Since the size of the biomolecule is about 0.1 to 20 nm, the pore diameter formed by the concavo-convex structure is preferably 20 nm or more and 150 nm or less. If the entrance of the space generated by the concavo-convex structure is less than that, the specific surface area increases, but the biomolecule cannot be bonded to the probe, and the detection signal decreases. If the irregularities are rough, the surface area will not increase so much and the signal intensity will not increase that much. A more preferable range suitable for sensing biomolecules is 50 nm or more and 150 nm or less.

  According to a preferred aspect of the present invention, it is preferable to employ a pillar structure in which nanoscale pillars (pillars) are regularly arranged on the surface as the convex structure. Various production methods are known, but a method using anodized alumina having nanometer-scale pores as a template is common. There are a method of removing the alumina mold by etching after filling the mold with a ceramic sol and heat treatment, and a method of performing a heat treatment after releasing the filled ceramic sol from the mold. Examples of a method for producing a pillar-shaped nanostructure include a method for producing a nanostructure on a transparent substrate and a transparent conductive layer described in JP-A-2004-130171. Here, a method is used in which a mold of anodized alumina is filled with titania sol, heat-treated at 300 to 400 ° C., and then removed by etching. As a result, titania nanotubes and nanowires are formed as nanostructures.

According to a preferred embodiment of the present invention, it is preferable to employ pores generated as a concave structure by firing an inorganic-organic hybrid precursor containing a ceramic component to form a gas phase by oxidative decomposition of organic matter. The inorganic-organic hybrid precursor is a compound in which an organic polymer, a metal-oxygen network structure produced by oxidation of an organometallic compound (metal alkoxide) and subsequent polycondensation reaction coexist. In addition, there is a method of adding an organic polymer or the like to commercially available titanium oxide particles (for example, anatase type crystals manufactured by Teika Co., Ltd., trade name AMT-600 (average particle size 30 nm), etc.) or a titanium oxide dispersion. Various proposals have been made for these compositions. For example, JP-A-10-212120 discloses a glyme solvent (HO — (— CH ₂ CH ₂ O—) n—R, n is 1 to 10, and R is alkyl. A composition in which titanium oxide particles are dispersed in a group or an aryl group and an organic polymer is added as a dispersion aid has been proposed. A dispersion having this composition is applied onto a support by an appropriate method (dip coating method, spray coating method, spinner coating method, blade coating method, roller coating method, wiper bar coating method, reverse roll coating method), and 200 to When fired at 800 ° C., a specific surface area of 40 to 50 cm 2 per 1 cm 2 (thickness 1 μm) is achieved. Japanese Patent Laid-Open No. 2001-233615 discloses that a sol solution composed of tetraalkoxytitanium and ethylene oxide, propylene oxide, ethylene oxide block copolymer, stabilizer and solvent is dropped on the substrate, and the substrate is rotated at high speed. An organic-inorganic composite titania thin film having a three-dimensional structure obtained by evaporating and gelling the solvent is sintered at a high temperature to remove the block copolymer, thereby achieving a fine three-dimensional concave structure. Furthermore, a method using oligosaccharide (trehalose) as an organic polymer is also disclosed (Japanese Patent Laid-Open No. 2004-83376), and a ceramic porous membrane having a porosity of 38 to 56% is obtained.

  As described above, various methods for controlling a fine concavo-convex structure of ceramic have been proposed, and an electrode material having a large specific surface area can be created by applying to a ceramic electrode material suitable for the present invention.

  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 base material usable in the present invention is not only a material having conductivity on the support itself, such as a metal such as titanium, but also a material having a conductive material layer on the surface of a glass or plastic support. It's okay. When a conductive substrate having this conductive material layer is used, the electron accepting layer is formed on the conductive material 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. As a result, the cell is configured such that light is irradiated from the back side of the working electrode (that is, the conductive substrate), and the light transmitted through the working electrode (that is, the conductive substrate and the electron accepting layer) excites the sensitizing dye. can do. 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, 2,761,419, 2,761791, 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) 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 aspect of the present invention, when a single-stranded nucleic acid is used as the probe substance, an oxidized layer is formed on the surface of the working electrode, and the nucleic acid probe and the working electrode are bonded via the oxidized layer. Can be done. 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. Thereby, 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. According to a preferred embodiment of the present invention, a material that crosslinks between the working electrode and the nucleic acid probe can be used in order to firmly fix the nucleic acid probe to the working electrode. 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.

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 the electrolyte medium. Glass, plastic, ceramics, A substrate made of SUS or the like on which a metal or conductive oxide is deposited 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.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
The p53 gene was detected using the apparatus and method according to the present invention. A perfect match probe was immobilized on the working electrode side. Each base sequence was as follows.
Fully matched probe: 5'-TGTAGGAGCTGCTGGTGCA-3 '(SEQ ID NO: 1)
The target DNA to be hybridized with this probe was amplified by the PCR method. The primers used for target DNA amplification by PCR were as follows.
Forward Primer: 5′-Cy5-GATATTGAACAATGGTTCACTGAAGAC-3 ′ (SEQ ID NO: 2)

Reverse Primer: 5′-GCCCCTCAGGGCAACTGAC-3 ′ (SEQ ID NO: 3)
In order to amplify using a Forward Primer having a Cy5 label on the 5 ′ end side, one molecule of Cy5 dye is labeled per target DNA molecule.

   Fluorine-doped tin oxide (F-SnO2: FTO) coated glass (manufactured by AGC Fabrytec Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 75 mm x 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 a 0.5 M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking washing for 5 minutes in ultrapure water was performed three 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 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 0.2 vol%, and stir at room temperature for 5 minutes to obtain a solution for coupling treatment Was prepared. The glass substrate was dipped in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol for 3 minutes was performed three times by replacing methanol, and an operation for removing excess coupling treatment solution was performed. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After cooling the glass substrate at room temperature, it was adjusted to 10 μM, held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes to completely denature the probe DNA (19mer). Then, each spot was spotted with a microarrayer (LT-BA manufactured by Philgen Co., Ltd., spot pin diameter = 1.5 mm) so that the center distance between spots was 5 mm. Thereafter, the mixture was kept at 95 ° C. for 3 minutes to evaporate the solvent, and irradiated with 120 mJ ultraviolet light with a UV crosslinker (UVP CL-1000 type) to immobilize the probe DNA on the glass substrate. Then, shaking washing for 5 minutes in a 0.2% SDS solution was repeated 3 times, and shaking washing for 5 minutes in ultrapure water was further repeated 3 times. 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.

  The probe DNA-immobilized electrode was used as a working electrode and housed and installed in the working electrode case shown in FIG. 6A in the apparatus shown in FIG. A silicon sheet having a thickness of 0.5 mm having an opening inside was installed as the spacer 330. This spacer also has a function for preventing a short circuit due to contact between the working electrode and the counter electrode. In the vicinity of the fluid discharge port from the sensor cell, the flow path is narrower on the downstream side than the portion where the probe is fixed so that the air and the solution in the sensor cell 1 can be easily replaced. The counter electrode used was a platinum electrode formed by sputtering a platinum thin film on a stainless steel (SUS316) surface. Further, the pressing member 310 is provided with an opening 311 for irradiating the detection spot 323 with light. A probe-immobilized electrode serving as a counter electrode and a working electrode is arranged to face each other via the spacer, and a counter electrode, a sensor cell composed of the spacer and the working electrode are configured. The sensor cell has a flow cell structure having a channel formed by a spacer opening and both electrodes. In addition, a fluid supply port and a fluid discharge port provided in the counter electrode connected to the flow path are formed. A piping tube connected to a pump for supplying fluid into the sensor cell 1 is connected to the fluid supply port, and a piping tube for discharging the fluid supplied to the sensor cell 1 is connected to the fluid discharge port. Has been.

  Next, 50 μL of Cy5-labeled target DNA solution and 50 μL of 0.2 M NaOH alkaline denaturing solution containing 2 mM EDTA are mixed and held at room temperature for 5 minutes, and the double-stranded DNA is converted to single-stranded DNA by alkaline denaturation. To. To this solution, 350 μL of 5 × SSC / 0.1% SDS solution, which is a temperature-adjusted hybridization solution at the hybridization set temperature, and 50 μL of 0.2 M HCl neutralizing solution for neutralizing the alkaline solution are added. And mix. 450 μL of the mixed solution is fed into the sensor cell 1, and the temperature in the sensor cell 1 is maintained at 35 ° C. for 10 minutes to allow hybridization with the probe DNA immobilized on the working electrode. At this time, before sending the hybridization solution into the sensor cell 1, the temperature inside the sensor cell 1 is kept at the hybridization set temperature by using a ceramic heater, a temperature controller, or a resistance temperature detector as temperature adjusting members. . After 10 minutes, air is supplied to the hybridization solution using a pump to completely discharge the solution in the sensor cell 1. Next, a 0.5 × SSC / 0.01% SDS solution is fed into the sensor cell 1 in an amount 6 times the cell volume in order to wash away the excess target DNA and the like that have not undergone hybridization. Thereafter, the electrolyte-containing solution is fed into the sensor cell 1. As the electrolyte-containing solution, a 0.4 M tetrapropylammonium iodide (NPr4I) aqueous solution was used. The inside of the sensor cell 1 was temperature-controlled to a detection temperature, and an electrolyte-containing solution was fed to fill the sensor cell 1 to detect a photocurrent.

  Photocurrent detection is performed by immobilizing probe DNA on the working electrode while moving a light source (wavelength: 658 nm, light diameter: about 0.2 mm, output 40 mW) attached to a single-axis stage provided in parallel with the holding member of the sensor cell 1 Irradiate the control spot sequentially for 5 seconds each, and the current value observed between the openings of the pressing member (that is, the current value in the state where the light is blocked and not applied to the working electrode) is set as the base current value. The difference between the spot-derived photocurrent value and the immediately preceding base current value was recorded as the observed value as the photocurrent value of each spot.

  Table 1 shows the results (N = 3) of the differences between the photocurrent values derived from each spot and the base current value just before that for each probe DNA. Note that the photocurrent value and the base current value derived from each spot are calculated using the average of five points at each center as values. From the obtained current values in Table 1, accurate detection was confirmed.

Claims (7)

A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye by attaching a working electrode in which a probe substance capable of binding to the test substance is carried in a plurality of spots. ,
A plurality of light sources for irradiating the spot with light;
An ammeter for measuring a photocurrent generated by photoexcitation of the sensitizing dye,
The light source is provided on at least the number of columns or rows on an extension of the center line corresponding to the spot,
A measuring apparatus measuring apparatus comprising a light source moving mechanism for moving the light source in an extension line direction of the center line. The measurement apparatus according to claim 1, wherein the light source is movable to a position where light from the light source is not irradiated to the spot by the light source moving mechanism. The measuring apparatus according to claim 1, wherein the light source is a pigtail laser. On the opposite side of the surface on which the spot is formed of the working electrode, a pressing member is provided for pressing the sensor cells formed from the counter electrode provided via a spacer, and the pressing member is optically excited. The measuring device according to any one of claims 1 to 3, comprising a plurality of openings or light-transmitting portions for allowing light to pass through. The measuring device according to claim 4, wherein the pressing member has a light shielding property except for the opening and the light transmitting portion. A measurement method for performing specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye using the measurement apparatus according to any one of claims 1 to 5,
A first light irradiation step of turning on the first light source among the light sources and irradiating light to the spot at the corresponding position while moving in the first direction on an extension of the center line,
A second light irradiation step of illuminating the spot at the corresponding position while turning on the second light source among the light sources and moving in the second direction on the extension of the center line;
A measurement method including at least. The measurement method according to claim 6, wherein, in the first light irradiation step, the light source is turned on at a position where the light is not irradiated to the spot.

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