JP2010038813A - Sensor chip used for specific detection of substance to be inspected using photoelectric current, and measurement device using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor chip capable of using an electrolyte-containing sheet as a substitute for electrolyte and thereby considerably simplifying structure and detection procedures thereof, and a measurement device in the specific detection of a substance to be inspected using a photocurrent generated by the photo-excitation of sensitizing dye. <P>SOLUTION: The sensor chip used for the specific detection of the substance to be inspected using the photocurrent generated by the photo-excitation of the sensitizing dye includes a working electrode, at least one of a hollow and a projection for holding the working electrode at a predetermined position, and an opening for passing a positioning member to fix the working electrode at a predetermined position in the measurement device. The specific detection of the substance to be inspected is performed by mounting the electrolyte-containing sheet on the working electrode or keeping the electrolyte-containing sheet clamped between the working electrode and a sensor chip case. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention is used in a method for specifically detecting a test substance having specific binding properties, such as a nucleic acid, an exogenous endocrine disrupting substance, and an antigen, using a photocurrent generated by photoexcitation of a sensitizing dye. The present invention relates to a sensor chip and a measuring apparatus in which an electrolyte-containing sheet can be used instead of the above.

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

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

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

  Incidentally, gene detection and genotype determination are mainly performed by a DNA chip and a fluorescence detection apparatus. A general DNA chip is a nucleic acid probe having a base sequence complementary to a known gene is immobilized on a glass chip or silicon chip of several centimeters square.

  A configuration of a commonly used fluorescence detection apparatus is shown in FIG. Excitation light 9 from a light source 5 such as a laser is reflected by the beam splitter 4 and enters the objective lens 6. The excitation light 9 is collected by the objective lens 6 and strikes the nucleic acid probe fixing part 7 of the DNA chip 8. When hybridization is performed by a hybridization reaction, a nucleic acid probe based on a gene and a nucleic acid chain labeled with a fluorescent dye form a double strand, and the fluorescent substance remains on the DNA chip 8 even after the solution is washed away. The fluorescent label generates fluorescent light 10 with spherical emission by the excitation light 9. If it is not hybridized, it does not fluoresce. The fluorescence 10 and the excitation light 9 have a wavelength difference of about several tens of nanometers. Part of the fluorescence 11 and the reflected light of the excitation light 9 return to the objective lens 6 and enter the beam splitter 4. Most of the reflected light of the excitation light 9 is reflected by the beam splitter 4 and travels toward the light source side, and part of the fluorescence 11 passes through the beam splitter 4 and travels toward the light receiver 1 side. Part of the fluorescence 11 transmitted through the beam splitter 4 is removed from the reflected light of the excitation light 9 by the filter 3 that limits the wavelength. A part 11 of the fluorescence passes through the light receiver lens 2 and enters the light receiver 1 for measuring the fluorescence intensity. When no hybridization is performed by the hybridization reaction, light does not enter the light receiver 1 because it does not fluoresce.

  In addition, a single-stranded nucleic acid probe having a base sequence complementary to the target gene to be detected is immobilized on the electrode surface, reacted with a sample containing a gene denatured into a single strand, and then hybridized with the gene. There is known a gene detection apparatus that confirms the presence of the target gene by binding a double-stranded recognizer to the soy nucleic acid probe and detecting it by electrochemical measurement (see, for example, Patent Document 5). Here, the electrochemical measurement is performed by immersing the working electrode and the counter electrode in an electrolytic solution, and measuring an oxidation current by linear sweep voltammetry.

JP-A-7-107999 Japanese Patent Laid-Open No. 11-315095 JP 2006-119111 A Japanese Patent Publication No. 5-84860 JP 2000-83647 A 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)

  The present inventors can now use an electrolyte-containing sheet in place of an electrolyte in the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye, whereby a sensor unit and a measuring device can be used. It was found that the structure and detection procedure of can be greatly simplified. And the knowledge that a photocurrent can be detected with high sensitivity and accuracy by using this electrolyte-containing sheet, and a sensor chip and a measuring device using the same was also obtained.

  Therefore, the present invention can use the electrolyte-containing sheet instead of the electrolyte in the specific detection of the test substance using the photocurrent generated by photoexcitation of the sensitizing dye, thereby greatly increasing the structure and the detection procedure. An object of the present invention is to provide a sensor chip and a measuring apparatus that can be simplified.

That is, the sensor chip according to the present invention is a sensor chip that is detachably attached to a measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The sensor chip is
A working electrode;
A sensor chip case, and
The sensor chip case is
At least one of a recess and a protrusion for holding the working electrode at a predetermined position, and an opening for passing a positioning member for fixing the working electrode at a predetermined position of the measuring device And
The specific detection of the test substance is performed by placing the electrolyte-containing sheet on the working electrode or sandwiching the electrolyte-containing sheet between the working electrode and the sensor chip case.

The apparatus according to the present invention is a measuring apparatus used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The sensor chip;
An electrolyte-containing sheet that is placed on the working electrode or sandwiched between the working electrode and the sensor chip case; and
A counter electrode provided on the side opposite to the working electrode of the electrolyte-containing sheet;
A light source for irradiating the working electrode with light;
An XY movement mechanism for moving the light source in the XY direction;
An ammeter for measuring a current flowing between the working electrode and the counter electrode;
Control calculation for controlling the light source, the XY moving mechanism, and the ammeter, receiving a current signal from the ammeter, and determining at least one of the presence, type, and concentration of the test substance based on the electrical signal Means.

Sensor Chip The sensor chip according to the present invention is a sensor chip used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. This sensor chip includes a working electrode and a sensor chip case. The sensor chip case has at least one of a depression and a protrusion for holding the working electrode in a predetermined position. The sensor chip case has an opening through which a positioning member provided in the measuring device for fixing the working electrode at a predetermined position of the measuring device is passed when the sensor chip is attached. By positioning the working electrode by the positioning member provided in the measuring device, the working electrode can be accurately positioned regardless of the dimensional error of the sensor chip case. For this reason, the recess or protrusion provided in the sensor chip case for holding the working electrode in a predetermined position has some play in order to fix the working electrode in a predetermined position of the measuring device. Provided. Positioning that requires the highest accuracy in the sensor unit of the present invention is that the working electrode is fixed at a predetermined position of the measuring apparatus, and therefore positioning for placing the sensor chip case at the predetermined position of the measuring apparatus. Is provisional positioning. The positioning member has a pin shape or a support member that is a projection-shaped wall surface having a flat surface at least in contact with the working electrode, and the working electrode is pressed and fixed to the supporting member with an appropriate pressure. And an urging member disposed at a predetermined position. As the material of the support member, a material having sufficient strength and workability such as resin, metal, ceramic, glass and the like can be used. Examples of the urging member include elastic protrusions made of a material such as a plate-like or coil-like spring, rubber or polymer. And in this sensor chip, the test substance is placed by an extremely simple method of simply placing the electrolyte-containing sheet on the working electrode or sandwiching the electrolyte-containing sheet between the working electrode and the sensor chip case. It becomes a state that can be used for specific detection of.

  This electrolyte-containing sheet is a sheet-like electrolyte-containing body used as an electrolyte medium in the specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. And this electrolyte containing sheet | seat can typically comprise a water-containing base material and the electrolyte contained in a water-containing base material. The electrolyte can freely move in the hydrous substrate and participate in the transfer of electrons between the sensitizing dye, the working electrode, and the counter electrode. Therefore, light generated by photoexcitation of the sensitizing dye between the working electrode and the counter electrode is obtained by sandwiching the electrolyte-containing sheet between the working electrode and the counter electrode and keeping the surface of each electrode in contact with the electrolyte-containing sheet. A current can flow. This electrolyte-containing sheet is the same as a conventionally used electrolytic solution in terms of an electrolyte medium, but is a sheet-like material that can be handled alone, so that it can be easily sandwiched between a working electrode and a counter electrode. Can be removed or carried around, resulting in greatly simplified device construction and detection procedures. In fact, according to the sensor chip or the measuring apparatus of the present invention, the working electrode, the electrolyte-containing sheet, and the counter electrode can be accurately assembled by a very simple method of simply placing or sandwiching them as described above. is there. This is a conventional method in which an electrolytic solution is filled between a working electrode and a counter electrode, and a mechanism for feeding the electrolytic solution (for example, a pump, a valve, and a control mechanism thereof), a liquid leakage prevention mechanism (for example, In light of the fact that complicated mechanisms or processes such as packing) and waste solution treatment of the electrolyte are required, this has led to an increase in cost and an increase in the size of the apparatus. In addition, the use of the electrolyte-containing sheet eliminates the need for time to send the electrolyte solution, so it can be immediately measured by simply sandwiching the electrolyte-containing sheet between the working electrode and the counter electrode. Measurement time is also shortened.

  As described above, it is sufficient that the sensor chip of the present invention includes the working electrode and the sensor chip case, and the electrolyte-containing sheet may not be provided in advance. This is because it is sufficient that the sensor chip of the present invention is combined with an electrolyte-containing sheet at the time of use. An example of the sensor chip of this aspect is shown in FIG. The sensor chip shown in FIG. 2 includes a plate-like working electrode 21 and a sensor chip case 22 having a recess 22a formed to have substantially the same dimensions as the working electrode 21. Therefore, only by placing the working electrode in the depression, the working electrode is regulated by the edge of the depression and the working electrode is held. However, the recess 22a provided in the sensor chip case 22 has some play to fix the working electrode at a predetermined position of the sensor chip receiving portion of the apparatus. By such a mechanism, the working electrode is temporarily positioned with respect to the sensor chip receiving portion. Further, the sensor chip case 22 shown in FIG. 2 is provided with an opening for passing a positioning member provided in the apparatus. Examples of the opening include a support member opening 34 and a biasing member opening 35. Further, in the sensor chip shown in FIG. 2, an electrical connection for connecting the opening 36 and the counter electrode, which are adapted to an auxiliary member for placing the sensor chip case 22 at a predetermined position of the measuring device, to the ammeter. An opening 22f for passing the contact is provided.

  According to a preferred aspect of the present invention, an electrolyte-containing sheet may be further provided on the working electrode or between the working electrode and the sensor chip case. That is, it is sufficient that the sensor chip of the present invention is combined with an electrolyte-containing sheet at the time of use, but an electrolyte-containing sheet may be provided in advance as a constituent member of the sensor chip. An example of the sensor chip of this aspect is shown in FIG. The sensor chip shown in FIG. 3 includes a plate-shaped working electrode 21, a sensor chip case 22 having a recess 22a, a support member opening 34, a biasing member opening 35, and a sensor chip case 22 of the measuring device. Although it is the same as the structure of FIG. 2 in that an opening 36 adapted to an auxiliary member for mounting at a predetermined position is provided, an electrolyte-containing sheet 23 is further provided on the working electrode 21. Here, as shown in FIG. 3, the sensor chip case 22 may be further provided with a second recess 22 b for placing the electrolyte-containing sheet 23 at a predetermined position. The second recess 22b may not necessarily be required to have high positioning properties, but it is needless to say that the second recess 22b may be formed with substantially the same dimensions as the electrolyte-containing sheet. Therefore, only by placing the working electrode and the electrolyte-containing sheet in the recess and the second recess, respectively, the working electrode and the electrolyte-containing sheet are regulated by the respective edges of the recess and the second recess. Temporary positioning of the electrolyte-containing sheet can be easily performed. However, the second recess 22b provided in the sensor chip case 22 has some play in order to place the working electrode at a predetermined position of the sensor chip receiver. However, when the working electrode and the electrolyte-containing sheet have the same dimensions, the recess may be configured so that both the working electrode and the electrolyte-containing sheet can be placed thereon.

  According to a preferred aspect of the present invention, a counter electrode may be further provided on the opposite side of the electrolyte-containing sheet from the working electrode. That is, it is sufficient that the sensor chip of the present invention is combined with the counter electrode at the time of use, but it may have a counter electrode as a constituent member of the sensor chip in advance. An example of the sensor chip of this aspect is shown in FIG. The sensor chip shown in FIG. 4 includes a plate-shaped working electrode 21, a sensor chip case 22 having a recess 22a, a support member opening 34, a biasing member opening 35, and a sensor chip case 22 of the measuring device. 3 is the same as the configuration of FIG. 3 in that the opening 36 conforms to the auxiliary member to be placed at a predetermined position and the electrolyte-containing sheet 23, but the counter electrode 25 is further provided on the electrolyte-containing sheet 23. It is done. Here, as shown in FIG. 4, the sensor chip case 22 may further be provided with a third recess 22 c for placing the counter electrode 25 at a predetermined position. The third recess 22c may not necessarily be required to have high positioning properties, but it is needless to say that the third recess 22c may be formed to have substantially the same dimensions as the counter electrode. However, the third recess 22c provided in the sensor chip case 22 has some play in order to place the working electrode at a predetermined position of the sensor chip receiver.

  According to a preferred aspect of the present invention, the sensor chip case further includes a second opening or a light transmitting portion for allowing light for light excitation to pass through the surface of the working electrode, and at least the opening of the sensor chip case. It is preferable that parts other than the part and the light transmitting part have a light shielding property. The opening 22e corresponds to this in FIGS.

According to a preferred aspect of the present invention, it is preferable that the measuring device has a light shielding portion at least in a portion that is exposed to light other than the opening and the light transmitting portion.
According to this aspect, since the working electrode can be locally irradiated with light, a plurality of test substances can be individually attached by attaching the test substances to a plurality of spots on one working electrode. Can be measured with high accuracy. That is, according to a more preferred aspect of the present invention, the working electrode has a plurality of detection spots, and an opening or a light transmitting portion is formed at each position corresponding to the plurality of detection spots of the measuring apparatus. Is preferred.

  Such a light shielding mechanism is also employed in the sensor chip receiving portion 33 of the measuring apparatus of FIG. 6 shows an enlarged view of the sensor chip receiving portion 33 of FIG. As shown in FIG. 5 and FIG. 6, openings 60 at respective positions corresponding to a plurality of detection spots provided in the measurement apparatus are formed, and the openings are arranged through the regularly arranged openings. One of a plurality of detection spots formed on the working electrode in the same pattern can be selectively and individually irradiated with light. That is, at least the area where the openings are arranged in the measuring apparatus is composed of a light-shielding member, so that light is irradiated to the other openings by irradiating light with a light diameter passing through only one opening. Thus, only one detection spot on the working electrode can be individually irradiated with light through only one desired opening. That is, it is possible to effectively prevent light from leaking to a detection spot that should not be irradiated with light, and to improve the detection accuracy of the test substance for each detection spot.

  5 and 6 includes a support member 131 used for positioning the working electrode, a biasing member 132, an auxiliary member 133 for placing the sensor chip in a predetermined position, and a working electrode. Are provided with an electrical contact 27a for connecting to the ammeter, a counter electrode 25 and a resilient sheet-like member 28, and an electrical contact 27b for connecting the counter electrode 25 to the ammeter. The support member 131 may be a pin shape or a protrusion shape having a flat surface at least at a position where the working electrode contacts, or a wall surface of a recess provided in the sensor chip receiving portion. As the material of the support member 131 and the auxiliary member 133, those having sufficient strength and workability such as resin, metal, ceramic, glass and the like can be used, and as the material of the urging member 40, a plate shape, a coil An elastic protrusion shape made of a material such as a spring, rubber or polymer.

  According to still another preferred aspect of the present invention, the sensor chip case has a third opening in at least a part of the region where the working electrode is placed, and the specific detection of the test substance is performed, It is preferable that the counter electrode is brought into contact with the electrolyte-containing sheet through the third opening. That is, in this embodiment, the working electrode is placed and the first opening for allowing light to pass through and the third opening for allowing the counter electrode to pass through are provided. In this aspect, the third opening may also serve as an opening for passing an electrical contact for connecting the counter electrode to the ammeter. Also in this aspect, the measuring device is formed with openings or light transmitting portions at positions corresponding to a plurality of detection spots. An example of the sensor chip of this aspect is shown in FIG. 7, and an example of the sensor unit of the measuring apparatus is shown in FIG. The sensor chip shown in FIG. 7 includes a plate-like working electrode 61, a sensor chip case 62 having a recess 62a in which the working electrode 61 is formed to have substantially the same dimensions, and a working electrode 61 installed in a measuring device. The support member 141 opening 63, the biasing member 142 opening 64, and the opening 65 suitable for the auxiliary member 143 for placing the sensor chip case 62 at a predetermined position of the measuring device are provided. 2 is the same as the configuration of FIG. 2, but an opening 66 through which the counter electrode can pass from below is formed in at least a part of the region where the working electrode is placed. Therefore, the dimension of the opening 66 is a size through which the counter electrode can pass. The sensor chip shown in FIG. 9 includes a plate-like working electrode 71, a sensor chip case 72 having a dent 72a and a second dent 72b, an electrolyte-containing sheet 70, and a working electrode installed in a measuring device. A support member opening 73 used for positioning 71, a biasing member opening 74, and an opening 75 adapted to an auxiliary member for placing the sensor chip case 72 at a predetermined position of the measuring device. 3 is the same as the configuration of FIG. 3, but a third opening 76 through which the counter electrode can pass from below is formed during measurement, as in FIG. Also in these aspects, the measurement apparatus is formed with openings or light transmitting portions 144 at positions corresponding to a plurality of detection spots.

  According to another preferred embodiment of the present invention, the depression, the second depression, and / or the third depression is replaced with a protrusion, a second protrusion, and / or a third protrusion. It is good also as a structure. Also in these protrusions, the working electrode, the electrolyte-containing sheet, and the end portions of the counter electrode can be regulated in the same manner as the above-described depression, so that these members can be accurately positioned. The shape and arrangement of these protrusions are not particularly limited as long as these members can be accurately positioned only by being placed, and for example, rib-like (linear) or warped (dot-like) May be provided. Further, the protrusions may be formed over the entire outline of the region on which the member to be positioned is placed, or may be formed only on at least a part thereof. In particular, when the working electrode is positioned on the measuring device, a biasing member may be provided at one end of a region where the working electrode is fixed, and a protrusion may be provided at the other end facing the biasing member and / or in the vicinity thereof. . In this case, at least one side of the working electrode placed along the protruding portion is pressed or biased toward the protruding portion by the biasing member, and can thereby be accurately positioned. For example, when the working electrode has a square shape (when viewed from above), the protruding portion has a shape (for example, two L-shaped apex shapes) surrounding both ends of the side facing the biasing member. It may be formed in a linear shape along the side or a combination thereof (for example, a shape in which two L-shaped apex shapes are connected by a straight line). Moreover, you may form a projection part in several dot shape instead of a linear shape.

Measuring device By using the sensor chip of the present invention, it is possible to construct an inexpensive and small sensor unit or measuring device whose structure is greatly simplified. This is because the sensor chip of the present invention uses an electrolyte-containing sheet, and therefore a mechanism for feeding an electrolyte solution, which is required in a conventional method performed by filling an electrolyte solution between a working electrode and a counter electrode ( This is because complicated mechanisms or processes such as a pump, a valve, and a control mechanism thereof, a liquid leakage prevention mechanism (for example, packing), and a waste liquid treatment of the electrolytic solution are unnecessary.

  The measuring apparatus according to the present invention is an apparatus for performing specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye using the sensor chip of the present invention. An example of the measuring apparatus of the present invention is shown in FIG. As shown in FIG. 10, the measuring device 80 of the present invention includes a sensor chip 81, a light source 82, an XY moving mechanism 83, an ammeter 84, and a control calculation means 85. As described above, the electrolyte-containing sheet 81b and the counter electrode 81c may be used as constituent members of the sensor chip in advance, or may be used as constituent members of the measuring device. In any case, in the measurement, the electrolyte-containing sheet 81b is provided on the working electrode 81a or sandwiched between the working electrode 81a and a sensor chip case (not shown). The counter electrode 81c should just be provided in the opposite side to the working electrode of the electrolyte containing sheet 81b. And the light source 82 should just be arrange | positioned in the position which can irradiate light to the surface of the working electrode 81a, and may be arrange | positioned in any of the downward direction and upper direction of a sensor chip. An XY moving mechanism 83 is connected to the light source 82, and the light source 82 is configured to be movable in the XY direction toward a desired test spot. An ammeter 84 is connected to the working electrode and the counter electrode, and a current flowing between them is configured to be measurable. In the control calculation means 85, the control unit 85a controls the light source 82, the XY movement mechanism 83, and the ammeter 84, receives a current signal from the ammeter 85, and the calculation unit 85b is based on the electric signal. And at least one of the presence, type, and concentration of the test substance. The measuring device 80 preferably further includes a display device 86 for displaying the result obtained by the control calculation means 85. Furthermore, it is preferable that the measuring device 80 further includes an input device 87 for inputting conditions for measurement.

  According to a preferred aspect of the present invention, the sensor chip receiving part of the measuring device is preferably provided with a concave or flat sensor chip receiving part to which the sensor chip is detachably mounted. In this aspect, it is preferable that the sensor chip receiving portion includes a positioning mechanism for fixing the working electrode attached to the sensor chip case at a predetermined position, whereby the working electrode is easily and accurately loaded into the measuring device. can do. Therefore, the sensor chip case has a support member that is a positioning member for fixing the working electrode at a predetermined position of the measuring device, and an opening through which the urging member is passed when the sensor chip is attached. By positioning the working electrode with the support member and the biasing member provided in the measuring device, the working electrode can be accurately positioned regardless of the dimensional error of the sensor chip case. As an example of such a positioning mechanism, after the sensor chip is installed, the working electrode is pressed or urged by the urging member toward the pin or hole which is a support member formed in the sensor chip receiving portion, and thereby the working electrode There is a mechanism for accurately positioning.

  According to a preferred aspect of the present invention, the sensor chip receiving part has an auxiliary member for placing the sensor chip at a predetermined position, so that the sensor chip can be easily attached to and detached from the sensor chip receiving part. It is preferable to be made. That is, the auxiliary member provided in the sensor chip receiving portion is brought into contact with an opening that matches the auxiliary member provided in the sensor chip case. However, as described above, the positioning for which the highest accuracy is required in the sensor unit of the present invention is that the working electrode is placed at a predetermined position of the measuring device, so that the sensor chip is placed on the measuring device. Is provisional positioning. Positioning the working electrode after placing the sensor chip on the measuring device requires the highest accuracy. Therefore, it is possible to form the sensor unit of the present invention without using an auxiliary member, but it goes without saying that the operability is improved by having the auxiliary member. As the material of the auxiliary member, a material having sufficient strength and workability such as resin, metal, ceramic, glass and the like can be used. As a form of the auxiliary member, a pin shape or a protrusion shape having a flat surface at least at a portion where the sensor chip abuts, a wall surface of a hollow portion provided in the sensor chip receiving portion, and an opening provided in the sensor chip case It is possible to use a pin shape that can be fitted to the portion or a protrusion shape that at least the opening abuts.

  According to a preferred aspect of the present invention, the measuring device has a lid portion that can be opened and closed with respect to the sensor chip receiving portion, and a convex pressing member that is provided at a position where the measuring device can contact the working electrode of the lid portion. However, it is preferable that the light source is provided above the pressing member. In this aspect, the pressing member is composed of a light-shielding member that blocks light from the light source, and further includes an opening or a light-transmitting portion for allowing light for light excitation to pass through the surface of the working electrode. Thus, it is preferable that light can be irradiated only on a specific portion of the working electrode. According to a more preferred aspect of the present invention, the working electrode has a plurality of detection spots, and the opening or the translucent portion can be formed at each position corresponding to the plurality of detection spots.

  According to a preferred aspect of the present invention, the sensor chip body has an opening in at least a part of the region where the working electrode is placed, and the measuring device has a convex portion that can be inserted into the opening. It is preferable that the counter electrode is provided on the convex portion, and that the counter electrode is in contact with the electrolyte-containing sheet by inserting the convex portion into the opening when the sensor chip is attached to the apparatus. .

An example of a measuring device according to the present invention in which the light source of the lower light source type measuring device is arranged below the sensor chip and the sensor chip 30 of the present invention shown in FIG. 3 can be used is shown in FIGS. FIG. 11 is a perspective view showing the external appearance of the measuring device 90. As shown in FIG. 11, a sensor chip insertion port 93 for inserting and removing the sensor chip 30 is provided on the upper surface of the apparatus housing 91. As shown in FIG. The sensor chip insertion port 93 has a size that allows an operator's hand holding the sensor chip 30 to enter. A body lid 94 is attached to the sensor chip insertion opening 93 so as to be openable and closable. The main body lid 94 is configured to prevent leakage of light irradiated from the light source 98 to the outside and intrusion of external light into the apparatus. A display device 96 and an input device 97 are further provided on the upper surface of the device housing 91. The display device 96 can display the detected photocurrent value as a numerical value or a time-dependent change graph of real-time display, and also displays the difference between the current value at the time of non-light irradiation and light irradiation read from an appropriate data point. The result of the gene test or genotype determination obtained based on this can be displayed. The input device 97 is configured to perform input operations such as keys and buttons for inputting operating conditions. FIG. 12 is an internal perspective view of the measuring apparatus 90. As shown in FIG. 12, the light source 98 is configured to irradiate each detection spot on the working electrode of the sensor chip 30 and the XY moving mechanism 99. Configured to be movable. The ammeter 100 is configured to measure the current flowing between the working electrode of the sensor chip 30 and the counter electrode of the device 90. Control of the light source 98, the XY moving mechanism 99 and the ammeter 100 and reception of the current signal are performed by the computer 102 via the interface board 101 in the control calculation unit. In addition, the computer 102 performs processing such as control and various calculations, stores data and processing results, and displays the results on the display device 96. FIG. 13 is a perspective view showing the main part of the measuring device 90. As shown in FIG. 13, the sensor chip receiving port 93 is a sensor chip holder which is a concave part where the sensor chip 30 is detachably mounted. A portion 103 is provided, and the counter electrode 92 is attached to the convex portion 104 of the main body lid 94. That is, the sensor chip receiving portion 103 and the counter electrode 92 are provided in the device housing 91.

  As shown in FIG. 13, the sensor chip used in the measuring apparatus 90 is the sensor chip 30 shown in FIG. 3, and the configuration thereof is as already described. The assembly method of the sensor chip 30 is as follows. First, the working electrode 21 is placed along the recess 22 a of the sensor chip case 22. Thereafter, the electrolyte-containing sheet 23 is placed along the second recess 22 b of the sensor chip case 22 to assemble the sensor chip 30. The electrolyte-containing sheet 33 is disposed so as to contact all the detection spots on the working electrode 21. The light irradiation port 22 e passes through the openings 105 at each position corresponding to a plurality of detection spots provided on the bottom of the sensor chip receiving portion 103, and the light emitted from the light source 98 passes through the back side of the working electrode 21. It is provided so that light may be irradiated. The opening 105 is provided for each detection spot provided in the working electrode 21, and when the number, arrangement, and shape of the detection spots are changed, the light irradiation port processed into a corresponding shape is provided. And For this reason, it is preferable that a member having openings corresponding to a plurality of detection spots of the sensor chip receiving portion be configured to be removable. As shown in FIGS. 14 and 15, in actual photocurrent detection, light is irradiated to the detection spot 21a of the working electrode to obtain the photocurrent 120 of the detection spot, and then the light source is placed at the light source standby position 21b. And the photocurrent 121 at the light source standby position 21b is obtained. The photocurrent 121 at the light source standby position 21b becomes the background photocurrent. However, since the member having openings corresponding to the plurality of detection spots of the sensor chip receiving portion is provided with the openings 105 processed in the number, arrangement, and shape corresponding to the detection spots, the sensor electrode receiving portion is actually mounted on the working electrode. The light source standby position 21b is not irradiated with light, resulting in a very small background photocurrent. The portion 103a between the light irradiation ports on the member having openings corresponding to the plurality of detection spots of the sensor chip receiving portion corresponding to the light source standby position 21b does not leak light to the adjacent detection spots. The diameter of the light emitted from the light source 98 is adjusted to be larger.

  As shown in FIGS. 13 and 14, when attaching the sensor chip 30 to the sensor chip receiving portion 103, the auxiliary member opening 22 d provided on the lower surface of the sensor chip case 22 is aligned with the auxiliary member 107. The working electrode 21 is in contact with the support member 108 and fixed by the biasing member 109. The main body lid 94 is provided with a working electrode contact probe 110. When the main body lid 94 is closed, the counter electrode 92 attached to the convex portion of the main body lid 94 is replaced with the electrolyte-containing sheet 23 and the counter electrode contact probe. 111 is contacted.

FIGS. 16 to 19 show examples of the measuring device according to the present invention in which the upper light source type measuring device light source is disposed above the sensor chip and the sensor chip 112 of the present invention shown in FIG. 7 can be used. FIG. 16 is a perspective view showing the external appearance of the measuring device 140. As shown in FIG. 16, a sensor chip insertion port 143 for inserting and removing the sensor chip 112 is provided on the side surface of the device housing 141. The sensor chip insertion port 143 is interlocked with the opening / closing button 144, and when the opening / closing button 144 is pressed, the sensor chip insertion port 143 is put in and out by driving means (not shown). When the sensor chip insertion port 143 is accommodated in the apparatus housing 141, the sensor chip insertion port 143 is configured to prevent leakage of light emitted from the light source 145 to the outside and entry of external light into the apparatus. A display device 146 and an input device 147 are further provided on the side surface of the device housing 141. The display device 146 can display the detected photocurrent value as a numerical value or a time-dependent change graph of real-time display, and also displays the difference between the current value at the time of non-light irradiation and the time of light irradiation read from an appropriate data point. The result of the gene test or genotype determination obtained based on this can be displayed. The input device 147 is configured to perform input operations such as keys and buttons for inputting operating conditions. FIG. 17 is an internal perspective view of the measuring apparatus 140. As shown in FIG. 17, the light source 145 is configured to irradiate each detection spot on the working electrode of the sensor chip 112, and the XY moving mechanism 148. Configured to be movable. The ammeter 149 is configured to measure the current flowing between the working electrode of the sensor chip 112 and the counter electrode of the device 140. The control of the light source 145, the XY moving mechanism 148 and the ammeter 149 and the reception of the current signal are performed by the computer 151 via the interface board 150 in the control calculation unit. Further, the computer 151 performs processing such as control and various calculations, stores data and processing results, and displays the results on the display device 146. FIG. 18 is a perspective view showing a main part of the measuring apparatus 140. As shown in FIG. 18, the sensor chip is a concave part in which the sensor chip 112 is detachably attached to the sensor chip insertion port 143. A receiving portion 152 is provided, and the counter electrode 159 is attached to a convex portion formed at the bottom of the sensor chip receiving portion 152. As shown in FIG. 18, the main body lid 154 is provided with a light passage port 161 provided so that light emitted from the light source 145 is emitted to the back side of the working electrode 113. That is, the sensor chip receiving portion 152, the counter electrode 159, the main body lid 154, and the light passage port 161 are provided in the device housing 141.

  As shown in FIGS. 18 and 19, the sensor chip used in the measuring apparatus 140 is the sensor chip 112 shown in FIG. 7, and the configuration thereof is as already described. The method for assembling the sensor chip 112 is as follows. First, the electrolyte-containing sheet 115 is placed along the second recess 114b of the sensor chip case 114. A third opening is formed at the bottom of the sensor chip 112 so that the counter electrode 159 provided on the sensor chip receiving portion 152 of the device housing 141 comes into contact with the electrolyte-containing sheet 115. Thereafter, the working electrode 113 is placed along the recess 114 a of the sensor chip case 114 to assemble the sensor chip 112. The electrolyte-containing sheet 115 is disposed so as to contact all detection spots on the working electrode 113. The main body lid 154 is provided with a pressing member 162 for pressing the counter electrode 159, the electrolyte-containing sheet 115, and the working electrode 113. The pressing member 162 is provided with a light irradiation port 160 so that light emitted from the light source 145 passes through the light passage port 161 of the main body lid 154 and light is irradiated to the back side of the working electrode 113. The light irradiation port 160 is provided for each detection spot provided on the working electrode 113. When the number, arrangement, and shape of the detection spots are changed, the light irradiation port 160 has a light irradiation port processed into a corresponding shape. Replace the holding member. Further, as shown in FIGS. 19 and 20, in actual photocurrent detection, the detection spot 113a on the working electrode 113 is irradiated with light to obtain the detection spot photocurrent 164, and then the light source standby position 113b. Light is irradiated to obtain a photocurrent 166 at the light source standby position 113b. The photocurrent 166 at the light source standby position 113b becomes the background photocurrent. However, since the pressing member 162 is provided with the light irradiation ports 160 processed into the number, arrangement, and shape corresponding to the detection spots, the light source standby position 113b on the working electrode is not actually irradiated with light. A small background photocurrent. The portion 167 between the light irradiation ports on the pressing member corresponding to the light source standby position 113b is larger than the diameter of the light emitted from the light source 145 so that light does not leak to the adjacent detection spot. Adjusted to

  FIG. 21 shows an enlarged view of FIG. As shown in FIGS. 18, 19, and 21, when the sensor chip 112 is attached to the sensor chip receiving portion 152, the auxiliary member opening 114 c provided on the lower surface of the sensor chip case 114 is used as the auxiliary member 169. The working electrode 113 is brought into contact with the support member 116 and fixed by the urging member 117. The urging member 117 is made of an elastic body such as a metal, resin, rubber, or the like, which has a restoring force in the direction of the support member 116, such as a metal, resin, rubber, etc., and is elastic when the sensor chip is attached. The working electrode is securely fixed by applying a restoring force such as a spring. When the sensor chip is attached, the fixed state of the working electrode can be easily released by applying a force against the restoring force of the biasing member in the direction opposite to the support member. When the sensor chip 112 is attached, the counter electrode 159 provided on the convex portion of the sensor chip receiving portion 152 comes into contact with the electrolyte-containing sheet 115, and the working electrode 113 comes into contact with the electrolyte-containing sheet 115. When the main body lid 154 is closed, the pressing member 162 presses the working electrode 113, and an appropriate arrangement is formed such that each of the light irradiation ports 160 provided in the pressing member 162 corresponds to each detection spot of the working electrode 113. Is done. The working electrode 113 is connected to the ammeter 149 by the working electrode contact probe 118, and the counter electrode 159 and the ammeter 149 are connected by the counter electrode contact probe 119. The sensor chip case 114 has a counter electrode opening portion so that the working electrode contact probe opening portion 175 and the counter electrode 159 can contact the electrolyte sheet 115 so that the working electrode contact probe 118 can contact the working electrode 113. 114d is provided.

Electrolyte-containing sheet The electrolyte-containing sheet used in the present invention is a sheet-like electrolyte-containing body used as an electrolyte medium in specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye. And this electrolyte containing sheet | seat contains a water-containing base material and the electrolyte contained in a water-containing base material.

In the present invention, the electrolyte is not limited as long as it can freely move 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. It can be used. 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).

(1) Gel sheet According to a preferred embodiment of the present invention, the hydrous substrate is a gel matrix comprising at least one selected from natural gels and synthetic gels, and the electrolyte is dispersed in the gel matrix. It is preferable that That is, in this aspect, the electrolyte-containing sheet is configured as a gel sheet. When a gel sheet is used as the electrolyte medium, a higher detection current can be obtained for a sample having the same analyte concentration than when an electrolyte is used, and the concentration of the detection current in a wider analyte concentration range. Dependency is obtained. That is, the use of the gel sheet can greatly improve the photocurrent detection sensitivity and accuracy.

According to a preferred embodiment of the present invention, the gel sheet preferably has a gel strength of 100 g / cm ² or more, more preferably 120 g / cm ² or more, and further preferably 150 g / cm ² or more. With such a gel strength, the gel sheet can be easily handled by itself, so that it can be easily sandwiched or removed between the working electrode and the counter electrode, and as a result, the sensor unit structure and detection procedure can be greatly simplified. .

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

  According to a preferred embodiment of the present invention, the gel sheet preferably has a thickness of 0.1 to 10 mm, more preferably 0.5 to 3 mm, and still more preferably 1 to 3 mm. With such a thickness, it is easy to obtain a strength suitable for handling the gel sheet alone, so it can be easily sandwiched, removed or carried between the working electrode and the counter electrode. As a result, the sensor unit structure and detection procedure can be greatly simplified. Moreover, it does not adversely affect the photocurrent measurement.

  In the present invention, the gel matrix is not limited as long as it contains at least one selected from natural gels and synthetic gels and exhibits a suitable strength and adhesion to electrodes. These gels can be formed by gelation of a gelling agent together with a solvent such as water, in the same manner as general gels. The concentration of the gelling agent in the gel matrix does not have a great influence on the photocurrent measurement, and therefore may be appropriately determined according to the type of the gelling agent from the viewpoint of securing strength that enables single handling.

  According to a preferred embodiment of the present invention, the gel matrix preferably comprises a natural gel mainly composed of polysaccharides and proteins. Preferred examples of such natural gels include gels of agarose, alginic acid, carrageenan, roast bean gum, gellan gum, gelatin, and mixtures thereof, more preferably agarose gel. A preferable addition amount of agarose is 0.5 to 25% by weight.

  According to another preferred embodiment of the present invention, the gel matrix preferably comprises a synthetic gel. Examples of preferred synthetic gels include polyacrylamide, polyvinylpyrrolidone, sodium polyacrylate, polyacrylamide with PVA, polyethylene oxide, N-alkyl-modified (meth) acrylamide derivatives, N, N-dimethylacrylamide, N, N-diethylacrylamide. , Acryloylmorpholine, N-methylacrylamide, N-ethylacrylamide, N- (iso) propylacrylamide, N-butylacrylamide, N-hydroxymethylacrylamide, N-hydroxyethylacrylamide, N-hydroxypropylacrylamide, N-hydroxybutylacrylamide , (Meth) acrylic acid, t-butyl (meth) acrylamide sulfonic acid, sulfopropyl (meth) acrylate, (meth) acrylic acid, Taconic acid, (poly) alkylene glycol (meth) acrylate, hydroxyethyl (meth) acrylate, polyethylene glycol (meth) acrylate, hydroxypropyl (meth) acrylate, polypropylene glycol (meth) acrylate, glycerin (meth) acrylate, methylene bis (meth) ) Acrylamide, ethylenebis (meth) acrylamide, (poly) ethylene glycol di (meth) acrylate, (poly) propylene glycol di (meth) acrylate, glycerin di (meth) acrylate, glycerin tri (meth) acrylate, tetraallyloxyethane , And mixtures thereof, more preferably polyacrylamide gels.

  According to a preferred embodiment of the present invention, the gel sheet of the present invention comprises: (1) a method of adding an electrolyte and a gelling agent to water and dissolving by heating to prepare a gel; ) After forming a gel only with a gelling agent and processing it into a desired sheet shape, it can be produced by a method of immersing it in an electrolyte solution and dispersing the electrolyte in the gel. In particular, depending on the combination with the electrolyte used, the gelling agent may not be gelled by mixing, heating, or cooling. Even in such a case, the gel sheet is produced according to the method (2) above. can do.

(2) Water-absorbent sheet According to another preferred embodiment of the present invention, the water-containing substrate is preferably a water-absorbent substrate. That is, in this aspect, the electrolyte-containing sheet is configured as a water absorbent sheet. And when a water absorbing sheet is used as an electrolyte medium, detection sensitivity and detection accuracy equivalent to the case where an electrolytic solution is used can be obtained. That is, the photocurrent can be accurately detected by using the water absorbent sheet.

According to a preferred embodiment of the present invention, the water absorbent sheet preferably has a moisture content of 20% or more, more preferably 30% or more, and further preferably 40% or more. When the moisture content is such, a high photocurrent can be detected when the photocurrent is detected, and the detection sensitivity is improved. The water content is determined from (water content around 1 mm ³ ) / (density of water-absorbing substrate). The moisture content referred to here is the moisture content of the water-absorbent sheet when detecting the photocurrent, and it does not have to satisfy the above moisture content during storage, as will be described later.

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

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

  In the present invention, the water-absorbing base material is natural fibers such as cotton, hemp, wool, silk, and cellulose; pulp fibers used in filter paper and papermaking; regenerated fibers such as rayon; glass fibers used in filter paper; felts and sponges It is preferable to contain at least one kind of fiber selected from synthetic fibers used for the above, and it is not limited as long as it is a water-absorbing substrate exhibiting an appropriate strength, water content, and adhesion to an electrode. In addition, the processing method of the fiber used for the water absorbing base material of the present invention is not limited to a specific processing method.

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

  According to a preferred aspect of the present invention, the electrolyte-containing absorbent sheet of the present invention is used after (1) being processed into a desired sheet shape and immersed in a water-based electrolyte, or (2) a desired sheet. After being processed into a shape, immersed in an electrolytic solution and dried, water can be dropped before use.

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

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

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

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

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

  According to a more preferred embodiment of the present invention, it is preferable that the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid having complementarity to the nucleic acid. More preferably, the probe substance has a complementary portion of 15 bp or more to the nucleic acid. The specific binding process of the test substance to the working electrode in this embodiment is shown in FIGS. 22 (a) and 22 (b). As shown in these figures, a single-stranded nucleic acid 221 as a test substance is hybridized with a single-stranded nucleic acid 224 having complementarity as a probe substance carried on the working electrode 223, and A double-stranded nucleic acid 227 is formed.

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

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

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

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

  In the present invention, the test substance and the probe substance may indirectly bind specifically. That is, according to another preferred embodiment of the present invention, a substance having specific binding properties is used as a test substance, a substance that specifically binds to the test substance is used as a mediator, and It is preferable to carry a substance capable of binding to the working electrode as a probe substance. Thereby, even a substance that cannot specifically bind to the probe substance can be detected by specifically binding indirectly to the working electrode via the mediator substance. In this embodiment, preferred examples of the combination of the test substance, the mediator, and the probe substance include a ligand, a receptor protein molecule that can accept the ligand, and two molecules that can specifically bind to the receptor protein molecule. A combination of nucleic acids in a strand is mentioned. Preferable examples of the ligand include exogenous endocrine disrupting substances (environmental hormones). An exogenous endocrine disrupting substance is a substance that binds to DNA via a receptor protein molecule and produces toxicity by affecting its gene expression. According to the method of the present invention, the receptor produced by a test substance is used. It is possible to easily monitor the binding of a protein such as a body to DNA. The specific binding process of the test substance to the working electrode in this embodiment is shown in FIG. As shown in FIG. 23, a ligand 230 as a test substance first specifically binds to a receptor protein molecule 231 that is a mediator. Then, the receptor protein molecule 233 to which the ligand is bound specifically binds to a double-stranded nucleic acid 234 as a probe substance.

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

(2) Sensitizing dye In the present invention, in order to detect the presence of a test substance by photocurrent, the test substance is directly or indirectly specifically bound to the probe substance in the presence of the sensitizing dye. Then, the sensitizing dye is fixed to the working electrode by the binding. Therefore, in the present invention, the test substance 221 or the mediating substance 231 can be previously labeled with sensitizing dyes 222 and 232 as shown in FIGS. In addition, when using a sensitizing dye 228 that can be intercalated with a conjugate 227 (for example, a double-stranded nucleic acid after hybridization) of a test substance and a probe substance as shown in FIG. By adding a sensitizing dye to the solution, the sensitizing dye can be fixed to the probe substance.

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

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

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

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

  According to a preferred embodiment of the present invention, the working electrode has an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation, and a probe material is provided on the surface of the electron accepting layer. Is preferably provided. According to a more preferred aspect of the present invention, it is preferable that the working electrode further comprises a conductive substrate, and an electron accepting layer is formed on the conductive substrate. An electrode of this embodiment is shown in FIGS. 22 and 23 includes a conductive substrate 225 and an electron-accepting layer 226 formed on the conductive substrate and containing an electron-accepting material. A probe substance is carried on the surface of the electron accepting layer 226.

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

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

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

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

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

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

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

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

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

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

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

  Preferred examples of the application method of the electron acceptor dispersion or colloidal solution include the roller method as the application system, the dip method, the air knife method as the metering system, the blade method, etc., and the application and metering in the same part. The wire bar method disclosed in Japanese Patent Publication No. 58-4589, the slide hopper method, the extrusion method, the curtain method, the spin method, and the spray method described in US Pat. Nos. 2,681,294, 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) in which tin oxide is doped with fluorine (FTO), the thickness of the electron-accepting layer is 1 nm. It is preferable that it is above, More preferably, it is 10 nm-1 micrometer.

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

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

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

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

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

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

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

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

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

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

(5) Electrode unit According to the preferable aspect of this invention, you may use the electrode unit by which a working electrode and a counter electrode are patterned on the same plane. A preferred electrode unit includes an insulating substrate, and a working electrode that is provided locally on the insulating substrate and includes an electron-accepting layer that includes an electron-accepting material that can accept electrons emitted by a sensitizing dye in response to photoexcitation. And a counter electrode provided on the same plane as the working electrode of the insulating substrate and spaced apart from the working electrode. An example of such an electrode unit is shown in FIG. The electrode unit 271 shown in FIG. 24 includes an insulating substrate 272, a working electrode 273, and a counter electrode 274. The insulating substrate 272 is a substrate having insulating properties so that the working electrode 272 and the counter electrode 273 are not short-circuited. The working electrode 273 is provided locally on the insulating substrate 272 and includes an electron accepting layer containing an electron accepting material capable of accepting electrons emitted by the sensitizing dye in response to photoexcitation. The counter electrode 274 is provided on the same surface as the working electrode 273 of the insulating substrate 272 and spaced from the working electrode 273. Lead wires 272 ′ and 274 ′ are provided so as to extend from the working electrode 272 and the counter electrode 274, respectively.

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

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

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

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

  The working electrode thus fixed with the sensitizing dye is brought into contact with the electrolyte together with the counter electrode, and the sensitizing dye is photoexcited by irradiating the working electrode with light, from the photoexcited sensitizing dye to the working electrode. The photocurrent flowing between the working electrode and the counter electrode due to the electron movement is detected. As a sensor unit at that time, a sensor chip using the electrolyte-containing sheet of the present invention is used.

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

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

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

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

  According to a more preferred embodiment of the present invention, the test substance and the second test substance are preferably antigens, and the probe substance is preferably an antibody. FIG. 25 shows the step of immobilizing the test substance and the second test substance on the probe substance in this embodiment. As shown in FIG. 25, the antigen 241 labeled with the sensitizing dye and the antigen 242 not labeled with the dye compete with each other and specifically bind to the antibody 243. Therefore, since the amount of the dye-labeled antigen 243 that specifically binds to the antibody decreases as the amount of the antigen 242 that is not dye-labeled increases, the detection current value decreases as the second analyte concentration increases. You can get a line.

Example A1: Photocurrent measurement using gel sheet (1) Preparation of dye-labeled DNA-immobilized working electrode Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (AI Special) as a glass substrate for the working electrode (Manufactured by Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50 mm × 26 mm) was prepared. This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, in order to remove sodium hydroxide, shaking 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 and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2 vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was dipped in this coupling treatment solution and slowly shaken for 15 minutes. Next, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling treatment solution by changing methanol three times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, the adjusted (100 nM) rhodamine-labeled ssDNA (25mer) was kept at 95 ° C. for 10 minutes, then immediately transferred onto ice and kept for 10 minutes to denature the DNA. The denatured DNA was filled in 5 μl each in 9 openings of the previously prepared seal on the glass, and kept at 95 ° C. for 10 minutes to evaporate the solvent. Then, 120 mJ of ultraviolet light was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the labeled ssDNA on the glass substrate. The seal was peeled off from the glass substrate, the glass substrate was shaken in 0.2% SDS solution for 15 minutes × 3 times, and rinsed with 3 changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. Thus, a dye-labeled DNA-immobilized working electrode was obtained.

(2) Preparation of gel sheet An aqueous solution containing 0.1 M, 0.2 M, and 0.5 M tetrapropylammonium iodide (NPr ₄ I) was prepared. Agarose (Agarose Standard Low-mr for electrophoresis, BIO-Rad) having a final concentration of 1% was added to this aqueous solution and heated in an autoclave at 121 ° C. for 20 minutes to dissolve the agarose. The liquid in which agarose was dissolved was poured between two glass plates sandwiching a 1.0 mm-thick packing and allowed to cool and solidify at room temperature to obtain a gel sheet. The obtained gel sheet was cut into an appropriate size with a cutter and processed into a photocurrent measurement size.

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

(Ii) Measurement using electrolytic solution (comparison)
For comparison, the same measurement as in the above (i) was performed using an electrolytic solution instead of the gel sheet. Specifically, the dye-labeled DNA immobilization working electrode obtained in the above (1) and a counter electrode formed by depositing platinum on a glass plate were prepared. A gasket having a thickness of 1 mm was sandwiched between both electrodes, and the gap was filled with the 0.2M NPrI ₄ solution prepared in the above (1). At this time, the working electrode was placed so that the surface on which the ssDNA was immobilized was opposed to the platinum deposition surface of the counter electrode. With both electrodes connected to the electrochemical analyzer, the working electrode was irradiated with a laser light source (output 60 mW, diameter of irradiated region 1 mm, wavelength 530 nm green laser), and the current value observed at that time was recorded.

  The results are shown in FIG. As shown in FIG. 26, when the gel sheet was used, a much higher photocurrent was detected over the entire electrolyte concentration range of 0.1 to 0.5M than when the electrolyte was used.

Example A2: Influence of gel concentration (1) Preparation of dye-labeled DNA immobilization working electrode Dye-labeled DNA immobilization action in the same manner as in Example A1, except that the ssDNA concentration to be immobilized was changed to two concentrations of 10 nM and 100 nM. An electrode was produced.
(2) Preparation of gel sheet A gel sheet was prepared in the same manner as in Example A1, except that the agarose concentration was changed to 0.5%, 4%, 8%, and 10%.
(3) Photocurrent measurement Using each working electrode with different adsorption and immobilization concentration prepared in (1) above and each gel sheet with different agarose concentration prepared in (2) above, photocurrent was measured in the same manner as in Example A1. It was measured.

  The results are shown in FIG. As shown in FIG. 27, almost no influence of the agarose concentration on the photocurrent value was observed over the entire region of the agarose concentration of 0.5% to 10%. Therefore, it is presumed that there is no adverse effect on photocurrent detection even if a relatively large amount of gel is used from the viewpoint of obtaining sufficient strength.

Example A3: Effect of gel sheet thickness (1) Preparation of dye-labeled DNA-immobilized working electrode Dye-labeled in the same manner as in Example A1 (1) except that the ssDNA concentration to be immobilized was changed to two concentrations of 10 nM and 100 nM. A DNA-immobilized working electrode was prepared.
(2) Production of gel sheet A gel sheet was produced in the same manner as in Example A1, except that the agarose concentration was fixed at 1% and the thickness of the gel sheet was 1 mm, 2 mm, and 3 mm.
(3) Photocurrent measurement Using each working electrode with different adsorption and immobilization concentration prepared in (1) above and each gel sheet with different thickness produced in (2) above, photocurrent was measured in the same manner as in Example A1. It was measured.

  The results are shown in FIG. As shown in FIG. 28, almost no influence on the photocurrent value was observed over the entire range of the gel sheet thickness of 1 mm to 3 mm. Therefore, it is presumed that there is no adverse effect on photocurrent value detection even if the gel sheet thickness is increased from the viewpoint of obtaining sufficient strength.

Example A4: Examination of gel sheet preparation method When an electrolyte and a gelling agent (agarose) are added to water and dissolved by heating to prepare a gel (hereinafter referred to as mixed preparation), and after the gel is formed with only the gelling agent The influence on the photocurrent measurement when the electrolyte was dispersed in the gel by immersing it in the electrolyte solution (hereinafter referred to as immersion preparation) was examined. Specifically, in mixing preparation, a gel sheet having a thickness of 1 mm was prepared in the same manner as in Example A1, and cut into an appropriate size. On the other hand, in the case of immersion preparation, a gel sheet having a thickness of 1 mm was prepared in the same manner as in Example 1 except that no electrolyte was used, and the gel sheet was cut into an appropriate size and then placed in a 0.2M NPr ₄ I solution. The gel sheet was immersed overnight at room temperature. Subsequently, the gel sheet was taken out of the solution, and the residual reducing agent solution adhering thereto was washed away with pure water, and then drained. Each gel sheet was sandwiched between the working electrode and the counter electrode, and photocurrent measurement was performed in the same manner as in Example A1 (3). In addition, working electrodes prepared using solutions with rhodamine-labeled ssDNA concentrations of 0 nM and 10 nM were also prepared, and photocurrent measurement was performed in the same manner as described above.

The results are shown in FIG. As shown in FIG. 29, it can be seen that the gel sheet prepared by the immersion preparation can detect the same level of photocurrent as the gel sheet prepared by the mixing preparation, that is, the same performance can be obtained. Therefore, even when the combination of the electrolyte and the gelling agent is not suitable for mixing preparation (for example, when it is difficult to solidify by heating such as a combination of polyacrylamide gel and NPr ₄ I), the gel According to the dip preparation for impregnation after the formation, the gel sheet can be easily produced.

Example A5: Examination of gel strength of gel sheet (1) Preparation of gel sheet containing no electrolyte No electrolyte is used and the final concentration of agarose is 0.1%, 0.5%, 0.8%, 1%, 2 %, 4%, 8%, and 15%, and a gel sheet was prepared in the same manner as in Example A1.
(2) Strength measurement of gel sheet The gel strength (g / cm ² ) of the obtained gel sheet was measured by MODE-2 of “Rheometer CR200D” (manufactured by Sun Kagaku Co., Ltd.). FIG. 30 shows a schematic view of the “rheometer CR200D”. As shown in FIG. 30, this measuring apparatus includes a table 281 and a rod-shaped adapter 282. The rod-shaped adapter 282 used was an adapter NO. The diameter of the surface that contacts the gel sheet is 15 mm. As shown in FIG. 31, in this measurement, a gel sheet having a shape of 30 mm × 30 mm and a thickness of 1 mm is placed on a table 281 and the rod-shaped adapter 282 enters a depth of 0.8 mm at a speed of 100 mm / min. The gel was compressed and the maximum load applied at that time was measured (the load was set to HOLD). At that time, the gel sheet was installed so that the rod-shaped adapter did not protrude from the gel sheet. This measurement was performed three times by exchanging the gel sheet, and the average was converted to the load per surface area to obtain the gel strength (g / cm ² ).

The results are shown in FIG. As shown in FIG. 32, it can be seen that the gel strength increases as the added amount of agarose increases. In addition, gelation did not occur when the agarose concentration was 0.1%, but when the agarose concentration was 0.5%, a gel sheet that could be handled independently was obtained, and the gel strength at that time was 169.9 g. / Cm ² . From the above, in order to form a gel sheet that can be handled alone, it is desirable that the gel strength is at least 100 g / cm ² or more.

Example A6: Gel sheet using polyacrylamide A 7.5% polyacrylamide gel (thickness 1 mm) was cut into an appropriate size and immersed in a 0.2M NPr ₄ I solution at room temperature overnight to impregnate with a reducing agent. I let you. After the remaining reducing agent solution was washed away with pure water and drained, the photocurrent was measured in the same manner as in Example A1. For reference, a gel sheet of the same thickness was prepared using 1% by weight agarose, and similarly immersed in a 0.2M NPr ₄ I solution overnight at room temperature and impregnated with a reducing agent. Photocurrent was measured in the same manner as A1. A dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example A1, and working electrodes prepared using solutions with rhodamine-labeled ssDNA concentrations of 0 nM and 10 nM were also prepared, and photocurrent measurement was performed as described above. .

  The results are shown in FIG. As shown in FIG. 33, in the gel sheet using acrylamide, a photocurrent comparable to that of the gel sheet using agarose was measured.

Example A7: Examination of various electrolytes Photocurrent measurement was performed using various electrolytes. Specifically, 1% by weight agarose was used as a gelling agent, and the concentration of each reducing agent was fixed at 0.2 M to prepare a gel sheet. As the electrolyte, use _{NaI, KI, CaI 2, LiI} , NH 4 I, tetrapropylammonium iodide _(NPr 4 I), sodium thiosulfate _{(Na 2} S 2 _{O 3)} and sodium sulfite _(Na 2 SO ₃₎ It was. Agarose and an electrolyte were added to water, and the mixture obtained by heating and dissolving in an autoclave in the same manner as in Example A1 was allowed to cool at room temperature, solidified, and cut into an appropriate size to prepare a gel sheet. The photocurrent was measured in the same manner as in Example A1. A dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example A1, and working electrodes prepared using solutions with rhodamine-labeled ssDNA concentrations of 0 nM and 10 nM were also prepared, and photocurrent measurement was performed as described above. .

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

Example A8: Single nucleotide polymorphism (SNPs) detection In this example, a gel sheet was applied to detect a single nucleotide polymorphism of the p53 gene. A perfect match probe, a single nucleotide variant probe, and a perfect mismatch probe were immobilized on the working electrode side. Each base sequence was as follows.
Perfect match (PM) probe: 5'-NH2-AGGATGGGCCTCCAGGTTCATGCCCGC-3 '(SEQ ID NO: 1)
Single nucleotide variant (SNP) probe: 5'-NH2-AGGATGGGCCCTCCGGTTCATGCCCGC-3 '(SEQ ID NO: 2)
Complete mismatch (MM) probe: 5'-NH2-GCGGCATGAACCGGAGGCCCCATCT-3 '(SEQ ID NO: 3)

The base sequence of the target DNA to be hybridized with these probes was as follows.
Target DNA: 5'-Rhodamine-GCGGCATGAACCTGAGGCCCCATCT-3 '(SEQ ID NO: 4)

Fluorine-doped tin oxide (F-SnO ₂ : FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50 mm × 26 mm) is prepared as a glass substrate for the working electrode did. This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, in order to remove sodium hydroxide, shaking 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 and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2 vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was dipped in this coupling treatment solution and slowly shaken for 15 minutes. Next, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling treatment solution by changing methanol three times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, the probe DNA (25mer) having a perfectly matched strand, a single nucleotide variant, and a completely mismatched strand prepared to 1 μM was held at 95 ° C. for 10 minutes, and then immediately transferred to ice and held for 10 minutes to denature the DNA. . The denatured DNA was filled in 5 μl each in 9 openings of the previously prepared seal on the glass, and kept at 95 ° C. for 10 minutes to evaporate the solvent. Thereafter, UV light of 120 mJ was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the probe DNA on the glass substrate (three spots were immobilized for each probe). The seal was peeled off from the glass substrate, the glass substrate was shaken in 0.2% SDS solution for 15 minutes × 3 times, and rinsed with 3 changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. In this way, a probe DNA fixed working electrode was obtained.

  Thereafter, 5 × SSC, 0.5% SDS solution containing the target DNA was placed on the electrode on which the probe was fixed and sealed with a cover glass, and kept at 37 ° C. for 10 hours. Thereafter, the electrode was placed on a rack, immersed in a water bath containing 5 L of 0.2 × SSC, 0.2% SDS solution at 63 ° C. for 1 minute, and then rinsed twice with water.

  The working electrode thus obtained was incorporated into a measurement cell, and an XY stage for automatic light source movement was mounted on the measurement cell. In the case of using a gel sheet, the cell portion was configured such that the working electrode and the platinum counter electrode were opposed to each other to prevent a short circuit due to contact between both electrodes and sandwich the gel sheet. When using an electrolyte, a silicon sheet with a film thickness of 500 μm is used for the purpose of creating a space for filling the electrolyte by preventing the short circuit due to contact between the working electrode and the platinum counter electrode. Inserted. The silicon sheet has a sufficiently large hole for all the spots to enter, and the electrolyte solution sent here accumulates, so that the DNA immobilized on the working electrode comes into contact. Both the working electrode and the counter electrode were connected to a highly sensitive ammeter via a spring probe in electrical contact.

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

  FIG. 35 shows the result when the target DNA concentration is 1 μM, and FIG. 36 shows the result when the target DNA concentration is 100 nM. As shown in FIGS. 35 and 36, when the target concentration was 1 μM, single nucleotide polymorphisms (SNPs) could be detected as a difference in photocurrent values regardless of whether the electrolyte solution or the gel sheet was used. However, in the case of 100 nM, it was only the form of the gel sheet that could detect SNPs as a difference in photocurrent value.

Example B1: Photocurrent measurement using electrolyte-containing water-absorbent sheet (1) Preparation of dye-labeled DNA-immobilized working electrode Fluorine-doped tin oxide (F-SnO2: FTO) coated glass as a glass substrate for the working electrode (Manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50 mm × 26 mm) was prepared. This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking for 5 minutes in ultrapure water was performed 3 times with the water changed. After the glass substrate was taken out and air was blown to scatter the remaining water, the glass substrate was immersed in anhydrous methanol for dehydration.

Using 95% methanol and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was immersed in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling solution, and the methanol was changed 3 times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, 5′-end rhodamine-labeled ssDNA (25mer) adjusted to 100 nM and unlabeled ssDNA (24mer) adjusted to 100 nM were held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes. Then, the DNA was denatured. The denatured DNA was filled in 5 μl each in 9 openings of the seal on the glass prepared previously, and kept at 95 ° C. for 10 minutes to evaporate the solvent. Then, 120 mJ ultraviolet light was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the labeled ssDNA on the glass substrate. The seal was peeled off from the glass substrate, and the glass substrate was shaken in a 0.2% SDS solution for 15 minutes × 3 times and rinsed with three changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. Thus, a dye-labeled DNA-immobilized working electrode was obtained. The base sequence of the probe DNA used here is as follows.
5 'terminal rhodamine labeled ssDNA (probe 1): 5'-Rho-GCGGCATGAACCTGAGGCCCATCCT-3' (SEQ ID NO: 5)
Unlabeled ssDNA (probe 2): 5′-TTGAGCAAGTTCAGCCTGGTTAAG-3 ′ (SEQ ID NO: 6)

(2) Preparation of electrolyte-containing absorbent sheet An aqueous solution containing 0.2 M, 0.4 M, and 0.6 M tetrapropylammonium iodide (NPr ₄ I) was prepared. A 0.9 mm thick blotting filter paper (CB-13A; Ato Co., Ltd.) cut to 26 mm × 20 mm was immersed in this aqueous solution, and lightly drained to obtain an electrolyte-containing absorbent sheet.

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

  The results are shown in FIG. As shown in FIG. 37, an increase in photocurrent was observed depending on the concentration of tetrapropylammonium iodide, but it is clear that any concentration of tetrapropylammonium iodide can be used for measurement. Became.

Example B2: Examination of various electrolytes Photocurrent measurement was performed using various electrolytes. Specifically, filter paper was used as the water-absorbing substrate, and the concentration of each reducing agent was fixed to 0.2 M, to prepare an electrolyte-containing absorbent sheet. As the electrolyte, use _{NaI, KI, CaI 2, LiI} , NH 4 I, tetrapropylammonium iodide _(NPr 4 I), sodium thiosulfate _{(Na 2} S 2 _{O 3)} and sodium sulfite _(Na 2 SO ₃₎ It was. Prepare electrolytes containing these various electrolytes and water, immerse 0.9mm thick blotting filter paper (CB-13A; Ato Co., Ltd.) cut into 26mm x 20mm, drain lightly, and absorb electrolyte Sex sheet was obtained. The photocurrent was measured in the same manner as in Example B1. The dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example B1. Working electrodes prepared using solutions with a rhodamine-labeled ssDNA concentration of 10 nM and a rhodamine-unlabeled ssDNA concentration of 100 nM were also prepared, and light was applied as described above. Current measurement was performed.

  The results are shown in FIG. As shown in FIG. 38, any of the studied electrolytes showed an increase in photocurrent depending on the amount of immobilized ssDNA, and it was revealed that it can be used for measurement.

Example B3: Effect of thickness of electrolyte-containing absorbent sheet (1) Preparation of dye-labeled DNA immobilization working electrode The same as Example B1 (1) except that the concentration of ssDNA to be immobilized was changed to two concentrations of 100 nM and 1 μM. Thus, a dye-labeled DNA-immobilized working electrode was prepared.
(2) Preparation of electrolyte-containing absorbent sheet
Prepare an electrolyte solution with tetrapropylammonium iodide (NPr4I) at a concentration of 0.2 M and water, and stack one, two, and three sheets of 0.9 mm thick blotting filter paper (CB-13A; Ato Co., Ltd.) And each was immersed in electrolyte solution and the electrolyte containing absorptive sheet was produced.
(3) Photocurrent measurement Similar to Example B1, using the working electrode having spots with different immobilization concentrations prepared in (1) above and the electrolyte-containing absorbent sheets having different thicknesses prepared in (2) above. The photocurrent was measured.

  The results are shown in FIG. As shown in FIG. 39, almost no influence on the photocurrent value was observed over the entire thickness of the electrolyte-containing absorbent sheet of 0.9 mm to 2.7 mm. Therefore, it is presumed that there is no adverse effect on the detection of the photocurrent value even if the electrolyte-containing absorbent sheet thickness is increased from the viewpoint of obtaining sufficient strength.

Example B4: Examination of each absorbent substrate As a water-absorbent substrate, 0.9 mm thick blotting filter paper (CB-13A; Ato Co., Ltd.), felt (thickness: 1 mm, density: 0.00049 g / mm ³ ), Cardboard (thickness: 0.5 mm, density: 0.00071 g / mm ³ ), glass fiber filter paper (GF / D; Whatman: thickness: 0.68 mm), coated paper containing pulp fibers and synthetic fibers (thickness: 0.14 mm) , Density: 0.00111 g / mm ³ ), and photocurrent was measured in the same manner as in Example B1, except that a membrane filter mainly containing a fluorine resin (JCWP09025; MILLIPORE: thickness: 0.1 mm) was used. The dye-labeled DNA-immobilized working electrode was prepared in the same manner as in Example B1, and a working electrode prepared using each solution with rhodamine-labeled ssDNA concentrations of 100 nM and 1 μM and an unlabeled ssDNA concentration of 100 nM was also prepared. Photocurrent was measured in the same manner as B1. As the electrolytic solution, an aqueous solution containing 0.2 M tetrapropylammonium iodide (NPr ₄ I) was used.

  The results are shown in FIG. As shown in FIG. 40, in all the electrolyte-containing water-absorbent sheets, an increase in photocurrent was recognized depending on the amount of immobilized ssDNA, and it was revealed that the electrolyte can be used.

Example B5: Examination of Moisture Content of Electrolyte-Containing Absorbent Sheet (1) Measurement of Moisture Content First, 0.4M was added to a filter paper for blotting (CB-13A; Ato Co., Ltd.) with a thickness of 0.9mm cut into 26mm x 20mm. 500 μl of an aqueous solution containing tetrapropylammonium iodide (NPr ₄ I) was dropped, and the filter paper was completely immersed in the electrolytic solution. In this way, six filter papers impregnated with the electrolyte were prepared. Next, each impregnated filter paper was dried at 50 ° C. for 0 hour, 0.25 hour, 0.5 hour, 1 hour, 1.25 hour, and 1.5 hour, respectively. The weight of the filter paper after drying was measured, and after removing the weight of tetrapropylammonium iodide, the water content around 1 mm ³ was calculated. Thereafter, the ratio of (water content around 1 mm ³ ) / (filter paper density) was calculated and used as the water content of the electrolyte-containing absorbent sheet. The density of the blotting filter paper is 0.00049 g / mm ³ .
(2) Photocurrent measurement The photocurrent was detected in the same manner as in Example B1 using the electrolyte-containing absorbent sheet having each moisture content prepared in (1).

  The results are shown in FIG. As FIG. 41 shows, it turns out that a photocurrent is so high that the moisture content of an electrolyte containing absorptive sheet is high. Photocurrent could not be detected when the water content was 2.2%, but photocurrent could be detected when the water content was 25.3%. From the above, it is desirable that the electrolyte-containing absorbent sheet capable of detecting photocurrent has a water content of at least 20%.

Example B6: Single nucleotide polymorphism (SNPs) detection using various electrolytes In this example, an electrolyte-containing absorbent sheet was applied to detect a single nucleotide polymorphism of the p53 gene. A perfect match probe, a single nucleotide variant probe, and a perfect mismatch probe were immobilized on the working electrode side. Each base sequence was as follows.
Perfect match (PM) probe: 5'-AGGATGGGCCTCAGGTTCATGCCGC-3 '(SEQ ID NO: 1) Single nucleotide variant (SNP) probe: 5'-AGGATGGGCCTCCGGTTCATGCCGC-3' (SEQ ID NO: 2)
Complete mismatch (MM) probe: 5'-GCGGCATGAACCGGAGGCCCATCCT-3 '(SEQ ID NO: 3)

The base sequence of the target DNA to be hybridized with these probes was as follows.
Target DNA: 5'-Rhodamine-GCGGCATGAACCTGAGGCCCATCCT-3 '(SEQ ID NO: 4)

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

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

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

The working electrode and the electrolyte-containing absorbent sheet thus obtained were assembled in a measurement cell, and an XY stage for automatic light source movement was attached on the measurement cell. The structure of the cell part is a highly sensitive ammeter through a spring probe in which the working electrode and the platinum counter electrode are opposed to each other to prevent a short circuit due to contact between both electrodes and the working electrode and the counter electrode are in electrical contact with each other. Connected to. The electrolyte containing absorptive sheet used the filter paper as a water absorptive base material, and fixed the density | concentration of each reducing agent to 0.4M, and produced the electrolyte containing absorptive sheet. Tetrapropylammonium iodide (NPr ₄ I), sodium thiosulfate (Na _{2 S 2} O 3) and sodium sulfite (Na ₂ SO ₃ ) were used as the electrolyte. The manufacturing method is to prepare electrolytes containing various electrolytes and water, soak a 0.9mm thick blotting filter paper (same as in Example B1) cut into 26mm x 20mm, drain lightly, and electrolyte-containing absorbent sheet Got.

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

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

Example B7: Single nucleotide polymorphism (SNPs) detection using dried electrolyte-containing absorbent sheet After preparing an electrolyte-containing absorbent sheet, it was dried at 50 ° C. for 2 hours to obtain a working electrode and dried electrolyte-containing absorbent. Photocurrent was detected in the same manner as in Example B6 except that 300 μl of water was dropped onto the electrolyte-containing absorbent sheet immediately before photocurrent detection after the sheet was incorporated into the measurement cell (dry type). For comparison, an electrolyte-containing absorbent sheet prepared by immersing a water-absorbing substrate in an electrolyte just before photocurrent detection was used in the same manner as in Example B6 (immersion type).

  The results are shown in FIG. Even when the electrolyte-containing absorbent sheet dried from the results was used, single nucleotide polymorphisms (SNPs) could be detected as a difference in photocurrent values.

Example B8: Comparison between aqueous electrolyte and electrolyte-containing absorbent sheet Electrolyte containing 0.4 M tetrapropylammonium iodide (NPr ₄ I) and water, and a water-absorbing substrate just before photocurrent detection as in Example B6 SNPs were detected in each of the electrolyte-containing absorbent sheets prepared by immersing the membrane in the electrolyte solution, and the detection accuracy was compared between the case where the aqueous electrolyte solution was used and the case where the electrolyte-containing absorbent sheet was used. SNPs detection of the electrolyte-containing absorbent sheet was performed in the same manner as in Example B6. Detection of SNPs using an aqueous electrolyte was performed in the same manner as in Example B6 for preparing the electrode substrate, and a flow cell type measurement cell was used for measurement. 47 and 48 show a sectional view and an exploded view of the flow cell type measurement cell according to the first embodiment, respectively.

  The flow cell type measurement cell shown in FIGS. 44 and 45 is provided with a counter electrode 353 on a substrate 357, and an electrolyte solution or cleaning solution supply hole 362 and a discharge hole 363 are formed on the substrate 357, and on the counter electrode 353. Is provided with an insulating spacer 364 having a space 354 for containing an electrolytic solution, a working electrode 352 is provided on the insulating spacer 364, and a plurality of electron-accepting layers 355 are spaced apart from the surface of the working electrode 352 facing the space 354. Is formed. A working electrode contact 358 is provided through the substrate 357 so as not to interfere with the counter electrode 353. The photocurrent is taken out by this working electrode contact 358. A pressing member 359 is provided on the working electrode 352, and through holes 360 are formed in the pressing member 359 at positions corresponding to the plurality of electron accepting layers 355. The working electrode 352 is irradiated with light from the light source 356 through the through-hole 360. An ammeter 361 is connected between the working electrode 352 and the counter electrode 353, and the photocurrent flowing through the system by light irradiation is measured by the ammeter.

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

Example C1: Single nucleotide polymorphism (SNPs) detection using the apparatus of FIG. 18 In this example, single nucleotide polymorphism of the p53 gene was detected. A perfect match probe, a single nucleotide variant probe, and a perfect mismatch probe were immobilized on the working electrode side. Each base sequence was as follows.
Exact match (PM) probe: 5'-AGGATGGGCCTCAGGTTCATGCCGC-3 '(SEQ ID NO: 1)
Single nucleotide variant (SNP) probe: 5'-AGGATGGGCCTCCGGTTCATGCCGC-3 '(SEQ ID NO: 2)
Complete mismatch (MM) probe: 5'-GCGGCATGAACCGGAGGCCCATCCT-3 '(SEQ ID NO: 3)

The base sequence of the target DNA to be hybridized with these probes was as follows.
Target DNA: 5'-Rhodamine-GCGGCATGAACCTGAGGCCCATCCT-3 '(SEQ ID NO: 4)

Fluorine-doped tin oxide (F-SnO2: FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50mm x 26mm) was prepared as the glass substrate for the working electrode . This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking for 5 minutes in ultrapure water was performed 3 times with the water changed. After the glass substrate was taken out and air was blown to scatter the remaining water, the glass substrate was immersed in anhydrous methanol for dehydration.

  Using 95% methanol and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was immersed in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling solution, and the methanol was changed 3 times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 9 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, the probe DNA (25 mer) having a perfectly matched strand, a single nucleotide variant, and a completely mismatched strand prepared to 1 μM was held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes to denature the DNA. . 5 μl of this denatured DNA was filled into 9 openings of the previously prepared seal on the glass and kept at 95 ° C. for 10 minutes to evaporate the solvent. Thereafter, UV light of 120 mJ was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the probe DNA on the glass substrate (three spots were immobilized for each probe). The seal was peeled off from the glass substrate, and the glass substrate was shaken in a 0.2% SDS solution for 15 minutes × 3 times and rinsed with three changes of ultrapure water. The glass substrate was immersed in boiling water for 2 minutes and taken out, and then air was blown to scatter the remaining water. Subsequently, the glass substrate was immersed in absolute ethanol at 4 ° C. for 1 minute for dehydration, and air was blown to disperse residual ethanol. In this way, a probe DNA fixed working electrode was obtained. Thereafter, a 5 × SSC, 0.5% SDS solution containing the target DNA adjusted to a concentration of 100 nM was placed on the electrode on which the probe was fixed and sealed with a cover glass for 10 hours at 37 ° C. After that, the cover glass is peeled off in 2 × SSC (room temperature), the electrode is placed on a rack, shaken in a 2 × SSC / 0.2% SDS solution set at 40 ° C. for 30 minutes, and then rinsed with water to dry the electrode. I let you.

The working electrode 71 thus obtained was assembled in the sensor chip case 72 shown in FIG. An electrolyte-containing sheet 73 is placed on the counter electrode 159 of the main body device shown in FIGS. 18 and 20, and the conductive surface of the working electrode 71, that is, the surface on which the probe DNA is fixed is in contact with the electrolyte-containing sheet 73. Placed on. Filter paper was used as a water-absorbent sheet used for the electrolyte-containing sheet. Tetrapropylammonium iodide (NPr ₄ I) is used as the electrolyte (concentration is 0.4M). This is dissolved in water to prepare an electrolyte, and a filter paper with a thickness of 0.9mm cut into 26mm x 20mm is immersed. And drained lightly to obtain an electrolyte-containing sheet.

  From the upper part of the sensor chip, a light source attached to the XY stage was sequentially irradiated to the probe DNA fixed spot on the working electrode. The photocurrent observed at each spot irradiation was recorded. Moreover, it stops temporarily between each opening part of the sensor chip corresponding to each spot, and sets the time when light is not irradiated to a working electrode. The current value observed at this time is defined as a base current value. The difference between the photocurrent value derived from each spot and the base current value immediately before or after that was taken as the observed value.

  The results are shown in FIGS. FIG. 47 shows the change over time in the photocurrent value obtained when each spot is irradiated. FIG. 48 summarizes the differences between each spot-derived current and the base current for each probe DNA, and shows the average and standard deviation (error bars are ± 1 SD). As is clear from FIG. 48, the difference between single bases (difference in current value observed between PM and SNPs) can be statistically significant.

Example C2: Detection of protein (streptavidin) using the apparatus of FIG. 18 This example is a detection example of streptavidin. Biotin-labeled synthetic oligo DNA (25 bases) was immobilized on the working electrode side. As a control, unlabeled synthetic oligo DNA (25 bases) was also immobilized. Rhodamine-labeled streptavidin was brought into contact with these oligo DNAs at different concentrations, and rhodamine-labeled streptavidin was captured by specific binding of biotin-streptavidin to detect rhodamine-derived photocurrent.

  Fluorine-doped tin oxide (F-SnO2: FTO) coated glass (manufactured by AI Special Glass Co., Ltd., U film, sheet resistance: 12Ω / □, shape: 50mm x 26mm) was prepared as a glass substrate for the working electrode . This glass substrate was subjected to ultrasonic cleaning in acetone for 15 minutes and then in ultrapure water for 15 minutes to remove dirt and residual organic matter. The glass substrate was shaken in 5M aqueous sodium hydroxide solution for 15 minutes. Then, for removal of sodium hydroxide, shaking for 5 minutes in ultrapure water was performed 3 times with the water changed. After the glass substrate was taken out and air was blown to scatter the remaining water, the glass substrate was immersed in anhydrous methanol for dehydration.

Using 95% methanol and 5% ultrapure water as a solvent, add 3-aminopropyltrimethoxysilane (APTMS) to 2vol% and stir at room temperature for 5 minutes to prepare a solution for coupling treatment did. The glass substrate was immersed in this coupling treatment solution and slowly shaken for 15 minutes. Subsequently, the glass substrate was taken out and shaken in methanol about 10 times to remove excess coupling solution, and the methanol was changed 3 times. Thereafter, the glass substrate was held at 110 ° C. for 30 minutes to bond the coupling agent to the glass substrate. After the glass substrate was cooled at room temperature, an adhesive seal (thickness: 0.5 mm) having 6 spots of openings with a diameter of 3 mm was placed and brought into close contact therewith. Subsequently, biotin-labeled synthetic oligo DNA (25 bases) or non-labeled synthetic oligo DNA (25 bases) adjusted to 1 μM is held at 95 ° C. for 10 minutes, then immediately transferred to ice and held for 10 minutes to denature the oligo DNA. I let you. 5 μl of this denatured oligo DNA was filled into 6 openings of the previously prepared glass seal (fixed 3 spots for each oligo DNA) and kept at 95 ° C. for 10 minutes to evaporate the solvent. Then, 120 mJ of ultraviolet light was irradiated with a UV crosslinker (UVP CL-1000 type) to immobilize the oligo DNA on the glass substrate. The glass substrate was thoroughly washed with 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween20.
In this way, five working electrodes were prepared in which three spots each of biotin-labeled synthetic oligo DNA and unlabeled synthetic oligo DNA were immobilized. Thereafter, 5 μl of a rhodamine-labeled streptavidin solution was dispensed in an amount of 5 μl into the opening of the seal affixed to the working electrode, sealed with a cover glass, and left at 37 ° C. for 1 hour. Thorough washing was performed using 10 mM HEPES buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween20. At this time, the concentration of rhodamine-labeled streptavidin was changed to 1, 10, 100, 1000, 10000 ng / ml, and one working electrode was used for each concentration.

The working electrode thus obtained was assembled in the sensor chip case shown in FIG. 18 to obtain a sensor chip. An electrolyte-containing sheet is placed on the counter electrode of the main body device shown in FIGS. 16 and 18, and the conductive surface of the working electrode, that is, the surface on which the probe DNA is fixed is placed on the counter-electrode in contact with the electrolyte-containing sheet. did. Filter paper was used as a water-absorbent sheet used for the electrolyte-containing sheet. Tetrapropylammonium iodide (NPr ₄ I) is used as the electrolyte (concentration is 0.4M). This is dissolved in water to prepare an electrolyte, and a filter paper with a thickness of 0.9mm cut into 26mm x 20mm is immersed. And drained lightly to obtain an electrolyte-containing sheet.

  From the upper part of the sensor chip, a light source attached to the XY stage was sequentially irradiated to the oligo DNA-immobilized spot on the working electrode. The photocurrent observed at each spot irradiation was recorded. Moreover, it stops temporarily between each opening part of the sensor chip corresponding to each spot, and sets the time when light is not irradiated to a working electrode. The current value observed at this time is defined as a base current value. The difference between the photocurrent value derived from each spot and the base current value immediately before or after that was taken as the observed value.

  The results are shown in FIG. The difference between the current from each spot and the base current is summarized for each concentration of rhodamine-labeled streptavidin, and shows the average and standard deviation (error bars are ± 1 SD). In spots where unlabeled oligo DNA that does not specifically bind to rhodamine-labeled streptavidin is immobilized, almost no photocurrent is observed, whereas in spots where biotin-labeled oligo DNA is immobilized, it increases in a concentration-dependent manner. A photocurrent was observed.

It is a figure which shows the structure of the fluorescence detection apparatus generally used. It is a disassembled perspective view which shows an example of the sensor chip by this invention. It is a disassembled perspective view which shows another example of the sensor chip by this invention. It is a disassembled perspective view which shows another example of the sensor chip by this invention. It is a disassembled perspective view which shows an example of the structure of the sensor chip receiving part of the apparatus by this invention, and its vicinity. It is an enlarged view of an example of a structure of the sensor chip receiving part of the apparatus shown in FIG. 5 and its vicinity. It is a disassembled perspective view which shows another example of the sensor chip by this invention. It is a disassembled perspective view which shows an example of the structure of the sensor chip receiving part of the apparatus by this invention, and its vicinity. It is a disassembled perspective view which shows another example of the sensor chip by this invention. It is a conceptual diagram which shows the basic composition of the measuring apparatus by this invention. 1 is an external perspective view showing an example of a lower light source type measuring apparatus according to the present invention. It is a perspective view which shows the internal structure of the apparatus shown by FIG. It is a perspective view of the sensor chip receiving part of the apparatus shown by FIG. 11, its vicinity, and the sensor chip with which it is mounted | worn. It is an enlarged view of the sensor chip receiving part shown in FIG. 13 and its vicinity. It is a figure which shows the time-dependent change of the photocurrent which can be detected in the apparatus shown by FIG. 1 is an external perspective view showing an example of an upper light source type measuring apparatus according to the present invention. It is a perspective view which shows the internal structure of the apparatus shown by FIG. It is a figure explaining the structure of the sensor chip receiving part of the apparatus shown by FIG. 16, and its vicinity. It is a figure explaining the main body lid | cover of the apparatus shown by FIG. 16, a pressing member, and a sensor chip. It is a figure which shows the time-dependent change of the photocurrent which can be detected in the apparatus shown by FIG. It is a figure explaining the structure of the sensor chip receiving part of the apparatus shown by FIG. 16, and its vicinity. FIG. 4 is a diagram showing a process of immobilizing a test substance on a probe substance when the test substance is a single-stranded nucleic acid and the probe substance is a single-stranded nucleic acid complementary to the nucleic acid. (A) shows the case where the test substance is previously labeled with a sensitizing dye, and (b) shows the case where a sensitizing dye capable of intercalation is added to a double-stranded nucleic acid. It is a figure which shows the immobilization process to the probe substance of a test substance in case a test substance is a ligand, a mediator is a receptor protein molecule, and a probe substance is a double-stranded nucleic acid. It is a figure which shows an example of an electrode unit. It is a figure which shows the fixation process to the probe substance of a test substance in case the test substance and the 2nd test substance which have the specific binding property which mutually compete are antigens, and a probe substance is an antibody. The photocurrent measured at each tetrapropylammonium iodide (NPr ₄ I) concentration when using the gel sheet (1% agarose gel) obtained in Example A1 and when using the electrolytic solution (aqueous solution). It is a figure which shows a value. It is a figure which shows the relationship between a gel concentration (agarose concentration) and a photocurrent value in each rhodamine labeled ssDNA density | concentration of 10 nM and 100 nM which were obtained in Example A2. It is a figure which shows the relationship between the thickness of a gel sheet, and a photocurrent value in each rhodamine labeled ssDNA density | concentration of 10 nM and 100 nM which were obtained in Example A3. It is a figure which shows the photocurrent value when the gel sheet is prepared by mixing adjustment and when it is prepared by immersion adjustment at the rhodamine labeled ssDNA concentrations of 0 nM, 10 nM, and 100 nM obtained in Example A4. It is the schematic of "Rheometer CR200D" (made by Sun Scientific) used in Example A5. It is a figure explaining the measuring method of the gel strength performed in Example A5. It is a figure which shows the relationship between the gel concentration (agarose concentration) and gel strength which were obtained in Example A5. Photocurrent values obtained in Example A6 when using a gel sheet containing 1% by weight agarose and using a gel sheet containing 7.5% by weight acrylamide at each rhodamine-labeled ssDNA concentration of 0 nM, 10 nM, and 100 nM FIG. It is a figure which shows the photocurrent value at the time of using each electrolyte in each rhodamine labeled ssDNA density | concentration of 0 nM, 10 nM, and 100 nM obtained in Example A7. It is a figure which shows the photocurrent value whose target DNA density | concentration is 1 micromol in the case of using the perfect match (PM) probe, the single nucleotide variation | mutation chain | strand (SNP) probe, and the perfect mismatch (MM) probe obtained in Example A8. . It is a figure which shows the photocurrent value whose target DNA density | concentration is 100 nM at the time of using the perfect match (PM) probe obtained in Example A8, a single nucleotide variation | mutation chain | strand (SNP) probe, and a perfect mismatch (MM) probe. . Photocurrent measured at each tetrapropylammonium iodide (NPr ₄ I) concentration in 100 nM ssDNA and 100 nM rhodamine-labeled ssDNA using the electrolyte-containing absorbent sheet (filter paper) obtained in Example B1 It is a figure which shows a value. It is a figure which shows the photocurrent value at the time of using each electrolyte in each rhodamine labeled ssDNA density | concentration of 0 nM, 10 nM, and 100 nM obtained in Example B2. It is a figure which shows the relationship between the thickness of an electrolyte containing absorptive sheet, and a photocurrent value in each rhodamine labeled ssDNA density | concentration of 100 nM and 1 micromol which were obtained in Example B3. It is a figure which shows the photocurrent value at the time of using each electrolyte containing absorptive sheet in each rhodamine labeled ssDNA density | concentration of 100 nM ssDNA obtained in Example B4, 100 nM, and 1 micromol. It is a figure which shows the relationship between the moisture content of an electrolyte containing absorptive sheet | seat obtained in Example B5, and a photocurrent value. It is a figure which shows the photocurrent value of each electrolyte at the time of using the perfect coincidence (PM) probe, the single nucleotide variation | mutation chain | strand (SNP) probe, and the perfect mismatch (MM) probe obtained in Example B6. In the case of using the single-base mutant chain (SNP) probe and the complete mismatch (MM) probe obtained in Example B7, a case in which the water-absorbing substrate was immersed in an electrolyte immediately before measurement, It is a figure which shows a photocurrent with the case where a sheet | seat is immersed in electrolyte solution, is dried, and water is dripped just before use. It is sectional drawing of an example of the cell for a flow cell type | mold measurement. FIG. 45 is an exploded perspective view of the measurement cell shown in FIG. 44. The light obtained in Example B8 when using an aqueous electrolyte and when using an electrolyte-containing absorbent sheet when using a single nucleotide variant (SNP) probe and a complete mismatch (MM) probe It is a figure which shows an electric current. It is a figure which shows the time-dependent change of the photocurrent value obtained when irradiating each spot obtained in Example C1. It is the figure which arranged the difference of each spot origin current obtained in Example C1, and base current about each probe DNA, and shows a mean and standard deviation (error bar is ± 1SD). It is the figure which arranged the difference of each spot origin current obtained in Example C2, and base current for every density | concentration of rhodamine labeled streptavidin, and has shown the average and the standard deviation (error bar is +/- 1SD).

Claims (19)

A sensor chip that is detachably attached to a measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
The sensor chip is
A working electrode;
A sensor chip case, and
The sensor chip case is
At least one of a recess and a protrusion for holding the working electrode at a predetermined position, and an opening for passing a positioning member for fixing the working electrode at a predetermined position of the measuring device When,
With
A sensor chip in which the specific detection of the test substance is performed by placing an electrolyte-containing sheet on the working electrode or sandwiching the electrolyte-containing sheet between the working electrode and the sensor chip case.   The sensor chip according to claim 1, further comprising an electrolyte-containing sheet on the working electrode or between the working electrode and the sensor chip case.   The sensor according to claim 2, wherein the sensor chip case further includes at least one of a second depression and a second protrusion for placing the electrolyte-containing sheet at a predetermined position. Chip.   The sensor chip according to any one of claims 2 to 4, further comprising a counter electrode on a side opposite to the working electrode of the electrolyte-containing sheet.   5. The sensor chip according to claim 4, wherein the sensor chip case further includes at least one of a third recess and a third protrusion for mounting the counter electrode at a predetermined position. .   The sensor chip case further includes a second opening or a light transmitting portion for allowing light for light excitation to pass through the surface of the working electrode, and at least the second opening of the sensor chip case and The sensor chip according to any one of claims 1 to 5, wherein a portion other than the light transmitting portion that is exposed to the light has a light shielding property.   The sensor chip case has a third opening in at least a part of a region where the working electrode is held, and specific detection of the test substance is performed through the third opening. The sensor chip according to claim 1, which is performed by bringing an electrode into contact with the electrolyte-containing sheet. Specific detection of a test substance using the photocurrent,
A probe electrode to which a test substance is directly or indirectly specifically bound is provided on the surface, and a working electrode having a sensitizing dye fixed by the binding and a counter electrode are brought into contact with the electrolyte-containing sheet;
Light is applied to the working electrode to excite the sensitizing dye, and a photocurrent flows between the working electrode and the counter electrode due to electron transfer from the photoexcited sensitizing dye to the working electrode. The sensor chip according to any one of claims 1 to 7, wherein the sensor chip is performed by a step that includes detecting. A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A sensor chip according to claim 1;
An electrolyte-containing sheet that is placed on the working electrode or sandwiched between the working electrode and the sensor chip case; and
A counter electrode provided on the side opposite to the working electrode of the electrolyte-containing sheet;
A light source for irradiating the working electrode with light;
An XY movement mechanism for moving the light source in the XY direction;
An ammeter for measuring a current flowing between the working electrode and the counter electrode;
Control calculation for controlling the light source, the XY moving mechanism, and the ammeter, receiving a current signal from the ammeter, and determining at least one of the presence, type, and concentration of the test substance based on the electrical signal And a device comprising means. A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A sensor chip according to claim 2;
A counter electrode provided on the side opposite to the working electrode of the electrolyte-containing sheet;
A light source for irradiating the working electrode with light;
An XY movement mechanism for moving the light source in the XY direction;
An ammeter for measuring a current flowing between the working electrode and the counter electrode;
Control calculation for controlling the light source, the XY moving mechanism, and the ammeter, receiving a current signal from the ammeter, and determining at least one of the presence, type, and concentration of the test substance based on the electrical signal And a device comprising means. A measuring device used for specific detection of a test substance using a photocurrent generated by photoexcitation of a sensitizing dye,
A sensor chip according to claim 4;
A light source for irradiating the working electrode with light;
An XY movement mechanism for moving the light source in the XY direction;
An ammeter for measuring a current flowing between the working electrode and the counter electrode;
Control calculation for controlling the light source, the XY moving mechanism, and the ammeter, receiving a current signal from the ammeter, and determining at least one of the presence, type, and concentration of the test substance based on the electrical signal And a device comprising means.   The device according to any one of claims 9 to 11, wherein the measuring device further includes a display device for displaying a result obtained by the control calculation means.   The device according to any one of claims 9 to 11, wherein the measuring device further includes an input device for inputting conditions for measurement.   The apparatus as described in any one of Claims 9-13 further provided with the sensor chip receiving part in which the sensor chip as described in any one of Claims 1-8 is detachably mounted | worn.   The sensor chip receiving portion includes a positioning member for fixing the working electrode at a predetermined position, and an auxiliary member for mounting the sensor chip case at a predetermined position of the measuring device. The device described.   16. The working electrode has a plurality of detection spots, and the sensor chip receiving portion is formed with the opening or light transmitting portion at each position corresponding to the plurality of detection spots. The device described in 1. The device includes a lid portion that is openable and closable with respect to the sensor chip receiving portion, and a convex pressing member that is provided at a position in contact with the working electrode of the lid portion. Provided above the holding member,
The pressing member is made of a light-shielding member that blocks light from the light source, and includes an opening or a light-transmitting portion for allowing light for light excitation to pass through the surface of the working electrode. The device according to any one of claims 14 to 16.   The apparatus according to claim 17, wherein the working electrode has a plurality of detection spots, and the openings or light-transmitting portions are formed at positions corresponding to the plurality of detection spots.   The sensor chip case has an opening in at least a part of a region where the working electrode is placed, and the sensor chip receiving part has a protrusion that can be inserted into the opening. The counter electrode is provided on the sensor chip. When the sensor chip is mounted on the sensor chip receiving portion, the counter electrode comes into contact with the electrolyte-containing sheet by inserting the convex portion into the opening. The device according to any one of claims 14 to 18.

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