JP4015561B2 - Electrochemical online biosensor - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrochemical on-line biosensor, and more specifically, an electric for continuously collecting biological material in a living body or in the vicinity of cultured cells, introducing the biological material into a flow cell, and continuously monitoring the concentration of the biological material. The present invention relates to a chemical online biosensor.
[0002]
[Prior art]
Many attempts have been made to measure biological molecules in real time for elucidation of biological functions and on-site monitoring of patients in the medical field. In continuous measurement of biomolecules using the electrochemical detection method, a solution near the cultured cells is continuously sucked using a syringe pump or the like (for example, see Non-Patent Document 1), or a microdialysis membrane called a microdialysis probe is used. There is known a method of continuously introducing biomolecules collected through a flow cell having electrodes (for example, see Non-Patent Document 2).
[0003]
In addition, in the flow cell, an electrode on which an enzyme that reacts with the target molecule is immobilized is arranged, and the generated hydrogen peroxide is detected by oxidizing or reducing, or the change in dissolved oxygen concentration due to the enzyme reaction, There has been reported a method of continuously observing the concentration of a biological substance by using an enzyme sensor (for example, see Non-Patent Document 3).
[0004]
When detecting biological components using these electrochemical biosensors, oxidizable substances such as ascorbic acid and uric acid, which are widely present in the living body, affect the sensor as contaminants, and it is difficult to accurately quantify them. Met. For this reason, a hollow platinum tube is connected immediately before introduction into the flow cell, and ascorbic acid and the like are oxidized and decomposed by applying a potential to the tube, and the influence of ascorbic acid and the like on the detection electrode is reduced to perform quantification. (For example, refer nonpatent literature 4).
[0005]
In recent years, in order to improve the temporal and spatial resolution of these electrochemical online biosensors, reports have been made on the miniaturization of the sensors. Specifically, a microelectrochemical on-line sensor is formed by forming a thin layer flow path on a silicon or glass substrate by anisotropic etching, dry etching, dicing saw, etc., and bonding it to a substrate having an enzyme-modified electrode. (See, for example, Non-Patent Document 5).
[0006]
[Non-Patent Document 1]
Niwa, Horiuchi, Torimitsu, Biosenser and Bioelectronics, Vol. 12, pp. 311 to 319
[0007]
[Non-Patent Document 2]
Lunte, Scott, Kissinger, Analytical Chemistry, Volume 63, pages 773A-780A
[0008]
[Non-Patent Document 3]
Reach, Wilson, Analytical Chemistry, Vol. 64, pages 381A to 386A
[0009]
[Non-Patent Document 4]
Osborne, Niwa, Kato, Yamamoto, Journal of Neuroscience, 77, 143-150
[0010]
[Non-Patent Document 5]
Niwa, Kurita, Horiuchi, Torimitsu, Electroanalysis, Volume 11, 356-361
[0011]
[Problems to be solved by the invention]
In an electrochemical online biosensor, an enzyme that reacts with a target biomolecule needs to be immobilized on an electrode or in a flow path. However, the anodic bonding method, the thermal fusion method, and the bonding method using hydrofluoric acid, which have been used in the conventional micromachine technology, have a drawback that the activity of the enzyme immobilized at the time of bonding is significantly reduced.
[0012]
In addition, it is possible to maintain enzyme activity in bonding using an adhesive, but it is difficult to form and bond an adhesive layer only to the desired shape and part, and the sample solution flows through the adhesive during bonding. It flowed into the flow path, resulting in a decrease in yield as a biosensor cell electrode and a lack of productivity.
[0013]
Further, when forming the thin layer flow path, it is necessary to use a powerful drug such as an acid or an alkaline solution in the anisotropic etching, and the dry etching method has a disadvantage that an expensive apparatus is required and a long time is required.
[0014]
On the other hand, in biometric measurement using electrochemical online biosensors, ascorbic acid and other substances that act as contaminants are removed, so connecting a platinum tube increases the internal volume of the tube. There was a drawback of leading to a decline in Also, immobilizing biomolecules such as enzymes that remove interfering substances in the tube has the problem that reproducibility and function decrease.
[0015]
The present invention has been made in view of such problems, and the object of the present invention is that it is not necessary to produce a thin layer flow path by etching or the like, and no acid or alkali is used at room temperature. Therefore, it is an object of the present invention to provide an electrochemical on-line biosensor that can be safely and productively produced as a flow cell without reducing the activity of enzymes and antibodies.
[0016]
[Means for Solving the Problems]
In order to achieve the above object, the present invention described in claim 1 An electrochemical on-line biosensor comprising an insulating substrate, a plurality of thin-film electrodes formed on the insulating substrate, a double-sided adhesive sheet that has been subjected to circular drilling, sample introduction means, and sample discharge means A flow cell having a thin layer flow path by bonding the insulating substrate formed with the plurality of thin film electrodes and the polymer substrate with the double-sided adhesive sheet. The plurality of thin film electrodes includes an electrode for removing the interfering substance, a working electrode disposed on the same circumference so as to surround the electrode for removing the interfering substance, and a reference electrode It is characterized by that.
[0023]
As described above, the present invention is achieved by sandwiching a double-sided adhesive thin film sheet that has been perforated in the shape of a target flow path at the time of making into a flow cell, between a substrate having electrodes and a substrate to be made into a flow cell. Make it. This eliminates the need to produce thin-layer channels by etching, etc., compared to conventional products, and does not use acids or alkalis at room temperature. It is possible to make a flow cell with good characteristics.
[0024]
In addition, by using a round thin film electrode and a plurality of thin film electrodes arranged concentrically surrounding the round thin film electrode, a thin layer flow cell is formed so that the solution flows radially from the center of the round electrode, thereby achieving a highly efficient center. It is possible to remove interfering substances such as ascorbic acid with the round electrode and detect the target substance with the surrounding electrode. As a result, it is possible to eliminate the influence of easily oxidizable substances without significantly increasing the dead volume as compared with the prior art.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[Embodiment 1]
FIG. 1 is a structural diagram of an electrochemical online biosensor according to the present invention.
This biosensor is mainly composed of an insulating substrate 1 having a thin film electrode, a double-sided adhesive sheet 2, a polymer substrate 3, a sample introduction tube 4, and a sample discharge tube 5. That is, the insulating substrate 1 on which the thin-film electrodes are formed and the polymer substrate 3 to which the sample introduction tube 4 and the sample discharge tube 5 are connected are bonded with the double-sided adhesive sheet 2 that has been subjected to drilling. Thus, a flow cell having a thin layer flow path is configured.
[0026]
2 (a) to 2 (c) are detailed views of components used in the production of the electrochemical online biosensor according to the present invention, FIG. 2 (a) is an insulating substrate, and FIG. 2 (b) is double-sided adhesive. The sheet, FIG. 2 (c) shows the polymer substrate. In the figure, reference numeral 14 is a platinum thin film electrode, 15 is a round thin film electrode, 16 is an electrode modified with a laminated film in which osmium polyvinylpyridine complex containing horseradish peroxidase is modified in the upper layer and glucose oxidase is modified in the upper layer, and 17 is a silver paste. The coated electrode, 18 is a ring-shaped groove, 19 is a sample introduction hole, and 20 is a sample discharge hole.
[0027]
First, the insulating substrate 1 on which the thin film electrode is formed is spin-coated with a positive photoresist (manufactured by Fuji Hunt) on a glass substrate to a thickness of 1 μm using a spinner (manufactured by Mikasa), and a mask aligner PLA501. (Canon) was used for exposure, and an electrode pattern was formed by alkali development. Thereafter, the glass substrate having the resist pattern described above was attached to a magnetron sputtering apparatus (manufactured by Nippon Seed Co., Ltd.), titanium was sputtered for 5 nanometers, vacuum was maintained, and platinum was sputtered at 45 nm. The glass substrate having a resist, titanium, and platinum was peeled off while applying ultrasonic waves in methyl ethyl ketone to form a platinum thin film electrode pattern 14 as shown in FIG.
[0028]
Then, it cut out using a dicing saw (made by Disco Corporation) at 12x23 mm along the electrode pattern. Further, 1 μl (l is liter) of osmium polyvinylpyridine complex containing horseradish peroxidase was modified on one of the electrodes surrounding the central round thin film electrode 15 by a casting method. After drying at room temperature for 1 hour, glucose oxidase was mixed with 2% bovine serum albumin so as to have a weight ratio of 2%, and glutaraldehyde was added to a final concentration of 0.2%. 1 μl (l is liter) was laminated on the platinum thin film electrode 14 modified with osmium polyvinylpyridine complex containing wasabi peroxidase.
[0029]
Furthermore, one of the electrodes surrounding the central round thin film electrode 15 was modified with a silver paste 7 dissolved in toluene to serve as a reference electrode. As the double-sided adhesive sheet 2, a sheet having a thickness of 50 μm (manufactured by Kimoto) was used. After cutting into a 12 mm square using a cutting plotter (manufactured by MIMAKI), a hole having a diameter of 6 mm was formed in the center of the double-sided PSA sheet cut out using a cutting plotter.
[0030]
The polymer substrate 3 for forming a flow cell was prepared by processing a ring-shaped groove 18 having a width of 1 mm, a diameter of 7 mm, and a depth of 0.3 mm from an acrylic substrate having a thickness of 1 mm using a sand blast method. Then, it cut out using a dancing saw at 12 mm square. Further, a 0.7 mm sample introduction hole 19 and a sample discharge hole 20 were formed on the ring center and on the ring by a drill.
[0031]
A sample introduction tube 4 was formed by connecting a hollow tube (manufactured by BAS) having an outer diameter of 0.65 mm and an inner diameter of 60 microns to the sample introduction hole 19 formed in the center of the ring. Similarly, a hollow tube having an outer diameter of 0.65 mm and an inner diameter of 60 microns was connected to the sample discharge hole 20 formed on the ring to obtain a sample discharge tube 5.
[0032]
Finally, the double-sided adhesive sheet 2 with a circular hole formed in the center is sandwiched between the enzyme-modified platinum thin film electrode substrate and the acrylic substrate on which the sample introduction and sample discharge tubes 4 and 5 are formed. A glicose sensor was obtained by making it into a flow cell.
[0033]
FIG. 3 is a schematic view of a measuring apparatus using the electrochemical online biosensor of the present invention shown in the first embodiment.
The prepared sensor was connected to the sample introduction tube 4 with a microdialysis probe 21 (manufactured by CMA) and further connected to a syringe pump 22 (manufactured by CMA). Moreover, the pad of the platinum thin film electrode substrate was connected to each ALS1000 potentiostat 23 (manufactured by CHI). Changes in the current value output from the potentiostat 23 were recorded in a personal computer (IBM) 24.
[0034]
For measurement, a syringe pump 22 was used, and phosphate buffered saline (PBS) having a pH of 7.4 was continuously introduced into the sensor through the microdialysis probe 21 at a flow rate of 4 μl / min. Moreover, the potential of the thin film electrode was a potentiostat 23, and the potential of the electrode modified with enzyme or the like was applied to the reference electrode modified with silver in the sensor at -50 mV.
[0035]
The sample is introduced into the sensor by immersing the microdialysis probe 21 in a PBS solution 26 containing the target substance after immersing the microdialysis probe 21 in the PBS 25 not containing the target substance to obtain a stable baseline. This was done through the membrane.
[0036]
FIG. 4 is a diagram showing changes in the response current value of the sensor when the microdialysis probe is immersed in a 10 mM glucose solution. After the microdialysis probe 21 was immersed in the sample solution, the reduction current value started to increase after about 1 minute, and reached a constant current value of about 100 nA after another minute. When the microdialysis probe 21 was immersed again in PBS containing no sample, it returned to the baseline after about 1 minute.
[0037]
FIG. 5 is a diagram showing changes in the response current value of the sensor with respect to the 10 mM glucose and 100 μM ascorbic acid mixed solution shown in the first embodiment. Next, when the microdialysis probe 21 was immersed in a mixed solution of 10 mM glucose and 100 μM ascorbic acid, an increase in reduction current was confirmed after about 1 minute, and reached a constant value in the vicinity of about 40 nA. The reduction current value obtained by introducing the mixed solution was significantly reduced as compared with a 10 mM glucose solution not containing ascorbic acid. This is because ascorbic acid reduces the electrode directly or the osmium complex, so that only the glucose concentration cannot be detected accurately.
[0038]
FIG. 6 is a diagram showing a change in response current value when a potential of 500 mV is applied to the central round thin film electrode shown in Embodiment 1 and a 10 mM glucose and 100 μM ascorbic acid mixed solution is introduced into the sensor.
[0039]
A potentiostat 23 was used to apply a potential of 500 mV to the central round thin film electrode 15 with respect to the reference electrode in the sensor, and 10 mM glucose and a mixed solution of 10 mM glucose and 100 μM alcorbic acid were introduced into the sensor. Both sensors showed 100 nA, and the glucose concentration could be detected with good selectivity even in the presence of ascorbic acid. This is because ascorbic acid could be oxidized and decomposed with high efficiency by the round thin film electrode 15 disposed in the thin layer flow path. As described above, the target substance can be detected with high quantitativeness without increasing the sensor volume so much as compared with the conventional sensor structure.
[0040]
When constructing the biosensor of the present invention, special equipment and facilities are required because there is no need for heating as in the anodic bonding method and there is no need to use a deleterious substance such as hydrofluoric acid. It was possible to produce it safely without any problems. In addition, it is possible to produce a highly sensitive sensor without reducing enzyme activity. In addition, compared to the case of using an adhesive, it can be manufactured in a short time, and since the adhesive does not leak, it can be confirmed that the manufacturing method is superior in productivity compared to the conventional technology. .
[0041]
[Embodiment 2]
In Embodiment 2, after preparing a platinum thin film electrode substrate in the same manner as in Embodiment 1 described above, one of the electrodes surrounding the round thin film electrode is 1 μl (1 is liter) of osmium polyvinylpyridine complex containing horseradish peroxidase, Modified by the casting method. After drying at room temperature for 1 hour, 1 μl of a solution containing lactate oxidase dissolved in a 2% bovine serum albumin aqueous solution to a weight ratio of 2% and glutaraldehyde added to a final concentration of 0.2% ( l is a liter) and was laminated on an electrode modified with an osmium polyvinylpyridine complex containing horseradish peroxidase. Further, as in the first embodiment, the above-described electrode substrate is made into a flow cell to obtain a lactic acid sensor.
[0042]
In the second embodiment, a microdialysis probe was connected as in the first embodiment, and PBS was continuously introduced into the sensor using a syringe pump 22 at a flow rate of 4 μl (l is liter) / min. Each pad of the thin film electrode was connected to the potentiostat 22, and a potential of -50 mV was applied to the reference electrode for the electrode modified with an enzyme or the like, and a potential of 500 mV was applied to the central round thin film electrode 15. . When the microdialysis probe 21 was immersed in a 10 mM lactic acid solution, the reduction current value increased after about 1 minute, and showed a constant value at about 90 nA. Similarly, when a mixed solution of 10 mM lactic acid solution and 100 μM ascorbic acid was introduced into the sensor via the microdialysis probe 21, a reduction current of about 90 nA was observed. These results indicate that various biosensors can be easily constructed by changing the enzyme immobilized on the thin film electrode surrounding the round thin film electrode 15. The present invention can be applied to various biomolecule sensors that are substrates for oxidase such as glutamic acid and monoamine.
[0043]
[Embodiment 3]
A carbon thin film was deposited to 100 nm on a quartz substrate by a thermal CVD method. In the CVD method, a quartz substrate was placed in a glass tube and kept at 1000 ° C., and a polycyclic aromatic compound such as perylene was sublimated at 400 ° C. as a starting material, deposited on the quartz substrate, and then thermally decomposed. A silicon-based photoresist (manufactured by NTT-AT) was spin-coated on a quartz substrate having a carbon thin film, exposed and developed. The developed quartz substrate is covered with a photoresist only at the portion to be an electrode pattern. The substrate described above was subjected to oxygen plasma etching using a reactive ion etching apparatus DEM451 (manufactured by Anelva). The etching was performed under the conditions that the oxygen flow rate was 100 sccm, the power was 70 W, the pressure was 2 Pascal, the time was 25 minutes, and all the carbon thin film not covered with the photoresist was etched. As a result, a carbon thin film electrode having the same pattern as that of the metal electrode of Embodiment 1 was obtained.
[0044]
As in Embodiment 1, an osmium polyvinylpyridine complex containing horseradish peroxidase and glucose oxidase were stacked on one of the electrodes surrounding the central round thin film electrode. Further, in the same manner as in Embodiment 1, a flow cell was formed using an acrylic substrate to which a double-sided adhesive sheet and sample introduction / sample discharge tubes 4 and 5 were attached, and a glucose sensor was obtained. PBS was introduced into the fabricated sensor by changing the flow rate from 1 μl (l is liter) / min to 20 μl (l is liter) / min. However, this method has sufficient adhesive strength even on the carbon thin film electrode, and there is no liquid leakage. There wasn't.
[0045]
Thus, the thin film electrode was effective as the sensor of the present invention not only with platinum but also with a carbon thin film or a gold electrode.
[0046]
[Embodiment 4]
7 (a) to 7 (c) are configuration diagrams of components of the electrochemical online biosensor according to the present invention. FIG. 7 (a) is an insulating substrate having a thin film electrode, and FIG. 7 (b) is a double-sided adhesive. FIG. 7 (c) shows a polymer substrate.
[0047]
This biosensor includes an insulating substrate 27 having a thin film electrode, a double-sided adhesive sheet 28, a polymer substrate 29, a sample introduction capillary 30, a sample discharge capillary 31, a first working electrode 32, a first working electrode 32, and a first working electrode 32. It is composed of a double working electrode 33, a reference electrode 34, and a counter electrode 35.
[0048]
As shown in FIG. 7A, the insulating substrate 27 having a thin film electrode was formed with a gold / titanium thin film electrode pattern by the lift-off method in the same manner as in the first embodiment. The thin film electrodes were the first working electrode 32, the second working electrode 33, the reference electrode 34, and the counter electrode 35 from the upstream where the sample was introduced. On the second working electrode 33, glucose oxidase was immobilized using polypyrrole.
[0049]
Immobilization of glucose oxidase was performed by immersing the electrode substrate in an aqueous solution in which 0.2 M pyrrole and 1 M potassium chloride and 5000 units / ml (l is liters) of glucose oxidase were dissolved. A voltage of 7V was applied for 0.5 seconds, and then a potential of 0V was applied. By repeating this cycle of potential change 10 times, polypyrrole was electrolytically polymerized on the electrode, and at the same time, glucose oxidase was incorporated into polypyrrole and immobilized. The reference electrode 34 was modified with silver paste.
[0050]
On the other hand, as shown in FIG. 7C, two capillary mounting grooves 36 were formed in the polymer substrate 29 using a dicing saw, and the sample introduction capillary 30 and the sample discharge capillary 31 were connected.
[0051]
Next, as shown in FIG. 7B, a double-sided adhesive sheet having a thickness of 50 μm was cut into a width of 10 mm and a length of 30 mm using a cutting plotter. Further, using a cutting plotter, a hole having a width of 1 mm and a thickness of 20 mm was formed in the center of the cut double-sided adhesive sheet 28. The double-sided pressure-sensitive adhesive sheet 28 subjected to drilling was sandwiched between an insulating substrate 27 having electrodes and a polymer substrate 29 having capillaries to form a flow cell.
[0052]
The sample discharge capillary 31 was connected to a syringe, and the syringe was aspirated using a syringe pump 22 at a flow rate of 2 μl (l is liter) / min. The tip of the sample introduction capillary 30 was immersed in PBS, and PBS was continuously introduced into the flow cell. The first working electrode 32, the second working electrode 33, the reference electrode 34, and the counter electrode 35 formed on the insulating substrate of the manufactured flow cell are connected to the potentiostat 23. 0.5 V was applied to the second working electrode 33 and 0.3 V was applied to the reference electrode 34 in the flow cell.
[0053]
After obtaining a stable current value baseline, when a glucose solution was added to PBS to a final concentration of 1 mM, the reduction current value gradually increased and showed a current value of 3 nA after about 1 minute. . Similarly, when PBS containing 1 mM glucose and 100 μM ascorbic acid was introduced into the flow cell, the reduction current value gradually increased, showed a current value of 3 nA after about 1 minute, and the effect of ascorbic acid addition could not be confirmed. . However, when a potential of 0.3 V is applied to the second working electrode 33 and no voltage is applied to the first working electrode 32, PBS containing 1 mM glucose and 100 μM ascorbic acid is introduced into the flow cell, and a current of about 100 nA is obtained. The value is shown.
[0054]
These results show that the production of a flow cell using a thin film adhesive sheet is effective not only in the production of a radial flow cell but also in the production of a channel flow cell and the like. As effective.
[0055]
[Embodiment 5]
FIG. 8 is a schematic diagram of the flow channel structure of the electrochemical immunoassay flow cell according to the present invention, in which 36 is an insulating electrode substrate, 37 is a Y-shaped thin layer flow channel made of a double-sided adhesive sheet, and 38 is alkaline phosphatase. (Alkaline phosphatase) labeled antigen mouse-IgE, 39 is an ImM 4-aminophenyl phosphate solution, 40 is an antibody (anti-mouse IgE) immobilization position, 41 is a working electrode, 42 is Reference electrode 43 is a counter electrode, 44 and 45 are sample introduction holes, and 46 is a sample discharge hole.
[0056]
The working electrode 41, the reference electrode 42, and the counter electrode 43 were produced by forming a platinum thin film electrode by photolithography as in the first embodiment. The reference electrode 42 was modified with a silver pace as in the first embodiment. Thereafter, a 10 mM n-octadecyltrichlorosilane benzene solution was cast into a flow path 40 upstream from the working electrode 41 to make it hydrophobic. Thereafter, the sample is immersed in 1 mg / ml (l is liter) protein A (manufactured by Wako Pure Chemical Industries, Ltd.) phosphate buffer solution (pH 7.4) for 30 minutes, and further 5 mg / ml (l is liter) of anti-mouse IgE antibody tris. The IgE antibody was immobilized by immersing in a buffer solution (pH 8.2) for 30 minutes.
[0057]
Then, as shown in FIG. 8, the flow cell which has the Y-shaped thin layer flow path 37 was formed using the double-sided adhesive sheet and the acrylic board which were punched into Y shape. In the same manner as in the first embodiment, the acrylic plate was processed with the sample introduction hole 44, the sample introduction hole 45, and the sample discharge hole 46 using a drill, and a hollow capillary was attached. A 10% albumin aqueous solution was introduced into a flow cell-made immunoassay flow cell for 10 minutes to suppress nonspecific adsorption in the cell.
[0058]
First, glutaraldehyde was added to an aqueous solution containing 1 pg / ml (l is liter) of the target sample mouse-IgE to a final concentration of 0.1%, and reacted at 5 ° C. for 24 hours to label the alkaline phosphatase enzyme. For the electrode in the flow cell, a potential was applied to the working electrode at 0.6 V with respect to the reference electrode using a potentiostat. The enzyme-labeled sample solution was introduced into the immunoassay flow cell through the sample introduction hole 44 at a flow rate of 1 μl (l is liter) / min for 1 minute.
[0059]
Thereafter, when 1 mM 4-aminophenyl phosphate was introduced from the sample introduction hole 45 at a flow rate of 1 μl (l is liter) / min, the oxidation current value increased by about 2 nA. This is because the target sample enzyme-labeled to the antibody 40 immobilized in the channel is immobilized by the antigen-antibody reaction, and at the same time, 4-aminophenyl phosphate is oxidized by the enzyme immobilized in the channel. This is because aminophenol was produced and 4-aminophenol was oxidized on the electrode.
[0060]
Next, when a similar experiment was performed using a sample containing mouse-IgE of 5 pg / ml (l is liter), an increase in oxidation current value of about 10 nA was observed, and the observed oxidation current value was It was almost proportional to the concentration of the antigen contained in the sample solution. Thereby, it was possible to know the antigen concentration in the sample solution.
[0061]
These results indicate that the flow cell using thin film adhesive sheets can be produced not only on-line biosensors using immobilized enzymes but also without reducing their activity when immobilizing antigens and antibodies. It was confirmed that the method was also effective for producing an immunoassay cell.
[0062]
【The invention's effect】
As described above, according to the present invention, a flow cell in which an insulating substrate having a thin film electrode and another substrate are bonded together with a double-sided adhesive sheet has the following effects.
1. Without changing the thickness of the double-sided adhesive sheet to be used, it is easy to reduce the depth and shape of the product without using dangerous reagents or expensive and time-consuming equipment such as wet etching with hydrofluoric acid and dry etching equipment. It is possible to form a layer flow path.
2. When the flow cell is formed, it is not necessary to perform heating like anodic bonding or heat fusion, and the bonding can be performed at room temperature. Therefore, the flow cell can be easily formed without reducing the activity of the enzyme. Further, since no adhesive is used, there is no leakage of the adhesive, and the manufacturing method is excellent in yield and productivity.
3. Since it is possible to produce cells without inactivating not only immobilized enzymes but also antigens and antibodies, it is possible to easily produce cells for electrochemical immunoassays.
4). A circular thin film electrode and an insulating substrate on which the electrode is placed so as to surround it are bonded to each other with a double-sided adhesive sheet, and a capillary or microdialyzer is used so that the sample is introduced radially from the center of the thin layer channel. By connecting a cis-probe, it is possible to oxidize and remove easily oxidizable substances that interfere with the sensor with a round thin film electrode located in the center of the thin layer flow path, increasing the volume in the sensor. The target substance can be detected with good quantitativeness by using the thin film electrode arranged around the thin film electrode.
[Brief description of the drawings]
FIG. 1 is a structural diagram of an electrochemical online biosensor according to the present invention.
FIG. 2 is a detailed view of each part used in the production of an electrochemical online biosensor according to the present invention, where (a) shows an insulating substrate, (b) shows a double-sided adhesive sheet, and (c) shows a polymer substrate. FIG.
FIG. 3 is a schematic diagram of a measuring apparatus using the electrochemical online biosensor of the present invention shown in the first embodiment.
FIG. 4 is a diagram showing a change in response current value of a sensor when a microdialysis probe is immersed in a 10 mM glucose solution.
5 is a graph showing changes in the response current value of the sensor with respect to the 10 mM glucose and 100 μM ascorbic acid mixed solution shown in Embodiment 1. FIG.
6 is a diagram showing a change in response current value when a potential of 500 mV is applied to the central round thin-film electrode shown in Embodiment 1 and a 10 mM glucose and 100 μM ascorbic acid mixed solution is introduced into the sensor. FIG.
FIG. 7 is a configuration diagram of each part of an electrochemical online biosensor according to the present invention, where (a) is an insulating substrate having a thin film electrode, (b) is a double-sided adhesive sheet, and (c) is a polymer substrate. FIG.
FIG. 8 is a schematic view of a channel structure of an electrochemical immunoassay flow cell according to the present invention.
[Explanation of symbols]
1 Insulating substrate
2 Double-sided adhesive sheet
3 Polymer substrate
4 Sample introduction tube
5 Sample discharge tube
14 Platinum thin film electrode
15 Round thin film electrode
16 Electrode modified with laminated film
17 Electrode coated with silver paste
18 Ring-shaped groove
19 Sample introduction hole
20 Sample discharge hole
21 Microdialysis probe
22 Syringe pump
23 Potentiostat
24 Personal computer
25 PBS
26 PBS containing the target substance
27 Insulating substrate
28 Double-sided adhesive sheet
29 molecular substrate
30 Capillary for sample introduction
31 Capillary for sample discharge
32 First working electrode
33 Second working electrode
34 Reference electrode
35 Counter electrode
36 Insulating electrode substrate
37 Thin layer channel made of double-sided adhesive sheet
38 Alkaline phosphatase labeled antigen mouse-IgE
39 1 mM 4-aminophenyl phosphate solution
40 Immobilization position of antibody (anti-mouse IgE)
41 working electrode
42 Reference electrode
43 Counter electrode
44, 45 Sample introduction hole
46 Sample discharge hole

Claims (1)

An insulating substrate;
A plurality of thin film electrodes formed on the insulating substrate;
Double-sided adhesive sheet that has been drilled in a circle,
A polymer substrate to which the sample introduction means and the sample discharge means are connected,
The insulating substrate on which the plurality of thin film electrodes are formed and the polymer substrate are bonded with the double-sided adhesive sheet to form a flow cell having a thin layer flow path. An electrochemical on-line biosensor comprising: an electrode to be removed; a working electrode disposed on the same circumference so as to surround the electrode to remove the interfering substance; and a reference electrode.

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