JP3515908B2 - Micro online biosensor and production method thereof - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a minute amount online biosensor for collecting a small amount of a sample from cultured cells or a minute portion in a living body and continuously quantitatively analyzing the sample.

[0002]

2. Description of the Related Art In researches such as neuroscience and molecular biology, many attempts have been made to measure a physiologically active substance contained in a microscopic region in a living body in real time.
Among them, the electrochemical analysis method is suitable for (1) measurement of a very small amount. (2) The sensitivity is relatively high. (3) Highly selective measurement is possible by modifying the electrode with a selective membrane or enzyme. (4) It has a feature that a simple and inexpensive sensor can be manufactured. As an attempt to directly measure a physiologically active substance while keeping the living body alive, a microelectrode such as a carbon fiber electrode is directly inserted into a specific region in the living body and in-situ measurement is performed, or a microdiameter is used. Insert a minute double tube of about 1 to 5 mm covered with a dialysis membrane called a lysis probe into the living body,
Biomaterials are measured by sending dialysate by a pump, sampling physiologically active substances contained in a living body through a membrane, and sending them online to an electrochemical sensor. By using an electrode modified with an enzyme membrane that selectively reacts with a target substance in a biological sample to produce an electrochemically active substance as a sensor, neurotransmission of sugars such as glucose and lactose, lactic acid and glutamic acid The substance is measured in real time.

On the other hand, in a system such as a cultured cell, measurement of an extremely minute amount of sample is required such as measurement of a physiologically active substance released from one cell. Since a carbon fiber electrode having a diameter of several μm can be easily used in the microelectrode method, single-cell level measurement is performed for neurotransmitters such as catecholamine that can be measured by an electrochemical reaction directly at the electrode. This is the case, for example, in T. J. Schroeder, J .;
A. Jankowski, K .; T. Kawagoe,
R. M. Weightman, C.I. Lefrouand
C. Amatore, Analytical Chem
istry, 64, pp. 3077-3083, 1992.
Listed in the year.

Further, it has been attempted to detect an enzymatic reaction or an immune reaction in a micro area of a micrometer order by utilizing a scanning electrochemical microscope. This is the case, for example, in H.264. S
hiku, T .; Matsue, I.M. Uchida, An
Alytical Chemistry, Volume 68, 12
76-78, 1996. Further, also in the online type sensor described above, a sensor in which a minute glass capillary or a microdialysis probe and a minute volume flow cell in which an enzyme-modified electrode is set are combined is manufactured. In addition, as a method for manufacturing a minute amount of online sensor, grooves are formed on a silicon or glass substrate by using micromachining technology, such as anisotropic etching or dry etching, and then fused with another substrate having a thin film electrode. It has been reported that a microcapacity sensor in which an enzyme is immobilized inside after forming a flow path. This is the case, for example, in Y. Murakami, T .;
Takeuchi, K .; Yokoyama, E .; Tam
iya and I. Karube, Analytic
al Chemistry, Volume 65, 2731-273.
P. 5, 1993. A sensor manufactured using such a micromachine technology can be expected to have fast response and high sensitivity measurement in a minute amount. This is described in, for example, Niwa, Horiuchi, Torikou, Chemical Sensor Workshop, 13
Vol. 73-76, 1997.

[0005]

When measuring a minute amount of a substance in a biological sample, a fiber type microelectrode can be easily miniaturized to a size of a single cell, and a compound which easily causes an electrochemical reaction can be obtained. Can be detected in extremely small amounts. However, the amount of enzyme that can be immobilized on the microelectrode is small in a system in which an enzyme reaction and an electrode reaction are combined without performing an electrochemical reaction, and the long-term stability is poor depending on the enzyme used. Further, in a system in which a coexisting substance is present, it is necessary to eliminate all of the influences on the electrode, and therefore, there is a drawback that it is difficult to obtain high selectivity. Further, when a substance such as coenzyme is required for the enzymatic reaction, it is necessary to add the substance to the culture system for measurement, and it is necessary to always consider the influence on the biological sample.

On the other hand, the on-line type sensor has high sensitivity and can be thickly modified with the enzyme, so that stable and long-term measurement is possible. Further, addition of a coenzyme or the like, which has been difficult with a fiber type sensor, can be easily performed by providing a plurality of flow channels and merging the channels upstream of the detector.

[0007] In the online type sensor, an interfering molecule in the biological sample that has an influence on the measurement is selectively removed on the upstream side of the electrode for detection in the flow channel, so that an electrochemically active interfering For the substance, the electrode for pre-electrolysis, and for the interfering substance that causes the enzyme reaction, the beads on which the enzyme is immobilized are packed in the pre-column and placed upstream, thereby improving the selectivity. However, when an electrode for pre-electrolysis or a precolumn is arranged, the internal volume of the sensor increases and the responsiveness decreases. On the other hand, although the online sensor manufactured by using the micromachine technology shows a faster response than the normal online sensor, in order to obtain high selectivity, (1) when performing pre-electrolysis, the If it is not large, 100% of interfering substances cannot be removed,
(2) There is a problem that it is difficult to immobilize the enzyme-immobilized beads for removing the interfering substance in the microchannel of the microsensor.

On the other hand, even when the target substance in the biological sample is reacted with the pre-column and the product is detected electrochemically instead of improving the selectivity, the target substance is reacted with high reaction efficiency in the pre-column. However, there is a problem in that the capacity increases, and as a result, the responsiveness of the sensor decreases.

[0009]

In order to solve such a problem, a minute amount online biosensor according to the present invention is provided with an insulating first substrate having two minute channels and a working electrode inside. A second substrate on which a thin-layer electrochemical cell having a reference electrode, a counter electrode, and a flow path through which a biological sample flows on these three electrodes is formed, and the two minute flow paths and the flow path are continuous. And a plurality of minute projections are formed in an upstream portion of the working electrode in the flow channel. In addition, the surface of the large number of microprotrusions is modified with a conductive material or with a biological substance such as an enzyme that reacts with a specific substance. The substance is removed using an electrochemical reaction or an enzymatic reaction, and the reaction efficiency of the target substance in the biological sample is increased.

In order to solve such a problem, the method for manufacturing an online biosensor according to the present invention is, for example, one in which two minute channels are formed on an insulating first substrate,
A sampling capillary or a microdialysis probe is connected to the first substrate, and a working electrode, a reference electrode, and a counter electrode made of a conductor thin film are formed on the second substrate by a photolithography method, and then photolithography is performed again. The thin-layer electrochemical cell flow path and a large number of microprotrusions by the method
The reference electrode and the counter electrode are formed so as to flow over the reference electrode and the counter electrode so that the large number of minute protrusions are arranged in the upstream portion of the working electrode, and the large number of minute protrusions are made of a conductive material or a specific substance. After being modified with a biological substance such as a reacting enzyme, the first substrate and the second substrate are bonded so that the two minute channels and the channels are continuous.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below with reference to the drawings. The present invention is not limited to the following examples.

[0012]

[Embodiment 1] FIG. 1 shows a structure of a minute amount online biosensor of the present invention (hereinafter, abbreviated as "sensor") viewed from above. The sensor includes a first substrate 1 (FIG. 1a) in which two microchannels 2 are formed by a micromachine technology, a working electrode 4 modified with an enzyme or the like, a reference electrode 5, a counter electrode 6, and these three electrodes 4 , 5, 6 is formed with a microprojection structure electrode 3 composed of a large number of microprojections 16 whose surface is modified with a conductive material.
It has a structure in which 5 and 6 and the second substrate 13 (FIG. 1b) in which the flow path 7 is formed on the microprojection structure electrode 3 are attached. At this time, the minute channel 2 and the channel 7 are bonded so as to be continuous. In addition, the microprojection structure electrode 3, the working electrode 4, the reference electrode 5, the counter electrode 6 and the flow path 7 constitute a thin layer electrochemical cell.

The pads 8, 9, 10 and 11 of the microprojection structure electrode 3, working electrode 4, reference electrode 5 and counter electrode 6 are connected to the terminal portions of the potentiostat, respectively. Capillaries used for sampling, microdialysis (MD) probes, or capillaries for connecting to syringes can be easily attached to the minute channels 2.

A conductive material is modified on the surfaces of the many small protrusions 16. When the interfering substance in the biological sample, which is injected into the thin-layer electrochemical cell of the sensor at the same time as the target substance in the biological sample, is an electrochemically active substance, the surface of a large number of microprotrusions 16 is conductive, so that the potential is When applied, the interfering substance can be removed by reacting on the micro-projection structure electrode 3. Since the micro-projection structure electrode 3 is composed of a large number of columnar projections, it has a large surface area and an improved reaction rate as compared with a normal flat thin film electrode. As a result, the electrochemical reaction can be completed in a very short time, and the channel 7 can be shortened to improve the responsiveness.

[0015]

[Embodiment 2] In this embodiment, instead of the microprojection structure electrode of Embodiment 1, an example of a sensor in which the surface of many microprojections is modified with a biological substance such as an enzyme will be described. The sensor is a first substrate 1 (FIG. 1a) in which microchannels 2 are formed by micromachining technology, a working electrode 4 modified with an enzyme, a reference electrode 5, a counter electrode 6 and these three electrodes 4, 5, 6 A large number of microprojection structure reactors 12, the surface of which is modified with a biological substance such as an enzyme, are formed upstream of the three electrodes 4,
It has a structure in which 5 and 6 and a second substrate 13 (FIG. 1b) in which a flow path 7 is formed on a large number of microprojection structure reactors 12 are bonded together. At this time, the minute channel 2 and the channel 7 are bonded so as to be continuous. Further, the microprojection structure reactor 12, the working electrode 4, the reference electrode 5, the counter electrode 6 and the channel 7 constitute a thin layer electrochemical cell. In addition, a biological substance such as an enzyme has a catalytic function of reacting with a specific substance.

It is very difficult for an online sensor manufactured by the micromachine technique to put beads on which a biological substance such as an enzyme is immobilized in a minute channel. However, when an enzyme is immobilized on a large number of microprotrusions 16, the large number of enzyme-immobilized protrusions behaves similarly to the case where the enzyme-immobilized beads are placed in the thin layer channel, and the enzyme is immobilized on the substrate at a high reaction efficiency. Can be reacted. High sensitivity is obtained when the immobilized enzyme reacts with the target substance in the biological sample, and the target substance can be selectively detected due to the high reaction rate when the immobilized enzyme reacts with the interfering substance in the coexisting biological sample. Further, even when the two solutions are combined, the two solutions are well stirred in the thin layer electrochemical cell having the microprojection structure reactor 12,
Since the mixing is completed in a short time, high sensitivity can be realized.

By using the thin-layer electrochemical cell having a large number of microprojections 16 as an enzyme reactor,
It has a feature that a sensor with high sensitivity and high selectivity can be realized. Further, since the gap between the minute projections is always constant, there is an advantage that even if pressure is applied to the flow path for a long time, it does not move like beads and no clogging occurs.

[0018]

[Embodiment 3] FIG. 2 shows an embodiment of a manufacturing process of a sensor having a microprojection structure electrode according to the present invention. A THB-516-L positive photoresist 14 was applied onto a quartz wafer 13 (FIG. 2a) by a spinner at 4000 rpm (FIG. 2b). After that, the photomask is applied to the wafer 13
And the mask aligner PLA-501 is used to form the microprojection structure electrode 3, that is, a large number of the microprojections 16 used as the electrodes for pre-electrolysis and the part to be the groove 15 of the flow path 7 (excluding the large number of the microprojections 16). The pattern was exposed. The exposure time was 15 seconds. After exposure, the wafer was developed in an alkaline developer for 30 seconds, washed with water and dried (FIG. 2c). In the wafer after development, the concave portions around the large number of minute projections 16 and the portions to be the grooves 15 of the flow path are exposed.
The substrate 13 with the resist 14 was wet-etched in a hydrofluoric acid buffer solution for 20 minutes to form a large number of irregularities of the minute projections 16 and grooves 15 of the flow path (FIG. 2d). The height of these fine protrusions 16 was 20 μm, and the diameter was 20 μm. After the remaining resist 14 is removed by plasma asher (FIG. 2e), a positive photoresist 17 is spin-coated (FIG. 2f), and then a photomask is overlaid on the wafer 13 and a micro-protrusion structure is formed using a mask aligner. The electrode 3 portion and the working electrode 4, the reference electrode 5, and the counter electrode 6 were exposed and alkali-developed (FIG. 2g).
After that, the wafer 13 having this resist pattern was attached to a magnetron sputtering device, titanium and gold 18 were sputtered in order (FIG. 2h), and then the resist 17 was removed by ultrasonic waves by immersion in methyl ethyl ketone, and the fine projection structure electrode 3 was formed. A working electrode 4, a reference electrode 5, and a counter electrode 6 were prepared (FIG. 2i). The channel 7 of the thin-layer electrochemical cell of the sensor is formed in a range in which the channel 15 (excluding a large number of microprojections 16) and the microprojection structure electrode 3 are combined.

Microprojection structure electrode 3 and three electrodes 4, 5, 6
FIG. 3 shows the configuration of the substrate 13 having the. Gold 18 becomes a thin film for microprojection structure electrode 3, working electrode 4, reference electrode 5, and counter electrode 6 from the left, and osmium-polyvinylpyridine complex (Os-gel) containing horseradish peroxidase (HRP) is formed on working electrode 4. 20 was modified in the lower layer, and the membrane 21 in which glutamate oxidase was mixed with collagen was modified in the upper layer. Further, silver 19 was plated on the reference electrode 5 as a reference material (FIG. 2j). The channel 7 of the thin-layer electrochemical cell is formed in a range that covers the microprojection structure electrode 3, working electrode 4, reference electrode 5, and counter electrode 6.

FIG. 10 shows a manufacturing process of a first substrate (lid substrate) to which a glass capillary is connected. 12 mm wide and 27 mm long using a dicing saw for the glass substrate 1.
(Fig. 10a). Furthermore, in order to connect the capillaries for sampling and outlet, a width of 0.4 mm and a depth of 0.4 mm are provided by a dicing saw in parallel with the long side.
A groove for the minute channel 2 having a length of mm and a length of 7 mm was prepared from both ends (FIG. 10b). Outer diameter of 375μ in the sampling groove
m, inner diameter 75 μm sampling capillary 22
The outer groove has an outer diameter of 375 μm and an inner diameter of 1
Connect the 50 μm syringe connecting capillary 23,
It was fixed with an instant adhesive (Fig. 10c).

The second substrate 13 having the microprojection structure electrode 3, the three electrodes 4, 5, 6 and the flow path 7 and the micro-flow formed by the steps of FIGS. 2a to 2j are connected to the capillaries 22 and 23. The first substrate 1 having the channel 2 is pressed and aligned so that the two minute channels 2 and the channel 7 face each other and are continuous, and then a photocurable adhesive is applied at room temperature. I let it infiltrate from the surroundings. When the adhesive had soaked in just before the flow path 7, light was irradiated using a high pressure mercury lamp to bond the two substrates 1 and 13 (FIG. 2k). In the past, since it was often pasted at a fairly high temperature, the activity of enzymes etc. was lost and it could not be applied, but it became possible to attach at normal temperature, and the sensor can be used without losing the activity of the enzyme. It can be made. Instead of the photo-curable adhesive, a polymer thin film dissolved with a solvent vapor or a low melting point glass thin film may be used for bonding.

FIG. 4 shows the structure of a measurement system in which the manufactured sensor is connected to a syringe. Also, potentiostat 2
The terminals 4 (LC4C) were connected to the pads 8, 9, 10, and 11 of the microprojection structure electrode 3, working electrode 4, reference electrode 5, and counter electrode 6 of the thin-layer electrochemical cell, respectively. First,
First, a syringe 25 was used to draw a solution from the sampling capillary 22 at a flow rate of 4 μl / min, and a potential of 0 mV was applied to the microprojection structure electrode 3 and the working electrode 4 for measurement.

FIG. 5 shows the response to 100 μM ascorbic acid. The vertical axis represents time and the horizontal axis represents current value.
After obtaining the baseline with the phosphate buffer solution, switching to the ascorbic acid solution caused a rapid increase in the oxidation current, and the sensor showed a steady current of 300 nA. Again, the phosphate buffer solution was introduced back into the sensor's thin layer electrochemical cell to return to baseline. Next, after applying a voltage of 500 mV to a large number of microprojection structure electrodes 3, 100 μM ascorbic acid was introduced into the thin-layer electrochemical cell of the sensor, but no oxidation current due to ascorbic acid was confirmed. This is the micro-projection structure electrode 3
In part, it was because all ascorbic acid was oxidized.

Further, by using the same process as in FIG. 2, a sensor having the same apparent area of the electrodes but no microprojection was prepared, and the responses were compared. 5 for thin film electrode for pre-electrolysis
When the measurement was performed by applying a potential of 00 mV and 0 mV to the working electrode, an oxidation current of 20 nA was observed regardless of performing pre-electrolysis, and in order to completely remove the influence of ascorbic acid, the microprojection structure was used. Electrode 3 has been shown to be extremely effective. Also, 10 μM glutamic acid and 100 μM
When the PBS solution containing ascorbic acid is flowed, an oxidation current of 255 nA flows when the microprojection structure electrode is not used,
The glutamate response was blocked by ascorbic acid. On the other hand, when the micro-projection structure electrode 3 is used, 9.5 nA
It was found that glutamic acid alone could be quantified without being affected by ascorbic acid.

[0025]

Fourth Embodiment A first substrate 1 having a minute channel 2 and a second substrate 13 having a minute projection structure reactor 12 and three electrodes 4, 5 and 6 of a gold thin film were prepared by the same method as in the third embodiment. It was made. However, gold was not sputtered on the microprojection structure reactor 12. Further, the capillaries 22 and 23 were connected to the first substrate 1 having the minute channels 2 as in the third embodiment. Microprojection structure reactor 12 and gold thin film 3
Enzymes and silver were modified on the electrodes 4, 5 and 6 as follows. That is, instead of the step of FIG. 2j, glutamate oxidase was immobilized on the microprojection structure reactor 12. The immobilization method was as follows. First, 12 parts of the microprojection structure reactor were dipped in a 1 mM ethanol solution of mercaptoundecylate for 10 minutes, and then dipped in a 0.1% glutaraldehyde aqueous solution for 5 minutes. Then, after washing with water, it was immersed in a 0.1% glutamate oxidase aqueous solution and left in a refrigerator (5 ° C.) for 20 hours to fix the enzyme. Also, on the working electrode 4,
Polyvinylpyridine osmium complex film containing HRP 1
μl was cast and dried, and the reference electrode 5 was plated with silver.

For the measurement, the pads 9, 10, 11 of the working electrode 4, the reference electrode 5, and the counter electrode 6 were set to the potentiostat 2.
4 was applied, and a voltage of 0 mV was applied to the working electrode 4 while sucking the solution from the sampling capillary 22 using the syringe 25. Flow rate 4 μl / mi
Phosphate buffered saline is continuously introduced into the thin layer electrochemical cell of the sensor using a syringe 25 at n. After obtaining the baseline in this way, from phosphate buffered saline, 1
By introducing a 0 μM glutamic acid solution into the thin-layer electrochemical cell of the sensor, a reduction current started to flow and after 10 seconds, a reduction current of 10 nA / μM and high sensitivity could be obtained. However, when using the same volume of enzyme reactor without microprojections and performing the same measurement, 2 nA
Only a reduction current of about / μM could be obtained. This is because the production of a large number of columnar microprotrusions 16 has a large surface area and an increased reactivity as compared with a normal flat thin film electrode.

[0027]

[Embodiment 5] A first substrate 1 having a minute channel 2 and a second substrate 13 having a minute projection structure electrode 3 and three electrodes 4, 5 and 6 of a gold thin film were prepared by the same method as in the third embodiment. It was made. In addition, the first substrate 1 having the minute channels 2 has the third embodiment.
The capillaries 22 and 23 were connected in the same manner as in. An enzyme was immobilized on the microprojection structure electrode 3 in the same manner as in Example 4. The enzyme used was a mixture of choline oxydose and catalase. In the working electrode 4, a polyvinylpyridine osmium complex containing HRP was fixed in the lower layer, and acetylcholinesterase and choline oxydose were similarly fixed in the upper layer.

When a 1 μM acetylcholine solution was introduced into the thin-layer electrochemical cell of the sensor at a flow rate of 4 μl / min, a reducing current flowed and a sensitivity of 5 nA / μM was obtained. However, no response was obtained with the introduction of 10 μM choline solution. This is because choline oxidase of the microprojection structure electrode 3 oxidizes choline and the generated hydrogen peroxide is decomposed by catalase. On the other hand, acetylcholine does not react at the microprojection structure electrode 3, becomes acetylcholinesterase on the working electrode 4, becomes choline, and is further oxidized by choline oxidase to obtain a reducing current. Also, even if acetylcholine and choline are simultaneously introduced into the thin-layer electrochemical cell of the sensor,
No significant difference was observed when only acetylcholine was introduced. Since the sensor has the microprojection structure electrode 3 portion, choline efficiently reacts at the microprojection structure electrode 3,
Since only acetylcholine reaches the working electrode 4, high selectivity can be obtained. However, when an enzyme reactor with the same volume and no microprojections was used, 5 nA /
A reduction current of about μM was confirmed. This is because a large number of cylindrical fine projections 16 are produced, so that the surface area is large and the reactivity is increased, as compared with a normal flat thin film electrode.
This is because the ability of the microprojection structure electrode 3 to remove choline is greatly improved.

[0029]

Sixth Embodiment A first substrate 1 having a fine channel 2 and a second substrate 13 having a fine projection structure electrode 3 and three electrodes 4, 5, 6 of a gold thin film were prepared by the same method as in the third embodiment. It was made. Further, the capillaries 22 and 23 were connected to the first substrate 1 having the minute channels 2 as in the third embodiment. As shown in FIG. 6, a polyvinylpyridine osmium complex 27 containing choline oxydose and HRP was fixed to the microprojection structure electrode 3. Furthermore, as in Example 3, on the working electrode 4,
Polyvinylpyridine osmium complex 26 containing acetylcholinesterase and choline oxidase was fixed on the upper layer and HRP was fixed on the lower layer.

The pad 8 of the microprojection structure electrode 3 and the pad 9 of the working electrode 4 are connected to the first and second electrode terminals of the potentiostat 24, respectively, and the reference electrode pad 10 and the counter electrode pad 11 are also potentiostat. A voltage of 0 mV was applied to both the microprojection structure electrode 3 and the working electrode 4 by connecting to 24 terminals. Flow rate of 10 μM choline solution 4
By introducing it into the thin-layer electrochemical cell of the sensor at μl / min, the reduction current could be confirmed only by the microprojection structure electrode 3. Further, by introducing 1 μM acetylcholine, the reduction current could be confirmed only by the working electrode 4. Furthermore, when choline and acetylcholine solutions were introduced at the same time, respective reducing currents could be obtained. In addition, no significant difference could be confirmed when compared to the case where they were individually introduced. By using the microprojection structure electrode 3 and the working electrode 4 as electrodes, simultaneous measurement of two components is possible.

[0031]

[Embodiment 7] A first substrate 1 having a minute channel 2 and a second substrate 13 having a minute projection structure reactor 12 and three electrodes 4, 5 and 6 of gold thin films were prepared by the same method as in the fourth embodiment. It was made. Further, capillaries 22 and 23 were connected to the first substrate 1 having the minute channels 2 as in the fourth embodiment. Gabbase and working electrode 4 on the microprojection structure reactor 12
A polyvinylpyridine osmium complex film containing glutamate oxidase and HRP was immobilized on the top.

100 μM at a flow rate of 4 μl / min
Of 10 μM GABA (γ
-Aminobutyric acid), a reduction current of 1 nA could be obtained. In order to compare the performance of the microprojection structure electrode 3, the same measurement was carried out in a flat enzyme reactor without microprojections in the microprojection structure reactor 12 part.
Only a reduction current of 0.5 nA could be obtained. This is because the microprojection structure reactor 12 has a large number of microprojections 1.
The reason for this is that since it has 6, it is possible to obtain a large surface area even in a thin-layer electrochemical cell of a miniaturized sensor, and it is possible to efficiently react even with an enzyme having low activity such as gabase.

FIG. 7 shows the measuring system used for the measurement of the sensor of this embodiment. The sampling capillary 22 is branched into two channels by a branch 28 in the upstream part of the thin-layer electrochemical cell of the sensor, and one of the branches is used as a target sample suction capillary and connected to a phosphate buffer solution 31 containing 1 μM GABA. Then, the syringe 29 containing 100 μM α-ketoglutaric acid and a concentration of 1 unit / ml gabase is connected to the other side. A syringe 29 containing α-ketoglutaric acid and gabase is extruded at a flow rate of 1 μl / min, and the capillary 2 for syringe connection at the downstream portion of the thin-layer electrochemical cell is extruded.
A syringe 30 was connected to 3 and suctioned at a flow rate of 5 μl / min to introduce the target sample of 10 μM GABA into the thin-layer electrochemical cell of the sensor at a flow rate of 4 μl / min. As a result, a reduction current of about 4 nA could be obtained. This is due to the mixing of the two pathways upstream of the sensor's thin layer electrochemical cell, mixing GABA with α-ketoglutarate and gabase to produce glutamate.

In order to compare the performance of the microprojection structure reactor 12, the same measurement was carried out in a flat enzyme reactor having no microprojections in the reactor part, and only a reduction current of 2 nA was obtained. This is because the two flow channels were mixed properly in the case where there were a large number of microprojections 16 as compared with the case where there were no microprojections, so that the enzyme reaction was carried out efficiently.
It is considered that the sensitivity has improved. As described above, the microprojection structure reactor 12 of the present sensor is also useful when appropriately mixing two solutions, and even an enzyme having low activity as in the present experiment can be efficiently reacted. .

[0035]

[Embodiment 8] FIG. 8 shows another embodiment of the manufacturing process of the sensor having the fine projection structure electrode 3. A positive photoresist 33 is placed on the quartz wafer 32 (FIG. 8a) at 4000 rpm.
Spin coated by spinning (Fig. 8b). After that, a photomask is overlaid on the wafer 32, and the mask aligner PLA-5 is used.
01 was used to expose the patterns of the three electrode portions of the working electrode 4, the reference electrode 5, and the counter electrode 6. The exposure time was 15 seconds. After the exposure, the wafer was developed in an alkaline developer for 30 seconds, washed with water and dried (FIG. 8c). Wafer 3 after development
In 2, the portions for producing the three electrodes 4, 5, 6 are exposed. The wafer 32 having this resist pattern was attached to a magnetron sputtering apparatus, and gold 34 was sputtered (FIG. 8d). The remaining resist 33 was peeled and removed in methyl ethyl ketone while applying ultrasonic waves (FIG. 8e). Again, the positive photoresist 35 is spin-coated (see FIG. 8).
f) After that, a photomask is overlaid on the wafer 32, and a mask aligner is used to form a flow path surrounding the portion of the resist 35 that is to become the concave portions of the numerous small protrusions 36 and the three electrodes 4, 5, and 6. The portion that should become a mask was exposed and alkali-developed (FIG. 8g). As a result, a large number of minute projections 36 made of resist and the side wall 37 of the flow path 7 are formed. After that, a large number of small protrusions 3 are formed using a metal mask.
Gold 38 is sputtered only on the 6th part, and the microprojection structure electrode 3
Was prepared (FIG. 8h). The flow path 7 of the thin-layer electrochemical cell of the sensor is a region surrounded by the side wall 37 made of a resist, and is formed in a range covering the microprojection structure electrode 3 and the three electrodes 4, 5, 6.

In the first substrate 1 having the two minute channels 2 connecting the capillaries 22 and 23, a capillary mounting groove to be the minute channels 2 is formed as in the third embodiment, and then two more capillary mountings are performed. A channel having a depth of 20 μm and a width of 400 μm was formed by a dicing saw so as to connect the grooves, and both substrates 1 and 13 were bonded in the same manner as in Example 3. As a result, a space in which the biological sample flows can be secured on the microprojection structure electrode 3.

Further, when an experiment similar to that of Example 3 was conducted, almost the same result as that shown in FIG. 5 was obtained, and the microprojection structure electrode 3 prepared by vapor-depositing gold on the resist pattern of the microprojections was produced. It was possible to efficiently remove the interfering substance. Further, in this step, the wet etching step with hydrofluoric acid as in Example 3 can be omitted, and the sensor can be manufactured without using a dangerous reagent.

[0038]

[Embodiment 9] FIG. 9 shows still another embodiment of the manufacturing process of the sensor having the microprojection structure electrode 3. 75m diameter
After positive spin photoresist 39 (TSR-V3) was spin-coated at 4000 rpm on a glass substrate 40 having a thickness of 0.5 mm and a thickness of 0.5 mm, and baked on a hot plate at 90 ° C. for 90 seconds (FIG. 9a). , Mask aligner (PLA-
In 501), the pattern of the three-electrode portion of the microprojection structure electrode 3, the working electrode 4, the reference electrode 5, and the counter electrode 6 was exposed.
Next, after developing for 90 seconds with a resist developing solution, it is washed with pure water for 2 seconds.
A rinse pattern was formed by rinsing for a minute (FIG. 9b).
Next, on the substrate 40, a 5 nm thick chromium film was formed on the substrate 40 without breaking the vacuum, followed by a 100 nm thick gold film 41.
Was vapor deposited (FIG. 9c). Then, the substrate 40 is immersed in methyl ethyl ketone, the resist is peeled off while applying ultrasonic waves, and the base of the gold microprojection structure electrode 3 and the three electrodes 4,
5 and 6 were produced (Fig. 9d). After cutting the substrate 40 into a predetermined size with a dicing saw, a positive thick film resist 42 (THB-516-L) 20 is formed on the substrate 40.
It is applied to a thickness of μm (FIG. 9e), and a pattern of a large number of minute projections 16 is exposed, developed and rinsed with a mask aligner to form a columnar resist pattern in which the holes 43 are formed in the areas where the minute projections are to be formed. (Fig. 9f). This substrate 40 is immersed in a gold plating solution, the gold pad 8 connected to the underlying gold portion exposed at the bottom of the hole 43 is connected to a constant voltage power source for electroforming, and the temperature is 60 ° C. and the current density is 50 mA /
Gold was plated for 15 minutes under conditions of cm ² and electrodeposition rate of 80 μm / h (FIG. 9g). After that, the substrate 40 was immersed in methyl ethyl ketone, the resist 42 was peeled off while applying ultrasonic waves, and a large number of cylindrical fine projections 44 of 20 μm thick were formed by gold electroforming (FIG. 9h). Thus, FIG.
A microprojection structure electrode 3 similar to that was produced. Next, in the same manner as in Example 1, the surface of the working electrode 4 was modified with a film composed of an osmium-polyvinylpyridine complex containing horseradish peroxidase and a film in which glutamate oxidase was mixed with collagen, and silver was applied on the reference electrode 5. Plated.

On the other hand, the first substrate (lid substrate) 1 having the capillaries 22 and 23 was manufactured by the following steps.
First, a glass substrate 1 having a diameter of 75 mm and a thickness of 0.5 mm was coated with a low melting point glass (# 7570) by a sputtering device.
It was sputter deposited to a thickness of 2 μm. Next, a positive thick film resist was applied to a thickness of 20 μm, and the pattern was exposed, developed, and rinsed to form a resist pattern in which the portions corresponding to the upper portions of the microprojection structure electrode 3 and the three electrodes 4, 5 and 6 were exposed. After cutting into a predetermined shape with a dicing saw, the substrate was immersed in a hydrofluoric acid buffer solution, and the low-melting glass and the glass substrate 1 thereunder were etched by 20 μm. After removing the resist with methyl ethyl ketone, the upper part of the channel 7 was processed with a dicing saw in the same manner as in Example 8.

First Example Having Minute Channels 2 Similar to Example 3
Insert the capillaries 22 and 23 into the groove of the substrate 1 of
The second substrate 13 having the microprojection structure electrode 3 and the three electrodes 4, 5 and 6 manufactured in the step of FIG. 9 and the first substrate 1 are aligned by an anodic bonding apparatus, and a voltage of 1 kV is applied for 5 minutes. Then, the two substrates 1 and 13 were joined. Example 3
Similarly to the sensor, the thin-layer electrochemical cell of the sensor is attached to the syringe 25.
, And pads 8, 9, 1 of each electrode 3, 4, 5, 6
0 and 11 were connected to potentiostat 24. 450 mV to the micro-projection structure electrode 3 and the working electrode 4, respectively
A potential of 0 mV was applied, and a mixed solution of 10 μM glutamate and 100 μM ascorbic acid was aspirated. As a result, a reduction current of 40 nA due to glutamic acid flowed through the working electrode 4. When a potential was not applied to the microprojection structure electrode 3, ascorbic acid was also oxidized in the working electrode 4, an oxidation current of 160 nA was observed, and the signal current due to glutamic acid alone could not be measured.

[0041]

Tenth Embodiment In the same manner as in the third embodiment, a microprojection structure electrode 3 having a large number of microprojections 16 as shown in FIG. 1b, a working electrode 4, a reference electrode 5, a counter electrode 5, and a channel 7 are provided.
The second substrate 13 having The pad 8 of the microprojection structure electrode 3 on the substrate 13 was connected to a constant voltage power source, and was immersed together with the gold wire in a platinum plating solution in which 10 mg / mL of choline oxidase was dissolved. Temperature 45 ° C, current density 50m
Gold plating was performed for 6 minutes under the conditions of A / cm ² and electrodeposition rate of 80 μm / h. After plating, the surface of many microprojections 16 was covered with platinum black and a choline oxidase complex. After that, the surface of the working electrode 4 was modified with a film made of an osmium-polyvinylpyridine complex containing horseradish peroxidase and acetylcholinesterase and choline oxidase were mixed with BSA and crosslinked with 0.2% glutaraldehyde. Further, a silver paste was applied on the reference electrode 5.

On the other hand, as the first substrate 1 having the minute flow paths 2, a substrate 1 similar to that shown in FIG. 1a of Example 3 was prepared by using a dicing saw. After that, the two substrates 1 and 13 were bonded with a UV curable resin.

As in Example 3, the syringe 25 was connected to the thin-layer electrochemical cell of the sensor, and the pads 8, 9, 10, 11 of the microprojection structure electrode 3 and the three electrodes 4, 5, 6 were potentiated. Connected to Stat 24. Micro-projection structure electrode 3
A potential of 450 mV and 0 mV was applied to the working electrode 4 and the mixed solution of 1 μM acetylcholine and 100 μM choline was sucked. As a result, a reduction current of 3.5 nA flowed through the working electrode 4. This value was almost equal to the value obtained by measuring only 1 μM acetylcholine without mixing choline (reduction current of 3.3 nA). On the other hand, when no potential is applied to the microprojection structure electrode 3, choline is oxidized at many microprojections 16 but the generated hydrogen peroxide reacts on the working electrode 4 and a large reduction current of 5 μA is observed. It was This is because choline also participates on the working electrode 4 at the same time as acetylcholine and only acetylcholine cannot be detected. From these results, it was found that by using the microprojection structure electrode 3 having a large number of microprojections 16 on which an enzyme is immobilized, only acetylcholine can be detected with high selectivity.

[0044]

As described above, the minute amount online biosensor having the microprojection structure electrode according to the present invention has the following effects.

First, when the surface of a large number of microprojections is modified with a conductive material, the surface of the microprojection structure electrode is conductive, so that a voltage is applied to the microprojection structure electrode to cause an electrochemical reaction. It is possible to remove the electrochemically active substance that interferes with the detection. In addition, since it has a large number of minute protrusions, it has a large surface area, and electrolysis is efficiently performed. Therefore, the reaction can be completed in a short time, and its effect is greater than that of a flat electrode.

Second, by modifying the surface of a large number of microprotrusions with a biological substance such as an enzyme having a catalytic function for reacting with a specific substance, the enzyme can be reacted more efficiently than a flat structure. Therefore, it is very useful when using an enzyme having low activity.

Thirdly, since the surface of the microprojection structure electrode is electrically conductive, by using the microprojection structure electrode 3 and the working electrode 4 as electrodes, simultaneous measurement of two components becomes possible.

Fourth, since the solution flows between a large number of minute projections, it is possible to efficiently mix a plurality of solutions joined upstream from this.

Fifth, since the gaps between the minute projections are always constant, there is an advantage that even if pressure is applied to the flow channel for a long time, it does not move like beads and no clogging occurs.

Sixth, it becomes possible to bond the first substrate and the second substrate at room temperature, and the sensor can be manufactured without losing the activity of the enzyme.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of a sensor according to a first embodiment of the present invention. 1a shows a first substrate having a microchannel, FIG. 1b shows a microprojection structure electrode, three electrodes, a second substrate having a channel, and FIG. 1c shows a microprojection structure reactor, three electrodes, a channel. Each structure of the second substrate included is shown.

FIG. 2 is a diagram showing a process of manufacturing a sensor according to a third embodiment of the present invention.

FIG. 3 is a diagram showing a configuration of a sensor according to a third embodiment.

FIG. 4 is a diagram showing a configuration of a measurement system using a sensor in Example 3.

FIG. 5 is a diagram showing the response of ascorbic acid measured using the sensor in Example 3.

FIG. 6 is a diagram showing a structure of a sensor in Example 6.

FIG. 7 is a diagram showing a configuration of a measurement system using a sensor in Example 7.

FIG. 8 is a diagram showing a process of manufacturing the sensor according to the eighth embodiment.

FIG. 9 is a diagram showing a process of manufacturing the sensor according to the ninth embodiment.

FIG. 10 is a manufacturing process diagram of a first substrate having minute channels according to the third embodiment.

[Explanation of symbols]

1. First substrate 2. Micro channel 3. Microprojection structure electrode 4. Working electrode 5. Reference electrode 6. Counter electrode 7. Channel 8. Micro-protrusion structure electrode pad 9. Working electrode pad 10. Reference electrode pad 11. Counter electrode pad 12. Microprojection structure reactor 13. Second substrate 14. Resist 15. Channel groove 16. Minute protrusions 17. Resist 18. Fri 19. Silver 20. Polyvinyl pyridine complex containing osmium 21. Membrane in which glutamate oxidase is mixed with collagen 22. Capillary for sampling 23. Capillary for syringe connection 24. Potentiostat 25. Syringe 26. The upper layer is a mixture of acetylcholinesterase and choline oxydose, and the lower layer is a bilayer of polyvinyl pyridine complex containing osmium. 2 layers of polyvinyl pyridine complex containing choline oxydose in the upper layer and osmium in the lower layer 28. Branch 29. Syringe filled with a phosphate buffer solution containing 100 μM α-ketoglutarate and gabase. Syringe 31.1 μM GABA in phosphate buffer solution 32. Quartz wafer 33. Resist 34. Money 35. Resist 36. Microprojections 37 made of resist Side wall of flow path made of resist 38. Gold thin film 39. Resist 40. Glass substrate 41. Gold 42. Resist 43. Hall 44. Micro-projection by gold electroforming

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Osamu Niwa 3-19-3 Nishishinjuku, Shinjuku-ku, Tokyo Inside Nippon Telegraph and Telephone Corporation (72) Inventor Tsutomu Horiuchi 3-19-3 Nishishinjuku, Shinjuku-ku, Tokyo No. 2 Japan Telegraph and Telephone Corporation (72) Inventor Keiichi Torizumi 3-19-3 Nishishinjuku, Shinjuku-ku, Tokyo Japan Nippon Telegraph and Telephone Corporation (72) Inventor Masao Morita Three Nishishinjuku, Shinjuku-ku, Tokyo Nihon Telegraph and Telephone Corporation (56) References JP-A-9-127039 (JP, A) JP-A-9-292360 (JP, A) JP-A-5-223772 (JP, A) JP-A-10-38844 (JP, A) JP-A-10-90214 (JP, A) JP-A-11-83784 (JP, A) JP-A-63-279153 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) G01N 27/28 321 C12Q 1/00 G01N 27/327

Claims (8)

(57) [Claims] 1. An insulative first in which two minute channels are formed.
And a second substrate in which a thin-layer electrochemical cell having a working electrode, a reference electrode, a counter electrode, and a flow path through which a biological sample flows on these three electrodes is formed. A minute amount online biosensor characterized by having a structure in which a channel and the flow channel are bonded so as to be continuous, and a large number of minute protrusions are formed in an upstream portion of the working electrode in the flow channel. . 2. The minute amount online biosensor according to claim 1, wherein the surface of the large number of microprotrusions is modified with a conductive material to function as an electrode. 3. The minute amount online biosensor according to claim 1, wherein the surface of the plurality of microprojections is modified with a biological substance such as an enzyme that reacts with a specific substance. 4. Two microchannels are formed on an insulating first substrate, a sampling capillary or a microdialysis probe is connected to the first substrate, and a photolithography method is used on the second substrate. After forming the working electrode, the reference electrode, and the counter electrode made of a conductive thin film by the photolithography method, the flow path of the thin-layer electrochemical cell and a large number of microprotrusions are again formed by the biological sample. The working electrode, the reference electrode, and the counter electrode are formed so as to flow over the working electrode, and the large number of microprojections are arranged in the upstream portion of the working electrode, and the surfaces of the large number of microprojections are made of a conductive material. ,
Alternatively, after being modified with a biological substance such as an enzyme that reacts with a specific substance, the first substrate and the second substrate are bonded so that the two minute channels and the channels are continuous. And a method for manufacturing a minute amount of online biosensor. 5. A microscopic flow path is formed on a second substrate by forming two minute channels on an insulating first substrate, connecting a sampling capillary or a microdialysis probe to the first substrate. And the etching method are used together to form the flow path of the thin-layer electrochemical cell and a large number of minute projections, and then the working electrode, the reference electrode, and the counter electrode made of the conductive thin film are formed again by the photolithography method to form the biological sample. The plurality of minute projections are formed so as to flow over the three electrodes and the plurality of minute projections are arranged in the upstream portion of the working electrode, and the surfaces of the plurality of minute projections are made of a conductive material. Alternatively, after being modified with a biological substance that reacts with a specific substance such as an enzyme, the first substrate and the second substrate are bonded so that the two minute channels and the channels are continuous. A small amount online Method of manufacturing a biosensor. 6. A microlithography method is formed on a second substrate by forming two minute channels on an insulating first substrate, connecting a sampling capillary or a microdialysis probe to the first substrate. And the etching method or the lift-off method are used together to form the working electrode, the reference electrode, and the counter electrode, and then the photolithography method and the electroforming method are used together to form the flow path of the thin-layer electrochemical cell and a large number of columns. The microprotrusions, the biological sample is a large number of columnar microprotrusions, the working electrode,
It is formed so as to flow on the reference electrode and the counter electrode, and the large number of columnar microprojections are arranged in the upstream portion of the working electrode, and the surface of the columnar microprojections is made of a conductive material or specified. After being modified with a biological substance such as an enzyme that reacts with a substance, the first substrate and the second substrate are bonded so that the two minute channels and the channels are continuous. Manufacturing method of micro amount online biosensor. 7. The method according to any one of claims 4 to 6.
In the step of laminating the first substrate and the second substrate, the step of laminating using a photo-curable adhesive, a polymer thin film dissolved in a solvent vapor, or a low melting point glass thin film. A small amount of online
Io sensor manufacturing method . 8. The method for producing a minute amount of online biosensor according to claim 6, wherein the surface of the columnar microprojections is modified with a biological material and the columnar microprojections are formed by electroforming at the same time. Method.