JPH1038844A - Online biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To attain quantitative measurement in a small area by incorporating a sampling portion of a capillary, an oxygen reactor, and an electrochemical detector in a flow cell and pumping them by a syringe pump. SOLUTION: A glass capillary of 0.7mm in inner diameter is fused and extended, a sampling portion of approx. 15μ in tip inner diameter is prepared, and a teflon tube of 100 micrometers in inner diameter is plugged from a non- extension side and sealed by epoxy resin. That tube is introduced into a radial flow type electrochemical detector through a fine platinum tube in approx. 3m. On a surface of a grassy carbon electrode of approx. 6mm in diameter, Os polymer containing Western horseradish per-oxydase, enzyme glutamic acid oxide, and bovine serum albumin cross-linked with glutaric aldehyde are qualified by the Os polymer. An output side of the flow cell is connected to a syringe pump through a teflon tube and absorbed. The sampling portion is fined to units of micrometer and is absorbed to measure in a small area.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for measuring a cultured cell for quantitative measurement in a small area such as a single cell.

[0002]

2. Description of the Related Art In recent years, with the development of neuroscience and molecular biology, many attempts have been made to measure a physiologically active substance contained in a minute region in a living body in real time. Among them, electrochemical analysis methods are (1) suitable for measurement of minute amounts, (2) relatively high sensitivity, and (3) high efficiency by modifying electrodes with selective membranes or enzymes. (4) A simple and low-cost sensor can be manufactured. As an attempt to directly measure a biologically active substance electrochemically while keeping a living body alive, insert a microelectrode such as a carbon fiber electrode directly into a specific region in the living body and perform in-situ measurement,
Or 1-5m called microdialysis probe
As shown in Fig. 2, a small dialysis membrane of about m is inserted into a living body, a dialysate is sent by a pump, and a physiologically active substance contained in the living body is sampled through the membrane and sent to an electrochemical sensor online. In this way, biological materials are measured in real time. By using a sensor with an electrode modified with an enzyme membrane that selectively reacts with the target substance to generate an electrochemically active substance, sugars such as glucose and lactose, and neurotransmitters such as glutamate are measured in real time. ing. That is, FIG. 2 is a diagram showing a basic structure of a general biosensor using a microdialysis film for the purpose of in vivo [in vivo] measurement. On the other hand, in the measurement of a cultured cell or the like, measurement of a micro area on the order of a micrometer, such as measurement of a single cell, is required. In the microelectrode method, since a carbon fiber electrode having a diameter of several μm can be easily used, a single-cell level measurement is performed for a neurotransmitter or the like which can be measured by an electrochemical reaction directly with an electrode such as catecholamine [eg, T . J. Schroeder, J. et al.
A. JA Jankowski, K.M. T. Kawagoe, R.K. M. Wightman (RMWightma
n), C.I. C. Lefrou, and C.L. Amatoa (C. Amatore), analytical chemistry (Analytical
Chemistry), Vol. 64, pp. 3077-3083 (1
992)]. Further, detection of an enzymatic reaction or an immune reaction in a micro area on the order of micrometers has been attempted by using a scanning electrochemical microscope [for example, H. H .; H.Shik
u), T.I. T. Matsue, and I. Uchida (I.Uchi
da), Analytical Chemistry, Vol. 68, No. 12
76-78 (1996)]. On the other hand, measurement of cultured cells has been performed using a sensor combining a microdialysis probe and electrochemical detection (for example, Niwa, Torimitsu, Morita, Osborne, Yamamoto, 63rd Annual Meeting of the Institute of Electrical Chemistry). Compared to the electrode method, it is superior in quantitative property and long-term stability, and is easy to calibrate.

[0003]

In the measurement of cultured cells, the cells usually have a size of about 10 μm, so that the electrodes or sampling probes used in the measurement need to be miniaturized to the same order as the cells. A method combining microdialysis and an electrochemical sensor has high sensitivity and excellent quantitativeness, but it is extremely difficult to prepare a dialysis tube on the order of a micrometer, so that measurement at a single cell level is difficult. In addition, when the probe is small, the recovery rate of the target substance (a numerical value indicating what percentage of the target substance can be dialyzed from the external solution and sampled) decreases, so that highly sensitive measurement is difficult. On the other hand, a fiber-type microelectrode can easily be miniaturized to the size of a single cell, and compounds that easily cause an electrochemical reaction can be detected with extremely high sensitivity. On the other hand, in a system in which measurement is performed by combining an enzyme reaction and an electrode reaction, the amount of enzyme that can be immobilized on a microelectrode is small, and the long-term stability is poor depending on the enzyme used. Further, a system in which a coexisting substance is present has a drawback such that it is difficult to obtain high selectivity because it is necessary to remove all the effects on the electrode. Further, when a substance such as a coenzyme is required for the enzymatic reaction, it is necessary to add the substance to the culture system for performing the measurement, and it is necessary to always consider the influence on the biological sample. An object of the present invention is to provide an on-line biosensor for solving quantitative problems in a minute area, which solves the above-mentioned problems.

[0004]

SUMMARY OF THE INVENTION To summarize the present invention, a first invention of the present invention relates to an online biosensor, and comprises a flow cell in which a capillary sampling portion, an enzyme reactor, and an electrochemical detector are arranged. And a syringe pump driven in a suction mode. The second invention of the present invention relates to another on-line biosensor, and is characterized by comprising a sampling portion of a capillary, a flow cell having an enzyme-modified electrode, and a syringe pump driven in a suction mode.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. FIG. 1 shows the structure of the online biosensor of the present invention. That is, FIG. 1 is a diagram showing the basic structure of the online sensor of the present invention for the purpose of measuring a small area, such as single cell measurement in a culture system. In the present invention, in an online biosensor which conventionally performed sampling through a dialysis membrane of millimeter order, the sampling portion is formed into a microcapillary whose tip can be miniaturized to micrometer order, and the sample is sampled by suction. Therefore, it is possible to perform measurement in a finer area as compared with the microdialysis method. Also,
The measurement can be performed with high sensitivity because there is no decrease in the recovery rate due to miniaturization as in the diamond lysis method. Next, by inserting a small reactor between the sampling capillary and the detection electrode, it is possible to (1) generate an electrochemically active substance by an enzymatic reaction of a target substance, and (2) remove an interfering substance. Since it can be performed on-line, higher reaction efficiency of the target substance and higher selectivity to interfering substances can be realized as compared with measurement using a modified microelectrode. Furthermore,
Since the substance necessary for the enzyme reaction or the electrode reaction can be easily added by the confluence circuit shown in FIG.
It has the characteristic that a complicated reaction can be performed. In the present invention, it is preferable to use a radial flow type thin film cell as the flow cell.

[0006]

Hereinafter, the present invention will be described more specifically with reference to examples and drawings. The present invention is not limited only to the following examples.

Embodiment 1 FIG. 3 is a schematic diagram illustrating a configuration of an online biosensor according to an embodiment of the present invention. In FIG. 3, the sampling portion is a glass capillary having an inner diameter of 0.7 mm, which is melt-drawn by a micropipette maker (Narimo, PP83) to produce a sampling portion having a tip inner diameter of 15 μm. (Micro Dialysis Co., Ltd.) was inserted all the way from the opposite side to the stretched side and sealed with epoxy.
As shown in FIG. 3, the tube was introduced into a radial flow type electrochemical detector through a small platinum tube of 3 cm.
The electrodes used for detection are shown in FIG. That is, FIG. 4 is a cross-sectional view of the detector modification electrode. In FIG. 4, reference numeral 1 denotes a glassy carbon (GC) electrode, 2 denotes an osmium-polyvinyl pyridine film containing horseradish peroxidase, and 3 denotes bovine blood albumin / glutamate oxidase / glutaraldehyde = 1/1 / 0.1. means.
As shown in FIG. 4, glassy carbon (G
C) Electrode [Bioanalytical Systems (Bioanaly
osmium (Os) polyvinyl pyridine redox polymer (Os polymer) containing horseradish peroxidase (HRP) having the property of reducing hydrogen peroxide at a low potential on the surface [for example, Analytical Chemistry, No. 64 volumes, 3084-309
P. 0 (1992)], and glutamate oxidase (Yamasa Shoyu Co., Ltd.) and bovine blood albumin (BSA) (Sigma) cross-linked to glutaraldehyde were modified into two layers on the Os polymer. The outlet side of the radial flow cell was connected to a syringe pump (manufactured by CMA Microdialysis) via a Teflon tube. The syringe pump was modified by a jig so that it was driven in the suction mode, contrary to the normal mode. This sensor, a platinum tube electrode and a modified GC electrode were connected to a bipotentiostat LC4C for liquid chromatography (manufactured by Bioanalytical Systems). The reference electrode uses a silver / silver chloride electrode and is arranged in a radial flow cell. The counter electrode used was a flow cell block (made of stainless steel). The measurement was performed by setting the potential of the platinum tube electrode to 400 mV and the potential of the modified GC electrode to 0 mV. First, phosphate buffered saline was sent at a flow rate of 16 μl / min, and then phosphoric acid containing 1 μmol / L of L-glutamic acid was used. The measurement of the buffer solution was performed. When the phosphate buffer solution was switched to a solution containing glutamic acid, the current started to increase after about 30 seconds, and -16
A steady-state current value of nA was obtained. This value is 70% or more of the theoretical value assuming that all the sampled glutamic acid was oxidized, and the electrochemical detector was arranged in a radial flow type thin layer cell having high reaction efficiency. It is due to. Next, L-glutamic acid 1
μmol / liter and L-ascorbic acid 20μmol
When a phosphate buffered saline solution containing 1 / liter was measured, a steady-state current due to an oxidation reaction of about 1.2 μA was observed at the platinum tube electrode. On the other hand, -11 nA for the modified GC electrode
A steady state current (reduction current) was observed. However, when the measurement was performed without applying a potential to the platinum tube electrode, the steady-state current value of the modified GC electrode was 2 nA, and it was difficult to detect glutamic acid. By performing the pre-electrolysis with the platinum tube electrode, L-ascorbic acid which is an interfering substance was almost removed, and L-glutamic acid could be selectively detected.

To perform cell counting, rat cerebral nerve cells were cultured on a petri dish for 20 days. As shown in FIG. 5, a capillary was brought close to one of the cultured cells using a manipulator to continuously sample a solution in the vicinity of the cell, and a 100 mM potassium chloride aqueous solution was sprinkled from the other capillary onto a very small amount of cells. , The current value decreased by -0.5 nA. This indicates that the glutamate release due to nerve cell stimulation could be measured in real time by the sensor. FIG. 5 is a schematic diagram showing measurement of neurotransmitter release from a single neuron by the sensor of the present invention.

Embodiment 2 FIG. 6 is a schematic diagram illustrating a configuration of an online biosensor according to an embodiment of the present invention. The apparatus used was basically the same as in Example 1, except that instead of a platinum tube electrode, an enzyme reactor filled with beads immobilized with two kinds of enzymes, choline oxidase and catalase, was connected. For the detection, an Os polymer film containing HRP on a 6-mm GC electrode, and a film obtained by modifying two kinds of enzymes, acetylcholinesterase and catalase, with a cross-linked film with glutaraldehyde were used. The measurement was aimed at highly sensitive and selective measurement of acetylcholine. 1 μM (M
= Mol / L), a reduction current began to flow 30 seconds after the start of the liquid supply, and a steady current value of -31 nA was obtained. next,
When a buffer solution containing 2 μM choline and 100 nM acetylcholine was measured, a value of −3.0 nA was obtained. Choline was completely oxidized by choline oxidase in the precolumn, and all the generated hydrogen peroxide was also converted by catalase. It was found to be decomposed. On the other hand, an electrode in which acetylcholinesterase and choline oxidase are immobilized on a carbon fiber electrode has the same
A value of 60 nA was obtained, making it difficult to evaluate the concentration of acetylcholine alone due to the choline reaction. The sensor of the present invention could quantitatively measure concentrations up to 2.5 nM in the presence of 2 μM choline.

Embodiment 3 FIG. 7 is a schematic diagram illustrating a configuration of an online biosensor according to an embodiment of the present invention. The apparatus used was basically the same as that in Example 1, and the oxygen-saturated phosphate buffered saline was merged immediately after the platinum tube electrode by a merging circuit. In the measurement, the potential of the platinum tube electrode was set to 400 mV, and the potential of the modified GC electrode was set to 0 mV. First, phosphate buffered saline was sucked at a flow rate of 16 μl / min with a syringe pump without attaching a merging circuit. A phosphate buffer solution containing 1 to 100 μmol / liter of glutamic acid was measured. When the phosphate buffer solution was switched to a solution containing glutamic acid, the current started to increase after about 30 seconds, and-
A steady-state current value of 16 nA was obtained. Next, L-glutamic acid 10 μmol / liter and 50 μmol / liter,
When a 100 μmol / liter solution was measured, it was -155 nA, -600 nA, and -930 nA, respectively, indicating that good linearity could not be obtained. On the other hand, when the merging circuit is connected and the solution is sent so that the solution volume on the sample side and the solution volume on the merging side becomes approximately 1: 1 and the measurement is performed, the glutamic acid concentration,
For 1, 10, 50, 100 μmol / liter-
8.2 nA, -79 nA, -390 nA, -760 nA
And the current value on the low concentration side was reduced to about half by dilution, but the linearity with respect to the measurement of glutamic acid of 1 to 100 μmol / liter was improved. This is because glutamate oxidase causes a decrease in response due to lack of oxygen when measuring high-concentration glutamate.However, by continuously flowing a solution saturated with oxygen, the enzyme reaction can be performed without oxygen shortage. This is because it has progressed. In the measurement of glutamate, glutamate dihydrogenase can be used instead of glutamate oxidase. In that case, it is necessary to supply NAD ⁺ as a coenzyme. Also in this case, the sensor for the purpose of measuring a micro area can be obtained by combining a solution containing NAD ⁺ immediately before the reactor (or electrode) on which glutamate dihydrogenase is immobilized without adding NAD ⁺ to the sample solution. Can be formed.

[0011]

As described above, the on-line biosensor according to the present invention comprises a sampling portion of a capillary, an enzyme reactor, a thin-layer electrochemical detector, a syringe driven in a suction mode, or an on-line biosensor. Alternatively, it is characterized by comprising a sampling portion of a capillary, an enzyme-modified electrochemical detector, and a syringe pump driven in a suction mode. In this sensor, since the sampling unit is a glass capillary, miniaturization is extremely easy as compared with a conventional online sensor using a microdialysis film as a sampling probe, and it is effective as a sensor for measuring a minute area. In addition, an enzyme reactor that reacts with interfering substances and removes it online between the sampling part and the detector can be inserted, and electrodes for pre-electrolysis can be inserted, achieving higher selectivity compared to fiber-type microelectrodes. can do. Further, since the size of the electrode may be somewhat large, more enzymes having a problem in life can be immobilized than the fiber-type electrode, and the long-term stability is excellent. Furthermore, even when a third chemical substance such as oxygen or coenzyme is continuously supplied for measurement of a target substance, it can be merged online from another solution reservoir without adding a reagent to the solution to be measured. It is extremely useful as a tool for neurochemistry and single cell level research such as measurement of transmitters in cultured neurons and measurement of single cells. In addition, the use of the radial flow type thin-layer cell achieves high reaction efficiency of the electrochemical reaction, and enables more sensitive measurement.

[Brief description of the drawings]

FIG. 1 is a diagram showing a basic structure of an on-line biosensor of the present invention for measuring a small area such as a single cell measurement in a culture system.

FIG. 2 is a diagram showing a basic structure of a general biosensor using a microdialysis film for the purpose of in vivo measurement.

FIG. 3 is a diagram showing the structure of an online glutamate sensor shown in Example 1 of the present invention.

FIG. 4 is a cross-sectional view of the detector modification electrode shown in FIG.

FIG. 5 is a schematic diagram of measurement of neurotransmitter release from a single neuron by the sensor of the present invention.

FIG. 6 is a diagram showing the structure of an online acetylcholine sensor shown in Example 2 of the present invention.

FIG. 7 is a view showing the structure of an on-line sensor for measuring high concentration of glutamic acid shown in Example 3 of the present invention.

[Explanation of symbols]

1: glassy carbon (GC) electrode, 2: osmium-polyvinylpyridine film containing horseradish peroxidase, 3: bovine blood albumin / glutamic acid oxidase / glutaraldehyde = 1/1 / 0.1

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Masao Morita, Inventor 3-19-2, Nishishinjuku, Shinjuku-ku, Tokyo Inside Nippon Telegraph and Telephone Corporation (72) Inventor Hisao Tabei 1-3-1, Gotenyama, Musashino-shi, Tokyo Inside NTT Advanced Technology Co., Ltd.

Claims (3)

[Claims] 1. An on-line biosensor comprising a capillary sampling part, an enzyme reactor, a flow cell in which an electrochemical detector is arranged, and a syringe pump driven in a suction mode. 2. An on-line biosensor comprising a capillary sampling portion, a flow cell having an enzyme-modified electrode, and a syringe pump driven in a suction mode. 3. The online biosensor according to claim 1, wherein a radial flow type thin layer cell is used as the flow cell.