JP2009236893A - Electrochemical biosensor for measuring ultratrace histamine (improved type) and ultratrace antibiotic measuring system using this sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide ultrasensitive electrochemical biosensor (improved type) for measuring histamine (hereinafter referred to as "histamine measuring sensor"), which detects histamine up to 0.005 nM, and also to provide an ultratrace antibiotic measuring system using this sensor. <P>SOLUTION: This sensor is an improved type of the already-patent applied histamine measuring electrochemical biosensor (Patent application No.2006-193388) having higher sensitivity than conventional ones. It consists of histamine dehydrogenase, tetrathiafulvalene, metal nanoparticle, naphion, gelatin and base material, and very sufficiently measures current generated by electrochemical oxidative reaction of hydrogen peroxide, a reactive product between the histamine dehydrogenase and histamine, through the tetrathiafulvalene as an electronic mediator. This determines a quantity of ultratrace antibiotic contained in water solution by utilizing the fact that the above enzyme reaction in this biosensor is significantly blocked and that this sensor has remarkable high sensitivity to the above histamine electrochemical oxidative reaction. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

Detailed description

The present invention relates to provision of a sensor for measuring trace amounts of histamine, which is a neurotransmitter contained in fresh foods such as fish meat and living tissues, and a system for analyzing trace amounts of various antibiotics using the characteristics of the sensors.

Construction of a system for measuring a very small amount of penicillin G, tetracycline and other various antibiotics remaining in a biological body fluid such as raw milk by a simple method is strongly demanded in the fields of medicine, physiology, dairy farming and a wide range of food industries. For the measurement of these trace antibiotics, a method using a combination of a high performance liquid chromatograph and a UV detector (for example, VF Samanidou et al., J. Sep. Sci., 29, 1550-1560, 2006), high performance liquid There are a method using a combination of a chromatograph and a mass spectrometer (for example, M. Kozono et al., Seikatsu Eisei, 49, 220-226, 2005) and a method using a combination of a gas chromatograph and a mass analyzer. In the method using a combination of a liquid chromatograph and a UV detector, the detection limit concentration is 3 ppb, and in the method using a combination of a liquid chromatograph and a mass analyzer, it is 1 ppb.

However, the above method is expensive in apparatus, requires skilled skills in its operation, and requires several days of measurement time. Furthermore, the measurement system is large and unsuitable for versatility.

On the other hand, as a smaller and cheaper method, utilizing the fact that the lactic acid oxidation reaction by lactic acid oxidase is inhibited by the presence of antibiotics such as penicillin G and chloramphenicol, the amount of antibiotics is reduced by lactate oxidase. Electrochemical oxygen sensor shows the rate of decrease of dissolved oxygen in lactic acid aqueous solution consumed by oxidation reaction and the rate of decrease of dissolved oxygen consumed in oxidative reaction by lactic acid oxidase in lactic acid aqueous solution added with any amount of the above antibiotics And developed a method for determining the antibiotic concentration from the difference in decrease rate (T. Rinken et al., J. Biochem. Biophys. Methods, 66, 13-21, 2006). Since this method measures the decrease rate of dissolved oxygen in the sample aqueous solution, high sensitivity cannot be expected, and the detection ranges are 0.2-1 ppm for penicillin G, 1-20 ppm for chloramphenicol, and a liquid chromatograph is used. The sensitivity is significantly inferior to the method.

Problems to be solved by the invention

The sensor for measuring histamine of the present invention is an improved version of the sensor for measuring histamine that has already been applied for a patent. Using this sensor, penicillin G, tetracycline, and various other ultra trace amounts that are extremely sensitive, inexpensive, small and easy to operate. The objective is to provide an antibiotic measurement system.

Means for solving the problem

The gist of the sensor for measuring histamine of the present invention is an electrochemical biosensor comprising a metal electrode support and a plurality of coatings containing histamine dehydrogenase, tetrathiafulvalene, gold nanoparticles, Nafion, and gelatin. .
The antibiotic measurement system utilizes the inhibitory effect of the enzyme function of antibiotics discovered by Rinken et al. Described in the section of the prior art, and has a very high sensitivity to histamine. The gist of this is to measure the amount of histamine oxidation current by the decrease of histamine oxidation current caused by the inhibition of ultra-small amount of antibiotics.

The sensor according to claim 1 is a tetrathiafulvalene which is an electronic mediator that promotes electrochemical oxidation of hydrogen peroxide, which is an oxidation product of histamine and histamine dehydrogenase, in order to measure microscopic histamine in an aqueous solution. It is a sensor for histamine measurement characterized by comprising a film.

The present invention according to claim 2 relates to a thin metal plate support as a sensor support and a film carried thereon. The coating consists of four layers, and in order from the bottom, the first layer is a coating of tetrathiafulvalene, which is generated by the reaction of histamine and histamine dehydrogenase, and reaches the tetrathiafulvalene layer by diffusion. An electronic mediator coating that mediates and promotes the reaction. The second layer is a mixture film consisting of Nafion, tetrathiafulvalene, and gold nanoparticles. Hydrogen peroxide that has reached this layer is promoted by the electrochemical oxidation reaction with tetrathiafulvalene and gold nanoparticles, and the electrons are converted into gold nanoparticles. Is transmitted to the support via. Nafion prevents a small amount of ascorbic acid, uric acid and other organic compound anions, which are active in electrochemical oxidation reactions, from being diffused to the support, and prevents poisoning by direct attachment of proteins such as enzymes to the support surface. prevent. The third layer is a coating of a mixture of histamine dehydrogenase, tetrathiafulvalene and gold nanoparticles, and the hydrogen peroxide generated in this layer is electrochemically mediated through tetrathiafulvalene on the gold nanoparticles close to the active center of the enzyme. Oxidized and generated electrons are transferred to the support through the gold nanoparticles close to each other as in the second layer. Gold nanoparticles function as an electrode that electrochemically oxidizes hydrogen peroxide, contribute to the increase in histamine detection sensitivity of this sensor, and also have the function of transmitting the generated electrons to the support (X. Ren, etc.) Biosensors and Bioelectronics, 21, 433-437 2005). The fourth layer is a crosslinked gelatin film that prevents the hydrogen peroxide generated in the third layer from escaping into the electrolyte solution.

The measurement system of the present invention according to claim 3 is a sensor for measuring various antibiotics using the histamine dehydrogenase inhibitory effect of histamine dehydrogenase by antibiotics, and is produced by adding a very small amount of antibiotics to an aqueous histamine solution. Antibiotic concentration is measured by a decrease in oxidation current.

The measurement system of the present invention uses a histamine dehydrogenase (produced by Kikkoman Co., Ltd.) having a function to oxidize histamine, and uses a sensor for measuring histamine electrochemically oxidized. Can quantitate antibiotics.

DESCRIPTION OF EMBODIMENTS Embodiments relating to a histamine measurement sensor of the present invention and an antibiotic measurement system using the same will be described below with reference to the drawings.

FIG. 1 is an example of a basic structural diagram of a sensor for measuring histamine according to the present invention.

A thin gold plate (11) is used for the support.

A tetrathiafulvalene layer (12) is formed on this support.

Further, a mixture film (13) of Nafion, tetrathiafulvalene and gold nanoparticles is formed on the upper part.

A mixture film (14) comprising histamine dehydrogenase, gold nanoparticles and tetrathiafulvalene is formed on this film.

A gelatin film (15) is formed on the uppermost layer.
(Manufacturing process of sensor for measuring histamine)

An example of the manufacturing process of the sensor for histamine measurement of this invention is shown.

A gold plate having a surface area of 0.5 cm ² and a thickness of 0.03 mm is polished with alumina, and then a gold wire having a diameter of 0.3 mm and a length of 7 cm is spot welded as a lead wire, followed by ultrasonic cleaning with acetone and purified water. A support is used.

The above support was dipped on a mixed solution of 7 mg of tetrathiafulvalene, 5 ml of 2-propanol, and 5 ml of purified water, then immediately washed with purified water and dried in the room to form a first layer of tetrathiafulvalene film (FIG. 1). (12)).

A coating solution of tetrathiafulvalene, Nafion, and gold colloid is applied on the coating by dipping and dried in the room for several hours to form a second layer coating that is a mixed coating of tetrathiafulvalene, Nafion, and gold fine particles (see FIG. 1 (13)). The above mixed solution is prepared by the following method. Add 7 mg of tetrathiafulvalene to 0.5 ml of 5% Nafion-117 solution (Sigma Aldrich), stir with Voltex, and heat in a water bath at 60 ° C. while shaking the solution well until the color of the solution changes from yellow to brown. The obtained solution and a 5 nm colloidal gold solution (Sigma Aldrich) are mixed at a volume ratio of 1: 1.

A mixture of histamine dehydrogenase, tetrathiafulvalene, gold colloid, and gelatin is applied to the resulting second layer coating by dipping and dried indoors all day and night to form tetrathiafulvalene, histamine dehydrogenase, gelatin, gold A third layer film, which is a mixture film of nanoparticles, is formed (FIG. 1 (14)). The mixed solution is prepared by the following method. Dissolve 16 mg of histamine dehydrogenase in 200 μl of purified water, mix 160 μl of the resulting solution, 1 ml of acetone solution containing 12 mg of tetrathiafulvalene, and 0.5 ml of 20% aqueous solution of 37% formaldehyde solution containing 1% gelatin. The obtained solution and a 5 nm colloidal gold solution are mixed at a volume ratio of 1: 1.

A 20% aqueous solution of 1% gelatin-37% formaldehyde solution is applied to the third layer coating by dipping to form a fourth layer coating, which is then dried in the room for several hours to complete the production of the sensor for measuring histamine.
(Construction of antibiotic measurement system)

FIG. 2 shows a measurement system including a histamine measurement sensor.

The measurement system includes a histamine measurement sensor (21), a reference electrode (22), an electrochemical cell comprising a counter electrode (23), an electrochemical analyzer (1200A manufactured by BAS) (26), and a PC (27). The reference electrode used was a silver / silver chloride electrode (manufactured by BIAS), and the counter electrode was a platinum wire electrode (manufactured by BIAS). When measuring, the potential of the measuring sensor is set to +650 mV with respect to the reference electrode, and when histamine or antibiotics are quantified, the concentration of histamine or antibiotics is gradually increased from 1 ppt to 1 ppm in the phosphate buffer solution in the electrolytic cell. In addition, the corresponding change in histamine oxidation current is measured sequentially. On the other hand, when quantifying antibiotics in raw milk, the raw milk concentration was diluted to 1/10 with a phosphate buffer to prepare a sample solution.
(Quantitative measurement of trace histamine)

FIG. 3 shows the relationship between histamine oxidation current and histamine concentration. The histamine oxidation current (I) and histamine concentration _{C his-following} relationship holds true in the following concentrations 4 ppb. Where k ₁ is a proportionality constant.
I = k ₁ logC _his formula 1

As shown in the figure, the detection limit of histamine of the sensor for measuring histamine of the present invention is 0.5 ppt, which is 10 times higher than the detection sensitivity of the electrochemical biosensor for detecting histamine previously applied for.

(Quantification of penicillin G)

The quantitative measurement method and measurement result of penicillin G using the histamine measurement sensor of the present invention are shown below. In the measurement, a phosphate buffer solution having a histamine concentration of 100 ppb is used as an electrolyte solution, and penicillin G is sequentially added to the solution from a concentration of 1 ppt to 100 ppb, and a decrease value of the histamine oxidation current corresponding to each penicillin G concentration is sequentially added. It was measured.

First, FIG. 4 shows the time course of histamine oxidation current when 1 ppt of penicillin G was added to a phosphate buffer solution containing 100 ppb histamine. As is apparent from the figure, after the addition of penicillin G, the oxidation current value first decreases with time, and becomes almost constant after 5-10 minutes.

A plot of the decrease in histamine oxidation current corresponding to a penicillin G concentration of 1 ppt-100 ppb is shown in FIG. The decrease value (−ΔI) of the histamine oxidation current by the addition of penicillin G has the following relationship when the concentration of penicillin G, C _PG is 50 ppb or less. Here k ₂ is a proportionality constant.
−ΔI = k ₂ log (C _PG ) Equation 2

As shown in this figure, the detection limit of penicillin G in the measurement system of the present invention is 0.5 ppt, which is about 2000 times higher than the detection limit of penicillin G by the previously reported high performance liquid chromatograph.

(Quantification of tetracycline)

Hereinafter, quantitative measurement and measurement results of tetracycline using the sensor of the present invention are shown. The measurement method is the same as in the first embodiment. The measured concentration range of tetracycline is 1ppt to 10ppb.

The measurement results are shown in FIG. The decrease value of the histamine oxidation current due to the addition of tetracycline is proportional to the logarithm of the tetracycline concentration up to a tetracycline concentration of 100 ppt, and is expressed by the same relationship as in Equation 2.

As shown in the figure, the detection limit of tetracycline in the measurement system of the present invention is 0.5 ppt, which is the same as the detection limit of penicillin G.

(Quantification of penicillin G in raw milk)

The quantitative measurement of penicillin G in raw milk and the measurement results are shown below using the sensor of the present invention. The measurement method is the same as in Examples 1 and 2. The measured concentration range of penicillin G is 6ppt to 15ppb.

The measurement results are shown in FIG. The decrease value of the histamine oxidation current due to the addition of penicillin G is proportional to the logarithm of the penicillin G concentration up to 200 ppt, and is expressed by equation 2 as in the case of Examples 1 and 3. The detection limit is 1 ppt. From this result, it was confirmed that this sensor can quantitatively measure antibiotics without being affected by protein, fat, sugar, etc. contained in raw milk.

The invention's effect

As described above, the measurement system of the present invention has the following effects.
1. The detection limit concentration is 0.5-1 ppt.
2. Compared with the previously reported measurement system using an electrochemical oxygen sensor, it has a sensitivity of 10 ⁶ -10 ⁷ times and about 2000 times that of a liquid chromatographic method combined with a UV detector.
3. Small and inexpensive and easy to operate.
4). Measurements can be taken under open air.

From the above characteristics, the electrochemical biosensor of the present invention is expected to be used in various fields of research such as food hygiene, medicine and physiology, and dairy farming, food processing, and many other production sites.

(Describe each figure according to the figure number in the drawing.)
It is a figure which shows sectional drawing of a sensor electrode. It is a figure which shows a sensor electrode and a measurement system. It is a figure which shows the relationship between the histamine density | concentration in a phosphate buffer solution, and a histamine oxidation current. It is a figure which shows the time passage of the histamine oxidation current change by 1ppt penicillin G addition in a histamine containing phosphate buffer solution. It is a figure which shows the relationship between the penicillin G density | concentration in a histamine containing phosphate buffer solution, and an electric current. It is a figure which shows the relationship between the tetracycline density | concentration in a histamine containing phosphate buffer solution, and an oxidation current. It is a figure which shows the relationship between the penicillin G density | concentration in 1/10 dilution raw milk with a phosphate buffer solution, and an oxidation current.

Explanation of symbols

11 Gold electrode support 12 Tetrathiofulvalene complex coating 13 Tetrathiofulvalene complex, gold nanoparticle, Nafion mixture coating 14 Histamine dehydrogenase, tetrathiofulvalene complex, gold nanoparticle, gelatin mixture coating 15 Gelatin coating 21 Electricity Chemical biosensor 22 Silver / silver chloride reference electrode 23 Platinum counter electrode 24 Teflon coating 25 Teflon cap 26 Potentiostat 27 Recorder

Claims (3)

A sensor for measuring histamine that measures the current generated by an electrochemical oxidation reaction in which tetrathiafulvalene mediates hydrogen peroxide, which is an oxidation reaction product of histamine and histamine dehydrogenase, as an electronic mediator. The sensor is composed of an electrode support and a four-layered support film, that is, a tetrathiafulvalene film (first layer) which is an electronic mediator, a mixture film (second layer) composed of tetrathiafulvalene, Nafion and gold nanoparticles, histamine dehydrogenation. 2. The sensor for measuring histamine according to claim 1, which has a structure comprising a mixture film (third layer) and a gelatin film (fourth layer) comprising an enzyme, gold nanoparticles, tetrathiafulvalene and gelatin. Ultrafine various antibiotic measurement system using the above sensor as a detector.

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