KR20100136159A - Creatinine microsensors with reduced interference from creatine - Google Patents

Abstract

PURPOSE: A creatine microsensor with reduced interfering effect of creatine using sensitive film composition is provided to fix the sensitive film on an operation electrode to effectively remove interfering effect of creatine. CONSTITUTION: A method for manufacturing a flat biosensor comprises: a step of laminating soluble polymer-fixed hydrolase and oxidase on a single electrode to form a layer; a step of introducing a porous thin film on the layer; a step of laminating soluble polymer-fixed transferring enzyme and triphosphate adenosine; and a step of gradually laminating polymer ion film and protection film.

Description

 Creatinine microsensors with reduced interference from creatine}

  The present invention relates to a biosensor sensitized membrane composition and an electrochemical biosensor comprising the same, and specifically, a biosensor sensitized membrane composition and an electrochemical including the same that can significantly reduce the interference of interference species in the measurement of creatinine It relates to a biosensor.

  Recently, the determination of creatinine in clinical samples is a very important problem for the determination and treatment of abnormalities in the kidney function. In addition, the development of a small clinical analyzer for the rapid increase in attention to health, rapid response in emergency and patient management is a development factor that has been strongly demanded in the medical device market. Until now, the development of a small measuring device and a small sensor suitable for handheld like a blood glucose meter have been commercialized. Based on the existing technology, if the development of clinical creatinine measuring sensors and small analyzers, which are currently in great demand, is urgently developed, the ripple effect on the domestic and overseas clinical analysis measuring equipment market is expected to be very large. The operating principle of the biosensor for measuring creatinine is based on colorimetric or electrochemical methods.

 Among them, the electrochemical method is illustrated by Scheme 1 below.

  In the above formula, CA represents creatininase, CI represents creatinase, and SO represents sarcosine oxidase. As shown in Scheme 1 (1) first creatinine in the blood is oxidized to creatine by creatinine hydrolase. Creatine then becomes urea with sacosine by creatine hydrolase, and sacosine is broken down by sacosine oxidase to form formaldehyde, glycine and hydrogen peroxide. Electrochemically active hydrogen peroxide is produced from creatinine and diffuses to the electrode surface. At this time, the concentration of creatinine in the blood is measured quantitatively by measuring the current generated by applying the oxidation potential of hydrogen peroxide on the surface of the working electrode.

  The biosensor using the above-mentioned electrochemical method as an operation principle is called an electrochemical biosensor. Such electrochemical biosensors can reduce the effects of oxygen, unlike conventional biosensors by colorimetric methods, and have the advantage that samples can be used without pretreatment. Such electrochemical biosensors can be used to monitor and control the amount of creatinine in the blood.

  In general, the accuracy of the sensor is greatly influenced by various interfering species such as ascorbic acid, uric acid and acetoaminophene, which are susceptible to oxidation in blood samples. In addition, the most serious error is caused by creatine produced by creatinine degradation. For those who frequently measure creatinine levels in the blood using disposable biosensor chips and strips, using creatinine sensors that are heavily influenced by interfering ion species is likely to give incorrect results. These inaccurate consequences can even pose a danger to people's lives.

  Therefore, in the present invention, creatine kinase and adenosine triphosphate were introduced to reduce the interference effect of creatine, which causes the largest error in creatinine measurement.

  The object of the present invention is not only to solve the problems caused by creatine, but also to manufacture a sensitized membrane composition and a sensor using the same, which can dramatically reduce the effect on interfering ion species in the measurement of creatinine in the blood. will be.

  In order to achieve the above object, the present invention provides a sensitized membrane composition comprising an enzyme, adenosine triphosphate, hydrophilic polymer, polyanion exchanger, polycation exchanger and the like. The present invention provides a biosensor including a substrate, an operating circuit connecting line, an auxiliary electrode connecting line, an operating electrode, an auxiliary electrode, and an insulating film. The present invention also provides a biosensor containing the sensitized membrane composition. That is, the sensitized membrane composition of the present invention can significantly reduce the influence on the interfering ions according to the composition of the membrane. In addition, the biosensor of the present invention has the advantages of excellent reproducibility and reliability, and can be mass-produced at low cost due to its simple structure.

  Hereinafter, the present invention will be described in detail.

 The present invention provides a sensitizing membrane composition comprising an enzyme, adenosine triphosphate, a hydrophilic polymer, a polyanion exchanger, a polycation exchanger, and the like, wherein the sensitizing membrane composition is selected according to an interfering substance in blood such as ascorbic acid, acetoaminophen and uric acid. It also reduces the effects on signal size changes and also significantly reduces the interference effects of creatine.

As shown in Equation 1, the metabolite is quantified by electrochemically measuring hydrogen peroxide generated by reaction with various metabolites. The water-soluble polymer serves to help stabilize and dispers the enzyme as a polymer support of the sensitized membrane composition. Biosensors that employ enzymes to fix hydrogels, such as water-soluble PVA, require an external protective film to minimize the outflow of enzymes and improve the life of the sensor. Various polymer materials with very fine pores were introduced into the biosensor as an outer protective film.

 Polymeric materials used for the outer protective film include cellulose acetate, poly (vinyl chloride), polycarbonate, and polyurethane. However, the above-mentioned materials have disadvantages of poor adhesion, inconsistent pore size of the membrane itself, or low permeability of the substrate. Polyurethanes, composed of hard-segments and soft-segments, have been used in biomedical applications for their outstanding biocompatibility as polymers with many uses in various fields. HPU consists of soft pieces (poly (tetramethylene oxide), poly (ethylene glycol), poly (propylene glycol)) that can dissolve or absorb water. In particular, the HPU has different capacities for water absorption and membrane pore size and excellent adhesion to various solids, depending on the ratio and nature of the soft pieces used. Compared with the introduction of these components, the time taken for the sensor to stabilize is relatively short. In the present invention, by using the advantages of the HPU to introduce an aromatic-based HPU as an outer protective film to study the creatinine sensor.

  In the present invention, the method used for measuring creatinine is amperometric. The measurement of hydrogen peroxide is a very important factor in the biosensor using the current method. This is because hydrogen peroxide is the final product of the oxidase reaction used to analyze biochemically important substances. Biosensors that use these hydrogen peroxide analytes are oxidized at the electrode together with other metabolites (eg, ascorbic acid, uric acid, acetoaminophen, dopamine, etc.) that can be oxidized at low potentials in the sample, causing significant measurement errors. . As a method for solving the measurement error caused by such interference materials, 1) using a membrane having selective permeability by particle size, 2) using a membrane having selective permeability due to repulsive force of negative charge, 3) electron transfer mediator Using a lower oxidation potential, 4) using a reduction potential using peroxidase, 5) introducing an oxidant layer, 6) reducing the oxidation potential of hydrogen peroxide by introducing Pt black to the electrode surface. Methods and the like have been studied.

 The method used in the present invention improves the sensor response by enlarging the area of the electrode by lowering the oxidation potential by introducing a Pt black layer on the electrode surface, which is the last method, and has the advantage of easily fixing the enzyme.

  As the substrate used in the present invention, a polymer material such as ceramic, plastic, glass plate, or silicon may be used, and organic polymer materials such as polyester, polyvinyl chloride, and polycarbonate are generally used.

  In the present invention, various electrode materials such as silver, palladium, copper, platinum, silver / silver chloride, mercury / mercury oxide, and carbon can be easily introduced onto the support using screen printing technology. Screen printing technology is economical in the construction of a potentiometric or voltammetric electrode system having a planar structure and a sandwich structure, and is easy to construct a disposable chemical sensor with excellent reproducibility and sensitivity. Electrode manufactured by introducing screen printing technology having this advantage is performed according to the movement while applying a constant force to the squeegee (squeegee) against the open pattern, which can sequentially introduce ink or dough onto a specific substrate in a pattern. It is a way.

  Surfactants used in the preparation of the enzyme solution were used for rapid movement of the sample, and the surfactants were nonionic interfaces such as polyethylene glycol, oxyethylated alkylphoenols (neonols), triton X-100, and polyethylated systems. It is classified as an active agent, anionic surfactant such as sodium decylsulfate family, or cationic surfactant of pyridinium family such as decylpuridinium chloride or tetradecylpyridininium, and Triton X-100 is generally used.

 As shown in Scheme 2, the introduction of creatine transferase and adenosine triphosphate produces phosphocreatin rather than creatine and creatine. That is, hydrogen peroxide is generated only from creatinine because hydrogen peroxide is not produced as shown in the above Reaction Scheme 1. Therefore, creatinine can be measured accurately without considering creatine interference.

  As described above, the present invention relates to a biosensor implemented in a planar shape, and the biosensor according to the present invention has a simple structure, is easy to manufacture, and does not require chemical or physical pretreatment of a small amount of sample, Even though it does not need to remove the obstacles, it is possible to introduce a constant and fast. In addition, the reproducibility and precision between the electrodes is excellent, the measurement time is fast, and the mass production can be made inexpensively. In addition, the patient can measure accurately at home or in the hospital without being influenced by interfering substances. It is a very useful biosensor that can measure creatinine more accurately by effectively removing creatine, which was considered to be the most severe interfering action in the world.

  Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the examples.

Manufacturing Example 1 Single Biosensor Electrode body  making

  As shown in FIGS. 1A, 1B, and 1C, screen printing was performed using silver paste on a polyester (PE) substrate to form an operating circuit connection line 11 and a reference electrode 12. Thereafter, screen printing is performed using a conductive carbon paste to form the working electrode 13 and the auxiliary electrode 14. Thereafter, an insulating polymer 15 was formed using an insulator polymer paste and heat-treated at 150 ° C. for 15 minutes.

≪ Example 1 > 3-electrode  Fabrication of Planar Biosensor

The planar constant voltage amperometric sensor used a printing method. First, the electrode was formed on a white polyester (PE) insulating support and an insulating layer was introduced thereon. The sensor consists of a reference electrode, a working electrode and an auxiliary electrode. The Ag layer formed was treated with 0.1 M FeCl ₃ solution for 10 minutes to form Ag / AgCl. Also, in order to form a Pt black layer on the carbon material of the working electrode, -0.1 V to 0.5 V (vs. Ag) in a mixed solution of 3 wt% chloroplatinic acid (H ₂ PtCl ₆ ), 0.029 wt% lead acetate, and 97 wt% water. / AgCl) was treated for 3 minutes at 50 mV / s in the potential range.

Example 2 Without Creatine Transferase Induction film  Sensor Preparation

  Pretreatment with surfactant (Triton-X100) at the working electrode and 10 units / 3 uL of creatininase, creatinase and sarcosine oxidase Proteins or water-soluble polymers capable of calibrating enzymes in a certain amount may be used. Generally, PVA is used, and a certain amount of a solution containing 12 mg / 300 uL of PVA is dropped. Then, after drying for about 20 minutes in a 40 ° C oven, the hydrogel layer on the enzyme can be covered with an outer protective layer, usually using HPU and mixed with HPU (AR-25-80A) 20 mg / 500 uL cyclohexanone A certain amount of the prepared solution is dropped. On top of that, the polyanion exchanger can be covered to reduce the interfering effect of other interfering ion species. Preferably, a solution containing 5 wt% of polyethlene imine is dropped and dried in a 40 ° C oven for about 10 minutes. Then drop the solution containing the polycation exchanger, preferably 5 wt% of nafion, and dry in a 40 ° C. oven for about 10 minutes.

Example 3 Using Creatine Transferase Induction film  Sensor Preparation

  Pretreatment with surfactant (Triton-X100) at the working electrode part Proteins or water-soluble polymers can be used to calibrate enzymes. Generally, PVA is used and a certain amount of 12 mg / 300 uL of PVA is dropped. Then, after drying for about 20 minutes in a 40 ° C oven, the hydrogel layer on the enzyme can be covered with an outer protective layer, usually using HPU and mixed with HPU (AR-25-80A) 20 mg / 500 uL cyclohexanone A certain amount of the prepared solution is dropped. In order to effectively remove creatine, which was considered to be the most severe interfering action in the conventional creatinine sensor, a solution of 10 units and 20 mM of creatine kinase and adenosine triphosphate, respectively, was dropped. On top of that, the polyanion exchanger can be covered to reduce the interfering effect of other interfering ion species. Preferably, a solution containing 5 wt% of polyethlene imine is dropped and dried in a 40 ° C oven for about 10 minutes. Then, drop the solution containing the polycation exchanger, preferably 5 wt% of nafion, and dry in a 40 ° C. oven for about 10 minutes.

 Figure 1a is a schematic view of the manufacturing process planar biosensor electrode of the present invention,

 Figure 1b is a perspective view of the planar biosensor combination of the present invention,

 1C is an exploded perspective view of the planar biosensor of the present invention.

 Figure 2a is a calibration curve for the hydrogen peroxide standard solution in the operating portion of the planar biosensor of the present invention,

 Figure 2b is a calibration curve for the hydrogen peroxide standard solution after the introduction of Pt black to the operating part of the planar biosensor of the present invention.

 Figure 3a is a calibration curve for creatinine when the creatine transferase and adenosine triphosphate are not introduced to the operating portion of the planar biosensor of the present invention,

 Figure 3b is a calibration curve for creatine without the introduction of creatine transferase and adenosine triphosphate to the operating portion of the planar biosensor of the present invention,

 3C is a calibration curve for creatinine when creatine transferase and adenosine triphosphate are introduced into an operation unit of the planar biosensor of the present invention,

Figure 3d is a calibration curve for creatinine when creatine transferase and adenosine triphosphate are introduced into the operating part of the planar biosensor of the present invention.

 4A is a schematic diagram of the planar biosensor of the present invention.

(Explanation of symbols about main parts of drawing)

       10: Board 11: Operation circuit connection line

       12: reference electrode 13: working electrode

14: auxiliary electrode 15: insulating film

Claims (8)

  Form a layer by placing hydrolyzed polymerase and oxidase immobilized by water-soluble polymer on a single electrode body, and then introducing a porous thin film into a pre-formed layer, and then transferase and triphosphate fixed by water-soluble polymer to reduce the interfering effect of creatine. After raising the adenosine to form a layer, the planar biosensor and the sensitized membrane composition used for the biosensor, in which the polymer ion membrane and the protective layer are sequentially stacked.   The circuit board of claim 1, wherein the single electrode body comprises a substrate, a plurality of circuit connectors coated at regular intervals in a longitudinal direction on the substrate, a reference electrode applied to one end of the circuit connector, a working electrode, and an auxiliary electrode. Flat bio-sensor, characterized in that the insulating film is coated on the substrate except for the reference electrode, the working electrode and the auxiliary electrode.   The method of claim 1, wherein the water-soluble polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, polyfluorosulfonate, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose acetate and polyamide Sensitive film composition for sensors.   The planar biosensor according to claim 1, wherein the interference ion component having a diameter smaller than the pore diameter of the porous thin film among the sample components injected from one end of the porous thin film can be removed preliminarily during the movement and the biosensor used. Membrane composition.   The planar biosensor of claim 4, wherein the porous thin film is a paper, a hydrophilic organic polymer, or a hygroscopic ceramic polymer having a plurality of pores having a diameter of 5 μm to 10 μm formed on a surface thereof.   The method of claim 1, wherein the polymer ion membrane is a biosensor sensitive membrane composition, characterized in that selected from the group consisting of polyethyleneimine, polylysine, polydiglutamic acid, Nafion.   The sensitizing membrane composition for biosensor according to claim 1, wherein the hydrolase and the oxidase are creatinine hydrolase, creatine hydrolase, and sacosin oxidase.   The biosensing membrane composition according to claim 1, wherein the transferase comprises adenosine triphosphate as a urea creatine kinase.

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