TWI408371B - Enzyme electrode and method for producing the same - Google Patents

Abstract

The present invention discloses an enzyme electrode and its producing method, which comprises: providing a substrate with a carbon surface; forming a gold surface on the carbon surface and thus forming an electrode; covalently bonding a mediator containing an aldehyde group to the electrode; and covalently bonding a glucose oxidase to the electrode.

Description

Enzyme electrode and its manufacturing method

The present invention relates to an enzyme electrode and a method of manufacturing the same and an application thereof.

Methods for the analysis of carbohydrates include optical rotation, colorimetry, high-performance liquid chromatography (HPLC), and enzyme electrodes (Qiu, J.-D., Zhou, W.). -M., Guo, J., Wang, R., Liang, R.-P., 2009. Anal Biochem. 385, 264-269; Sun, Y., Wang, H., Sun, C., 2008. Biosens. Bioelectron .24, 22-28; Zhang, S., Wang, N., Yu, H., Niu, Y., Sun, C., 2005. Bioelectrochemistry 67, 15-22). Most scientists use HPLC to simultaneously analyze several carbohydrates for high accuracy and sensitivity. However, HPLC also has a number of disadvantages, its apparatus is bulky, expensive, and has a long analysis time; therefore, HPLC is not the most desirable method for many applications, such as glucose analysis in clinically diabetic patients. In recent years, enzyme electrodes have become a better choice than HPLC for the analysis of specific carbohydrates due to their specific material selectivity, ease of operation, low cost, easy portability, and instant and rapid analysis.

Clark and Lyons developed the first generation of enzyme electrodes. Schlapfer (Wang, J., 2008. Chem. Rev. 108, 814-825) has since been modified to replace oxygen with a mediator to avoid the disadvantage of oxygen fluctuations in the air. Since then, ferrocene and its derivatives have been subjected to various desirable properties such as low molecular weight, reversibility, regeneration at low potential, and stable redox reactions (Fernández, L., Carrero, H., 2005. Electrochim. Acta 50, 1233-1240), an artificial vehicle widely used as a glucose enzyme electrode (Escorcia, A., Dhirani, A.-A., 2007. J. Electroanal. Chem. 601, 260-268; Qian, WL, Khan, Z., Watson, DG, Fearnley, J., 2008. J. FoodComposit. Anal. 21, 78-83; Wang, S., Du, D., 2004. Sens. Actuators B 97, 373-378). Typically, the vehicle is dissolved in an electrolyte that analyzes glucose. However, ferrocene and its derivatives are carcinogenic substances, and the electrolyte after the test cannot be disposed of arbitrarily, so the application range is limited. To overcome this disadvantage, Qiu et al. (2009) proposed an amperometric sensor for measuring glucose in which a vehicle, ferrocene carboxaldehyde, is adsorbed on the wall of a carbon nanotube. In addition, Sun et al. (Yang, W., Zhou, H., Sun, C., 2007. Macromol. Rapid Commun. 28, 265-270) propose an enzyme carbon electrode for the analysis of glucose in which cross-linking is effected. The ferrocene vehicle is immobilized on a chitosan substrate of the electrode.

Although conventional techniques suggest many methods, such as physical adsorption, cross-linking, encapsulation, etc., to improve the immobilization between an enzyme and an electrode or even a medium, it is still necessary to propose a high accuracy, high stability, and wideness. Measure the concentration range and the long-term reusability of the enzyme electrode.

On the other hand, the Danish scientist Ruzicka and Hansen (Zhi, Z.-L., 1998. Trends Anal. Chem. 17, 411-417) proposed a flow injection analysis (FIA) in 1976. Due to its advantages of rapid and continuous analysis, simple equipment, and low cost, the system has been effectively utilized in many fields such as environmental detection, process analysis, and clinical diagnosis. In operation, the flow injection analysis system can be on-line or off-line. The procedures for non-line operations are cumbersome, and the sample to be tested may also be lost during transmission. The way of online operation is expected to solve this shortcoming. In recent years, scientists have proposed some online operational FIA systems (Kumar, MA, Thakur, MS, Senthuran, A., Senthuran, V., Karanth, NG, Hatti-Kaul, R., Mattiasson, B., 2001. World J). .Microbio. Biotechnol. 17, 23-29; Nandakumar, MP, Lali, AM, Mattiasson, B., 1999. Bioseparation 8, 229-235), such as the use of enzyme electrodes to analyze the microbial hydrolysis of glucose biotransformation system of glucose (Gramsbergen, JB, Skj th-Rasmussen, J., Rasmussen, C., Lambertsen, KL, 2004. J. Neurosci. Methods 140, 93-101; Rhemrev-Boom, MM, Jonker, MA, Venema, K., Jobst, G., Tiessen , R., Korf, J., 2001. Analyst 126, 1073-1079; Yao, T., Yano, T., Nishino, H., 2004. Anal. Chimi. Acta 510, 53-59). However, in these existing applications, it is necessary to develop a more convenient sampling system, combined with an online flow injection analysis system, to make the analysis procedure easier.

The object of the present invention is to provide a method for producing an enzyme electrode, which is capable of producing a ferment with high accuracy, high stability, wide measurement concentration range, and long-term reusability. Prime electrode.

According to the above object, an embodiment of the present invention provides a method for manufacturing an enzyme electrode, comprising: providing a substrate having a carbon surface; forming a gold surface on the carbon surface to form an electrode; and covalently bonding an aldehyde An aldehyde group of the substrate is on the surface of the electrode; and a glucose oxidase is covalently bonded to the surface of the electrode.

According to the above object, an embodiment of the present invention provides a method for manufacturing an enzyme electrode, comprising: providing a substrate having a carbon surface; forming a gold surface on the carbon surface to form an electrode; and using cysteine ( L-cysteine) modifies the gold surface, the gold surface is covalently bonded to the thiol group in the cysteine, and forms a first electrode having a first modified surface; The medium is covalently bonded to the amine group of the cysteine to form a Schiff Base and forms a second electrode having a second modified surface; N, N' -N,N'-dicyclohexylcarbodiimide modifies the second modified surface, and N,N'-dicyclohexylcarbodiimide is dehydrated with the cysteic acid on the gold surface to form a total Valence bonding and forming a third electrode having a third modified surface; and the third modified surface is contacted with glucose oxidase to form a guanamine of the glucose oxidase and the carboxyl group of the cysteine group An amide bond is covalently bonded to form A fourth electrode having a fourth modified surface.

According to the above object, an embodiment of the present invention provides an enzyme electrode comprising: a substrate structure having a carbon surface; a gold surface formed on at least a portion of the carbon surface; and an amino acid comprising a An amino group, a carboxyl group, a thiol group, the amino acid is bonded to the gold surface through the thiol group; and a vehicle comprises an aldehyde group , the medium and the The amine group forms a Schiff Base; and a glucose oxidase is bonded to the amino acid through a peptide coupling agent having a diimide, wherein The glucose oxidase forms an amide bond with the peptide bond.

According to the above object, an embodiment of the present invention provides an enzyme electrode comprising: a substrate structure comprising a pencil core body, a carbon cover layer, a gold cover layer, and a modified structure, from the inside to the outside; The modified structure is chemically bonded to the gold coating layer, the modified structure comprising a cysteine group, a vehicle group comprising an aldehyde group, and a glucose oxidase group, the gold coating Covalently bonded to the cysteine group is gold-sulfur (Au-S), and the cysteine group and the glucose oxidase are covalently linked to an amide bond. The bond, the cysteine group and the vehicle group are covalently bonded to a carbon-nitrogen double bond.

10‧‧‧ pencil lead

11‧‧‧Carbon paste

12‧‧‧ Gold surface

12A‧‧‧First modified surface

12B‧‧‧Second modified surface

12C‧‧‧ Third modified surface

12D‧‧‧Four modified surface

13‧‧‧Electrode

13A‧‧‧First electrode

13B‧‧‧second electrode

13C‧‧‧ third electrode

13D‧‧‧fourth electrode

20‧‧‧Flow Injection Analysis System

21‧‧‧Bioreactor

22‧‧‧Electrochemical analysis subsystem

23‧‧‧Stop sampling system

30‧‧‧ Blood glucose test strips

31‧‧‧Capillary section

32‧‧‧Detection section

33‧‧‧Electrode contact section

34‧‧‧ sample hole

211‧‧‧pH electrode

212‧‧‧Temperature probe

213‧‧‧ Controller

214‧‧‧Sampling tube

221‧‧‧Three-electrode flow chamber

222‧‧‧Electrochemical Workstation

223‧‧‧peristal pump

231‧‧‧Sampling syringe

232‧‧‧ six-hole injection valve

233‧‧‧Peristal pump

234‧‧‧Standard syringe

235‧‧"Syringe filter

236‧‧‧glucose standard solution

237‧‧‧Deionized water

238A‧‧‧Three-hole valve

238B‧‧‧Three-hole valve

239A‧‧‧Three-hole valve

239B‧‧‧Three-hole valve

1 is a view showing a method of manufacturing an enzyme electrode according to a preferred embodiment of the present invention; FIG. 2A is a structural view of a flow injection analysis system according to an embodiment of the present invention; and FIG. 2B is a view showing a flow injection analysis system of FIG. 2A. Six-hole injection valve; Figure 2C shows a top view and a side view of a three-electrode flow chamber of the flow injection analysis system of Figure 2A; Figure 3A shows cyclic voltammetry analysis of three standard solutions of an enzyme electrode prepared in accordance with an embodiment of the present invention Figure FIG. 3B is a view showing a current-time curve when an enzyme electrode prepared according to an embodiment of the present invention is prepared by the above method, and a calibration curve is added; and FIG. 3C is a view showing the calibration of the standard electrode added to the enzyme electrode prepared by the embodiment of the present invention. Figure 4 shows the stability of the enzyme electrode prepared by the embodiment of the present invention after long-term use; Figure 5A shows the current signal generated by the enzyme electrode of the flow injection analysis system of the present invention at different flow rates; Figure 5B shows the present Inventive flow injection analysis system, the enzyme electrode continuously measures the current signal value generated 5 times at different flow rates; Figure 6 shows the glucose concentration of the hydrolyzate measured by HPLC and the flow injection analysis system of the present invention at the same sampling time. A linear regression analysis curve; Figure 7 shows a multiple use blood glucose test strip for a blood glucose meter in accordance with an embodiment of the present invention.

The embodiments of the present invention will be described in detail below with reference to the drawings. In addition to the detailed description, the invention may be widely practiced in other embodiments, and any alternatives, modifications, and equivalent variations of the described embodiments are included in the scope of the present invention, and the scope of the following patents is quasi. In the description of the specification, numerous specific details are set forth in the description of the invention. In addition, well-known program steps or elements are not described in detail to avoid unnecessarily limiting the invention.

1 is a view showing a method of manufacturing an enzyme electrode according to a preferred embodiment of the present invention, It consists of the following six steps. In step (I), a layer of carbon paste 11 is applied to the surface of a pencil lead 10 at a coating height of about 5 cm, and the process conditions are about 120 ° C for 10 min. The action of the carbon paste 11 prevents the activity of the enzyme from being disturbed by the impurities of the pencil lead 10. The higher the carbon purity, the higher the purity of the enzyme, the higher the enzyme activity is expected. In the step (II), the pencil lead 10 is placed in an aqueous solution of tetrachloroaurate, and a voltage of 0.2 V is applied to reduce the tetrachloroauric acid to gold particles deposited on the surface of the carbon paste 11 at a height of about 0.8 cm to form a The electrode 13 of the gold surface 12. The process conditions for this step are approximately 28 ° C for 2 hours. Next, the electrode 13 is washed with water at the same temperature to remove the unreduced gold compound on the surface of the electrode 13. In step (III), the electrode 13 is immersed in a solution of L-cysteine at a concentration of 20 mM at 25 ° C for 1 hour to make the gold particles and the thiol group in the cysteine (-SH, sulphydryl orthiol). Group) forms a covalently bonded first modified surface 12A and forms a first electrode 13A having a first modified surface 12A. Next, the first electrode 13A was washed with deionized water at 25 ° C to remove the cysteinolic acid physically adsorbed on the gold surface 12. In step (IV), the first electrode 13A having the first modified surface 12A is immersed in a solution of ferrocene carboxaldehyde (FcAld, dissolved in ethanol/hydrogen chloride in a volume ratio of 99.5/0.5) at a concentration of 0.1 mM at 75 ° C. 1 hour, the vehicle (FcAld) and the amino group of cysteine form a second modified surface 12B covalently bonded to a Schiff base, and formed with The second electrode 13B of the surface 12B is modified. Next, the second electrode 13B was washed with deionized water at 75 °C. In step (V), the second electrode 13B is immersed in a solution of N,N'-dicyclohexylcarbodiimide dissolved in methanol at a concentration of 5.0 mM for 1 hour to make N, N'-dicyclohexylcarbodiimide is dehydrated with a carboxyl group of cysteine to form a total The valence bond forms a third modified surface 12C and forms a third electrode 13C having a third modified surface. The third electrode 13C can then be washed with methanol and deionized water in sequence at 40 ° C for about 5 min to remove the physically adsorbed diimide. In step (VI), the third electrode 13C is immersed in a solution of 50 μM glucose oxidase (25 ° C) prepared in 0.1 M, pH 7.0 sodium phosphate buffer solution (NaPB) for 24 hours. The glucose oxidase is covalently bonded to the cysteine group of the third modified surface 12C to form a amide bond or peptide bond, and is fixed on the third modified surface 12C to form a fourth modified surface. 12D and a fourth electrode 13D having a fourth modified surface 12D. So far, the fourth electrode 13D is the desired enzyme electrode, and can be stored in a sodium phosphate buffer solution of 0.1 M, pH 7.0, and 4 ° C for use.

The above preferred embodiments can be modified as appropriate. For example, a part of the substrate has a carbon surface on some or all of its surface, which can replace the pencil core coated carbon paste. Step (IV) can be performed after step (V) or (VI). The shape of the substrate may be a rod shape, a sheet shape, or any other shape; the material may include any metal or non-metal or a combination thereof. The method of forming the gold surface 12 may include deposition, ink-injection, electroposition, screen-printing, or other methods. Other amino acids having an amino group, a carboxyl group, or a thiol group may be substituted for cysteine. Other peptide coupling agents having a diimine structure can be substituted for N,N'-dicyclohexylcarbodiimide. Other vehicles with an aldehyde group can replace ferrocene carboxaldehyde. In addition, the process temperature and the concentration of each solution can also be changed moderately.

The characteristics of the enzyme electrode prepared in the above preferred embodiment will be verified below, and the prepared enzyme electrode is applied to a one-line flow injection analysis system (FIA) for long-term, continuous The glucose concentration of a bioreactor is continuously analyzed.

2A is a block diagram showing a flow injection analysis system in accordance with an embodiment of the present invention. The flow injection analysis system 20 is coupled to the bioreactor 21 through an electrochemical analysis subsystem 22 and a stop-flow sampling subsystem 23 and analyzes the products. In the present embodiment, the bioreactor 21 hydrolyzes the discarded bamboo chopsticks with immobilized fiber hydrolyzing enzyme, and the product thereof contains glucose. In addition, the products were also analyzed by an online high performance liquid chromatography coupled with a refractive index detector (HPLC-RI) for comparison.

In this example, the glucose concentration is analyzed by a standard solution addition method to compensate for the decay of the enzyme electrode and to remove the matrix effect of the solution. During the hydrolysis of the bamboo chopsticks, the reaction of the first 16 hours, the hydrolyzate of the bioreactor 21 was sampled every 4 hours with a peristaltic pump 233, after which the sampling period was 8 hours. A sample of the hydrolyzed product of the immobilized hydrolyzed enzyme was filtered with a syringe filter 235. The procedures for sampling, loading, injecting, etc. of the hydrolyzed product sample are described as follows: (1) manually sampling 2 mL from the bioreactor 21 using the sampling syringe 231 of the stop flow sampling subsystem 23; (2) as shown in Fig. 2B As shown, the six-hole injection valve 232 of the stop flow sampling subsystem 23 is positioned at the "loading" position (as shown in Figure 2B), and a 1 mL sample is injected into the sample carrier tube of the six-hole injection valve 232. 50 μL; (3) Positioning the six-hole injection valve 232 in the "injection" position (as shown in Figure 2B), so that 50 μL of the sample located in the valve is injected into the electrochemical analysis subsystem 22 via the peristaltic pump 223; 4) Positioning the six-hole injection valve 232 in the "loading"position; (5) taking 1 mL of the glucose standard solution from the standard syringe 234; (6) passing the three-hole valve 238A and the three-hole valve 238B from the standard syringe 234 Injecting 0.1 mL of the glucose standard solution into the sample injector 231; (7) from the sample injector 231, through the three-hole valve 238B, injecting approximately 1.1 mL of the spiked sample to which the 0.1 mL standard solution has been added to the six-hole injection valve 232 (5) Positioning the six-hole injection valve 232 in the "injection" Positioning, 50 μL of the sample to which the standard solution was added in the valve was injected into the electrochemical analysis subsystem 22 via the peristaltic pump 223 for analysis; (9) When the analysis was completed, the six-hole injection valve 232 was positioned at the "loading" position. (10) The glucose standard solution 236 is replaced with deionized water 237 to clean the stop flow sampling subsystem 23 two injectors 231/234 and associated tubing for the next analysis. Note that the concentration of the above glucose standard solution depends on the glucose concentration of the hydrolyzate, and the standard concentration to be added is usually such that the test current intensity of the sample to which the standard is added is 1.5 times larger than that of the original sample. Accordingly, the content of glucose may be added to the hydrolyzate obtained by the standard equation (Harris, DC, 2007.Quantitative chemical analysis , 7 th ed., WHFreeman and Company, New York, p.87-90).

By way of example and not limitation, the system shown in FIG. 2A uses a CHI611B electrochemical workstation 222 manufactured by CH Instruments, Texas, USA, in conjunction with a specially designed three-electrode flow cell 221, and uses a peristaltic pump 223 to deliver samples. Perform an online glucose analysis. The three-electrode flow chamber 221 has a platinum wire as a counter electrode (CE), a silver/silver chloride (Ag/AgCl) as a reference electrode (RE), and an enzyme electrode prepared in the embodiment of the present invention. As a working electrode.

In addition, the stop flow sampling subsystem 23 utilizes a sample injection valve of the Hercules Bio-Rad Model MV-6 as the six-hole injection valve 232 of the above system. The sampling of the hydrolysate also involves the use of a peristaltic pump 233 and two three-hole valves 239A/B. The bioreactor 21 uses a 3L biological reaction of the Taiwanese Biotop process & Equipment company model BTF-A3L. The pH electrode 211 and the temperature probe 212 are respectively controlled by the controller 213 to control the pH and temperature of the reaction liquid in the bioreactor 21. Additionally, a sampling tube 214 is used to derive the product of the bioreactor 21.

Note that in other embodiments of the present invention, the above-described in-line flow injection analysis system 20 can be used to analyze other non-glucose analytes by simply replacing the enzyme electrode in the three-electrode flow chamber 22 with an enzyme electrode that can analyze the analyte. In addition, the three-electrode flow chamber 22 can also be designed as a two-electrode flow chamber.

Quantitative analysis of glucose by HPLC

During the hydrolysis of waste bamboo chopsticks to glucose, the hydrolyzed product was also analyzed on-line by HPLC-RI. The sampling period is the same as described for the previous enzyme electrode flow injection analysis.

Cyclic voltammetry

The oxidation potential of the enzyme electrode prepared in the examples of the present invention can be obtained by cyclic voltammetric. For analysis, the potential sweep ranged from 0.0 to 0.7 V and the scan speed was 50 mVs ^-1 . The samples were analyzed as three glucose standard solutions at concentrations of 0 mM, 4 mM, and 10 mM, respectively, using 0.1 M, pH 7.0 sodium phosphate buffer solution (NaPB).

Figure 3A shows a cyclic voltammetric analysis of the above three standard solutions, wherein curves (a), (b), and (c) are cyclic voltammetry curves of 0 mM, 4 mM, and 10 mM, respectively, and arrows indicate the direction of scanning.

It can be observed from the graph that curve (b)(c) has a significant anode current at 0.45 V and curve (a) has no anode current. This indicates that the oxidation potential of the vehicle FcAld is 0.45 V, and the vehicle and glucose oxidase are successfully immobilized on the modified surface of the carbon electrode.

Standard addition correction calibration line method

The present invention quantifies glucose by standard addition calibration method (Pijanowska, DG, Sprenkels, AJ, Olthuis, W., Bergveld, P., 2003. Sens. Actuators B 91, 98-102). . The calibration curve is added to the standard to obtain the sensitivity of the enzyme electrode for detecting glucose, the limit of detection (LOD), and the linear concentration detection range. A calibration calibration curve was added to the standard at 35 °C. The sodium phosphate buffer solution (NaPB) was deoxygenated using nitrogen for 10 minutes before each measurement. First, glucose standard solutions at concentrations of 50 mM and 250 mM were prepared at 0.1 M, pH 7.0 NaPB, and placed overnight to naturally mutate the two different optically active a- and b-forms of glucose. To add a calibration calibration line to the preparation standard, 8 mL of 0.1 M, pH 7.0 NaPB solution was placed in the electrochemical cell as the background solution. After 3 minutes, a volume of about 2 to 285 μL of the glucose standard solution was added every 1.5 minutes until the concentration of the solution was 55.6 mM. The current value was read for the last 3 seconds after each standard addition.

Figure 3B shows the current-time curve for the preparation of a standard additive plus calibration calibration line in an enzyme electrode prepared in accordance with an embodiment of the present invention. The amount of change in current when glucose is added each time indicates the amount of glucose oxidation corresponding to the added glucose. At 600 to 1800 seconds, the magnitude of the current change in the graph is small because the amount of glucose added at this stage is small, and the current change can still be clearly seen after the amplification is shown.

Fig. 3C is a view showing the standard addition correction calibration curve of the enzyme electrode prepared in the embodiment of the present invention, which was produced according to the above method and obtained a linear type calibration curve by linear least squares homing. The results showed a linear concentration range of 0 to 55.5 mM and a linear correlation coefficient (r ² ) of 0.9951. In the literature, the optimal linear concentration range for the calibration of the glucose standard is 0.05 mM to 26 mM, and the linear correlation coefficient (r ² ) is 0.9948 (Tan, X.-C., Tian, Y.-X. , Cai, P.-X., Zou, X.-Y., 2005. Anal. Bioanal. Chem. 381,500-507).

The slope of the calibration curve added to the standard is the detection sensitivity, which is 75.4nA mM ^-1 . This sensitivity is quite good for glucose analysis. In addition, the detection limit (LOD) is 15.0 μM, which is better than the glucose detection electrodes proposed in other literatures (Asav, E., Akyilmaz, E., 2010. Biosens. Bioelectron. 25, 1014-1018; Barsan, MM Klin Ar, J., Bati , M., Brett, CMA, 2007. Talanta 71, 1893-1900; Ghica, ME, Brett, CMA, 2006 Electroanalysis 18, 748-756; Tsai, M.-C., Tsai, Y.-C., 2009. Sens. Actuators, B 141, 592-598).

Enzyme electrode stability

Fig. 4 is a view showing the stability of the enzyme electrode prepared in the embodiment of the present invention after long-term use, and the linear correlation coefficient, sensitivity, and detection limit value of the calibration calibration line added to the glucose standard. After 38 days of testing, the sensitivity of the enzyme electrode was reduced to approximately 32.4 nA mM ^-1 , which was approximately 43% of the initial sensitivity; however, the enzyme electrode still had a good linear concentration range (0 to 44.4 mM) with a linear correlation coefficient of 0.9922. In addition, the detection limit (LOD) was increased from 15 μM to 90 μM. Compared with the glucose electrode proposed in the literature (Asav and Akyilmaz, 2010; Ghica and Brett, 2006; Sun et al., 2008; Yang et al., 2006), the sensitivity is after 10 days or 1 month of use. They were reduced to 0.15 μA mM ^-1 (50% reduction), 0.20 μA mM ^-1 (69% reduction), 4.86 μA mM ^-1 (85% reduction), and 6.7 μA mM ^-1 (90% reduction). This indicates that the attenuation of the sensitivity of the enzyme electrode is inevitable; however, as long as the enzyme electrode still has a broad linear concentration range and a good linear correlation coefficient, the analysis is still accurate. Therefore, in practical applications, the linear concentration range and detection limit of the glucose detecting electrode are the two most important specifications.

Optimal flow rate for flow injection analysis systems

In the flow injection analysis system, the enzyme electrode prepared in the foregoing examples was used as the working electrode of the three-electrode flow chamber. In addition, a 0.1 M, pH 7.0 NaPB solution was used as the carrier solution of the flow injection analysis system and the electrolyte of the three-electrode flow chamber. Since the current signal generated by the enzyme electrode is affected by the flow rate of the carrier solution, the flow rate of the carrier solution is set to 1.0, 1.5, 2.0, 2.5, 3.0, 4.5 mL min ^-1 , respectively, and the glucose standard solution having a concentration of 0.2 mM is tested. Trying to find the best flow value.

Figure 5A shows the current signal generated by the enzyme electrode at different flow rates of the flow injection analysis system of the present invention. Figure 5B shows the current signal value of the enzyme electrode continuously measured five times at different flow rates of the flow injection analysis system of the present invention, wherein the curves (a)(b)(c) are flow rates of 2.0 mL min ^-1 and 1.5 mL min, respectively. ^-1 , 5 mL measurement curves of 1.0 mL min ^-1 , and measured at an applied voltage of 0.45 V; a carrier solution of 0.1 M, a pH 7.0 NaPB solution; an injection volume of 50 μL; and a glucose concentration of 0.2 mM.

It can be seen from Fig. 5A that as the flow rate increases, the signal is weakened. When the flow rate exceeds 2.5 mL min ^-1 , the signal is too low, which is not conducive to the analysis procedure. Although Figure 5A shows a maximum signal value of 136.3 nC at a flow rate of 1.0 mL min ^-1 , Figure 5B shows that when the flow rate is 2.0 mL min ^-1 (curve a), the most stable signal is produced; therefore, 2.0 mL min is used. ^-1 as the optimum flow value for the flow injection analysis system.

Interference from flow injection analysis systems

When measuring glucose levels, it is often interfered with by other non-glucose substances, especially carbohydrates with a similar structure to glucose. For example, it is known from HPLC analysis that the bamboo chop hydrolysate contains at least three types of carbohydrates: glucose, xylose, cellobiose. However, the inventors' previous research (Cheng, C., Chen, C.-S., Hsieh, P.-H., 2010. J. Chromatogr. A 1217, 2104-2110; Cheng, C., Tsai, H .-R., Chang, K.-C., 2006. J. Chromatogr. A 1119, 188-196) indicates that the hydrolysate of plant fibers also contains some traces of sugars such as arabinose and mannose. ), galactose.

In order to analyze the effects of interfering substances, a 1.66 mM glucose standard solution and a 1.66 mM glucose solution having a carbohydrate interfering substance concentration of 3.33 mM (Barsan et al., 2007) were separately prepared and tested by the above flow injection analysis system. The selectivity coefficient of the enzyme electrode to the interfering substance is calculated.

Table 1 lists the selection coefficient (k) and the degree of interference of various interfering substances when the enzyme electrode prepared by the present invention is applied to a flow injection analysis system for measuring glucose. The results showed that for fiber disaccharide, xylose, and arabinose, the selection coefficient k was 0 and the degree of interference was 0%. In addition, mannose and galactose have a degree of interference of 7% and 5%, respectively.

^a measurement times (n) = 3

^b signal ratio = (total signal) _{glucose + interference} / (total signal) _glucose

^c k=(signal) _interferer / (signal) _glucose

Comparison of enzyme electrode and HPLC quantitative analysis results

In the example of the present invention, a flow injection analysis system using an enzyme electrode is connected with a high performance liquid chromatography apparatus to measure a refractive index detector (HPLC-RI), and the glucose concentration of the bamboo chopstick hydrolyzate is separately analyzed. Table 2 lists the comparison results of the two analytical methods. The results showed that, except for the fourth hour of the hydrolysis reaction, the glucose analysis results of the respective reaction times were greater than those measured by the high performance liquid chromatography with the glucose concentration measured by the flow injection analysis system. From this result, it seems that there is a systematic error between the two systems; however, if we consider mannose, galactose, the influence of unknown interfering substances contained in other hydrolysates on the enzyme electrode, and the matrix effect ( Matrix effect) For the effect of the high performance liquid chromatography refractive index detector using the external standard calibration quantification method, the results listed in Table 2 are reasonable test results.

^a measurement times (n) = 2

To verify whether there are systematic errors in the two analytical methods, a statistical linear homing method was used. Figure 6 shows the linear analysis of the glucose concentration of the hydrolysate by the two analytical methods at the same sampling time. A straight line is obtained with a slope of 1.0451, a Y coordinate intercept of 0.0110, and a linear enthalpy coefficient (r ² ). It is 0.9920. At 95% confidence level, the slope of the linear enthalpy line ranges from 0.9744 to 1.1158; this range covers an ideal slope value of 1.0. Similarly, at 95% confidence level, the Y coordinate intercept ranges from -0.0019 to 0.0239, and also covers the ideal intercept value of 0, which is the origin. Therefore, the results of the two analytical methods are comparable and there is no systematic error.

The above embodiment of the present invention prepares a novel enzyme electrode using chemical bonds The junction, the redox medium and glucose oxidase are attached to the modified surface of the electrode, and the enzyme electrode thus completed has the widest detection linear concentration range and extremely low detection limit value when used for detecting the glucose concentration. With long-term stability. The prepared enzyme electrode can be applied to a flow injection analysis system as a working electrode of the three-electrode flow chamber of the system, and the analysis system further comprises a stop flow sampling subsystem to make the analysis procedure easier. The flow injection analysis system can be connected to a bioreactor for on-line analysis of bioreactor bioconverter products. When used to measure glucose concentration, the analysis results of the flow injection analysis system can be compared with the results of the high performance liquid chromatography, and the relative standard deviation (RSD) of the analytical accuracy is less than 3.7%.

In addition, the enzyme electrode prepared in the embodiment of the present invention can be used to construct a blood glucose test piece which is connected to a blood glucose meter to read a signal generated by the blood glucose test piece, and thereby determine the glucose concentration of the blood sample on the test piece. Figure 7 shows a blood glucose test strip 30 for use in a blood glucose meter in accordance with an embodiment of the present invention. The blood glucose test strip 30 includes a capillary section 31, a detection section 32, and an electrode contact section 33. The detecting section 32 includes at least a working electrode (WE), a counter electrode (CE), and a reference electrode (RE), wherein the enzyme electrode of the present invention functions as a working electrode (WE). The capillary section 31 contains at least a sample well 34 which is inhaled by capillary force and delivered to the detection section 32 to bring the blood sample into contact with the electrode of the detection section 32. The working electrode (WE) has a part of its surface bonded with a redox medium and glucose oxidase. For example, the surface of the entire working electrode may be deposited with a gold surface, but only in the middle or end portion of the gold surface, a vehicle and glucose oxidase are bonded.

The detecting section 32 is electrically connected to the electrode contact section 33 through a wire (not shown) distributed under the capillary section 31. During testing, the electrode contact section 33 is inserted into the blood glucose meter, and the signal generated by the detection section 32 is read by the blood glucose meter via the electrode contact section 33, thereby determining the blood sample. Blood sugar concentration.

In another embodiment of the invention, the electrode contact segments 33 described above are omitted. Therefore, the detection section 32 is inserted into the blood glucose meter, which directly reads the signals of the electrodes of the detection section 32. Further, the sample hole 34 may be disposed at the end or side of the capillary section 31 in addition to being disposed at the intermediate portion.

Traditional blood glucose test strips are discarded after use. Since the long-term stability of the working electrode, i.e., the enzyme electrode, is excellent, the blood glucose test strip 30 of the present invention can be designed as a reusable blood glucose test piece. To this end, the capillary section 31 can be designed to be replaceable; after each blood glucose test is completed, the detection section 32 is immersed in one or more solutions or solvents to remove the blood sample and reaction remaining on the electrode. The product is then replaced with a new capillary section 31 to replace the existing capillary section 31 so that the next measurement can be taken.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the invention should be included in the following Within the scope of the patent application.

10‧‧‧ pencil lead

11‧‧‧Carbon paste

12‧‧‧ Gold surface

12A‧‧‧First modified surface

12B‧‧‧Second modified surface

12C‧‧‧ Third modified surface

12D‧‧‧Four modified surface

13‧‧‧Electrode

13A‧‧‧First electrode

13B‧‧‧Second electrode

13C‧‧‧ third electrode

13D‧‧‧fourth electrode

Claims (20)

A method for producing an enzyme electrode, comprising: providing a substrate having a carbon surface; forming a gold surface on the surface of the carbon, thereby forming an electrode; and having an amino group, a carboxyl group a thiol group branched amino acid to modify the electrode, wherein the amino acid is cysteine, and the gold surface is attached through the thiol group; covalently bonded a vehicle comprising an aldehyde group on the surface of the electrode, wherein the medium forms a Schiff Base with the amine group; and a peptide bond having a diimide The electrode is further modified by a peptide bond coupling agent, wherein the peptide bond agent and the carboxyl group of the amino acid pass through a nucleophilic reaction to form an O-acylisourea intermediate structure. And covalently bonding a glucose oxidase to the surface of the electrode, wherein an amino group of the glucose oxidase binds to the carboxyl group of the amino acid, and replaces the O-B through a nucleophilic substitution reaction The intermediate structure of thiol isourea forms a guanamine bond (ami De bond). A method for producing an enzyme electrode, comprising: providing a substrate having a carbon surface; forming a gold surface on the carbon surface to form an electrode; and modifying the gold surface with L-cysteine, The gold surface is covalently bonded to a thiol group in the cysteine and forms a first electrode having a first modified surface; a medium comprising an aldehyde group and cysteine Amine group Schiff base is covalently bonded and forms a second electrode having a second modified surface; N,N'-dicyclohexylcarbodiimide (N,N' -dicyclohexylcarbodiimide) modifying the second modified surface, the N,N'-dicyclohexylcarbodiimide is dehydrated by the dehydration reaction of the cysteine on the gold surface to form a third modified surface a third electrode; and the third modified surface is contacted with the glucose oxidase to cause the glucose oxidase to form a covalent bond with the carboxyl group of the cysteine group to form an amide bond A fourth electrode having a fourth modified surface. The manufacturing method of claim 2, wherein the material of the substrate is a non-metal. The manufacturing method of claim 3, wherein the material of the substrate is a pencil lead. The manufacturing method of claim 4, wherein the step of forming the carbon surface comprises coating a carbon paste on a surface of the pencil lead to form the carbon surface. The manufacturing method of claim 4, wherein the medium comprises ferrocene carboxaldehyde. An enzyme electrode comprising: a substrate structure having a carbon surface; a gold surface formed on at least a portion of the carbon surface; an amino acid comprising an amino group, a carboxyl group (carboxyl Group), a thiol group, the amino acid being cysteine, bonded to the gold surface through the thiol group; a vehicle comprising an aldehyde group, the medium and The amine group forms a Schiff Base; and a glucose oxidase is bonded to the amino acid through a peptide bond coupling agent having a diimide. Wherein the glucose oxidase forms an amide bond with the peptide bond linker. For example, in the enzyme electrode of claim 7, wherein the enzyme electrode is used to form a blood glucose test piece, which is connected to a blood glucose machine to read the signal generated by the blood glucose test piece, and thereby determine the blood on the blood glucose test piece. The glucose concentration of the sample; the blood glucose test piece comprises a detection section comprising a working electrode, a counter electrode, and a reference electrode, and the enzyme electrode serves as the working electrode. The enzyme electrode of claim 8, wherein the blood glucose test piece further comprises a capillary segment, the capillary segment comprising at least one sample hole, wherein the blood sample is sucked by capillary attraction and transmitted to the detecting segment, so that The blood sample is in contact with each electrode of the detection section. The enzyme electrode of claim 9, wherein the capillary segment is replaceable, and after each blood glucose test is completed, the existing capillary segment is replaced with an unused capillary segment. The enzyme electrode of claim 9, wherein the detecting section is electrically connected to an electrode contact section through a plurality of wires distributed under the capillary section, and the electrode contact section is inserted into the blood glucose meter, and the detecting section is inserted into the blood glucose meter. The generated signal is passed from the electrode contact segment by the blood glucose The machine reads, thereby obtaining the blood glucose concentration in the blood sample. For example, in the enzyme electrode of claim 11, wherein the detecting section and the electrode contacting section are reusable, and after each blood glucose test is completed, the detecting section is immersed in one or more solutions or solvents, The blood sample remaining on the electrode is removed with one or more reaction products. An enzyme electrode according to claim 7, wherein the amino acid is L-cysteine. An enzyme electrode according to claim 7, wherein the peptide bond connecting agent is N,N'-dicyclohexylcarbodiimide. An enzyme electrode according to claim 7, wherein the medium comprises ferrocene carboxaldehyde. An enzyme electrode comprising: a substrate structure comprising a pencil core body, a carbon coating layer, a gold coating layer from the inside to the outside; and a modifying structure chemically bonded to the gold On the cover layer, the modified structure comprises a cysteine group, a vehicle group comprising an aldehyde group, and a glucose oxidase group, the gold cap layer and the cysteine group The interstitial system is a gold-sulfur (Au-S) covalent bond, and the cysteine group and the glucose oxidase are covalently bonded to an amide bond, and the cysteine group is covalently bonded. The group is covalently bonded to the vehicle group as a carbon-nitrogen double bond. For example, in the enzyme electrode of claim 16, wherein the enzyme electrode is used to form a blood glucose test piece, which is connected to a blood glucose machine to read the signal generated by the blood glucose test piece, and thereby determine the blood on the blood glucose test piece. The glucose concentration of the sample; the blood glucose test piece contains A detecting segment includes a working electrode, a counter electrode, and a reference electrode, and the enzyme electrode serves as the working electrode. The enzyme electrode of claim 17, wherein the blood glucose test piece further comprises a capillary segment and an electrode contact segment; the capillary segment includes at least one sample hole, and the capillary sample is used to inhale and transmit the blood sample to the detect Measuring, the blood sample is in contact with each electrode of the detecting section; the detecting section is electrically connected to an electrode contact section through a plurality of wires distributed under the capillary section, and the electrode contacting section is inserted into the blood glucose meter, The signal generated by the detecting segment is read by the blood glucose meter via the electrode contact segment, thereby analyzing the blood glucose concentration in the blood sample. An enzyme electrode according to claim 18, wherein the capillary segment is replaceable, and after each blood glucose test is completed, the existing capillary segment is replaced with an unused capillary segment. An enzyme electrode according to claim 16, wherein the medium comprises ferrocene carboxaldehyde.