TWI644101B - A test strip for electrochemical biosensor with high stability and manufacturing method thereof - Google Patents

Abstract

The invention provides a high stability electrochemical biosensor test piece which has a ferricyanide stabilizer and a light shielding agent component, and can effectively overcome the disadvantage that the test piece is easily deteriorated. Therefore, the electrochemical biosensor test piece of the present invention has the advantages of stable manufacturing process, long shelf life, high temperature resistance and light resistance.

Description

Electrochemical biosensor test piece with high stability and manufacturing method thereof

The invention provides an electrochemical biosensor test piece with high stability, in particular to provide an electrochemical biosensor test piece with high temperature resistance and light.

The application of enzyme electrodes in electrochemical biosensors is the earliest and most widely used. At present, commercial electrochemical biosensors mainly use electrochemical biosensors that use electron transfer media to assist electron transfer. The reaction process is shown in Figure 1 (taking glucose detection as an example): the first step The glucose in the blood sample reacts with glucose oxidase to form gluconic acid and water, and the water reacts with potassium ferricyanide to become potassium ferrocyanide; in the second step, an external voltage is applied to the reaction system to oxidize potassium ferrocyanide. It is potassium ferricyanide and emits electrons. By detecting the amount of current released, and calculating the concentration of glucose.

The stability and lifetime factors of electrochemical biosensors that use electron transfer media to assist electron transport are most important for the stability of coated enzymes and electron transport media. Because enzymes are highly susceptible to temperature and other factors, and electron transfer media are susceptible to temperature, light and other factors affecting their chemical properties. Potassium ferricyanide is a very common electron transfer medium in various biosensors using redoxase, and its low cost is suitable for making disposable disposable test pieces; and potassium ferricyanide The solubility in water is high and there is sufficient relative reaction on the auxiliary electrode to balance the reaction on the working electrode. The relative reaction provides sufficient current and chemical potential energy so that the reference electrode is not required for the detection, and the auxiliary electrode larger than the working electrode is not required, indicating that the electrochemical biosensor using potassium ferricyanide as the electron transport medium can be simplified. The size of the two electrodes.

However, an electrochemical biosensor using potassium ferricyanide as an electron transporting medium has the following disadvantages, since potassium ferricyanide is converted into potassium ferrocyanide due to its vulnerability to light and heat, resulting in deterioration of the test piece. In particular, when potassium ferricyanide is mixed with an enzyme to form a reaction reagent, even if it is stored in a dry environment, the reaction with the enzyme or the auto-oxidation-reduction reaction of the enzyme proceeds. For example, in a test strip for detecting blood sugar, even if no glucose is involved, the potassium ferricyanide and enzyme mixed reaction reagent will still react with each other during storage and storage, so that the detected background value is increased and pseudo-hyperglycemia is high. value. Such misdiagnosis results in erroneous medical behaviors, such as blood glucose testing in diabetics, such as misdiagnosis of hyperglycemia and hypoglycemia treatment, which can lead to severe hypoglycemia coma and even death.

Furthermore, commercial test strips often require a shelf life of one year or longer, during which time they often encounter extremely cold, extremely hot or humid environments; and electronic transfer media for electrochemical biosensors. Most are sensitive to light, which results in inaccuracies in measurement. Therefore, an electrochemical biosensor test piece that is resistant to environmental changes and has a relatively long shelf life has its necessity.

In order to solve such a problem, the present invention relates to an electrochemical biosensor test piece incorporating a ferricyanide stabilizer and a light shielding agent to enhance electrochemical biosensing using oxidoreductase and using ferricyanide as an electron transport medium. The stability of the light and heat to provide process stability, extended shelf life, and high temperature environment.

An object of the present invention is to provide an electrochemical biosensor test piece for detecting a sample to be tested in a sample, comprising: a substrate; a circuit layer disposed on the substrate, wherein the circuit layer comprises at least a first electrode And a second electrode; a reagent layer disposed on the circuit layer, wherein the first electrode and the second electrode are formed to overlap with the reagent layer to form an electrode reaction zone; an adhesive layer is disposed on the electrode Above the reagent layer, partially exposing the electrode reaction zone underneath to facilitate detection of a test object; and a cover layer disposed on the adhesive layer and partially exposing the underlying reagent layer and the electrode reaction Wherein the reagent layer comprises a reagent layer composition, and the reagent layer composition comprises: a ferricyanide, a oxidoreductase, a ferricyanide stabilizer, and a light shielding agent; and the oxidoreductase The object to be tested is specific.

In one embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, cellulose. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.

In another embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.

In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), aluminum oxide (Al ₂ O ₃ ), a group consisting of cerium oxide (SiO ₂ ), kaolin, calcined kaolin, clay, and china clay.

In one embodiment of the present invention, wherein the analyte is glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and when the analyte is uric acid, the oxidoreductase is uricase; When the test substance is cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test substance is triglyceride, the oxidoreductase esterase, glycerol kinase or phosphoglycerol oxidase; When the test substance is creatinine, the oxidoreductase is the creatininase, creatase or sarcosine oxidase.

In an embodiment of the invention, the reagent layer composition further comprises a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and a Buffer.

In one embodiment of the invention, the buffer is a 0.1 M phosphate buffer; the nonionic surfactant is a ketol (Triron X-100).

In one embodiment of the invention, the enzyme stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose ), glycine, alanine, serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose A group consisting of xylose), fructose, and glutamic acid.

In an embodiment of the invention, the polymer enzyme structure stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly A group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate.

In an embodiment of the invention, the cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC).

In an embodiment of the invention, the reagent layer is disposed on the circuit layer by coating, and the coating method comprises an air brush, a doctor blade or a screen printing. ).

Another object of the present invention is to provide a method for manufacturing an electrochemical biosensor test strip, comprising: providing a substrate; and disposing a circuit layer on the substrate, wherein the circuit layer comprises at least a first electrode and a second An electrode is coated on the circuit layer, wherein the first electrode and the second electrode are alternately formed to overlap with the reagent layer to form an electrode reaction zone, and the reagent layer comprises a ferricyanide and an oxidation a reducing enzyme, a ferricyanide stabilizer and a light shielding agent; coating an adhesive layer on the reagent layer, partially exposing the underlying electrode reaction zone to facilitate detection of a test object; and coating a coating layer On the adhesive layer, the underlying reagent layer and the electrode reaction zone are partially exposed.

In an embodiment of the invention, wherein the coating comprises an air brush, a doctor blade or a screen printing.

In an embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, and fiber. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and combinations thereof.

In another embodiment of the present invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), and aluminum oxide (Al ₂ O ₃ ). a group consisting of cerium oxide (SiO ₂ ), kaolin, calcined kaolin, clay, and china clay.

Still another object of the present invention is to provide a reagent layer for detecting a sample to be tested in a sample, comprising: a ferricyanide; a oxidoreductase; a ferricyanide stabilizer; and a light shielding agent. In an embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, and fiber. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.

In an embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.

In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), aluminum oxide (Al ₂ O ₃ ), A group consisting of cerium oxide (SiO ₂ ), kaolin, calcined kaolin, clay, and china clay.

In one embodiment of the present invention, wherein the analyte is glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and when the analyte is uric acid, the oxidoreductase is uricase; When the test substance is cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test substance is triglyceride, the oxidoreductase esterase, glycerol kinase or phosphoglycerol oxidase; When the test substance is creatinine, the oxidoreductase is the creatininase, creatase or sarcosine oxidase.

In an embodiment of the invention, the reagent layer composition further comprises: a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and A buffer.

In one embodiment of the invention, the enzyme stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose ), glycine, alanine, serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose A group consisting of xylose), fructose, and glutamic acid.

In an embodiment of the invention, the polymer enzyme structure stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly A group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate.

In an embodiment of the invention, the cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC).

In one embodiment of the invention, the buffer is a 0.1 M phosphate buffer; the nonionic surfactant is a ketol (Triron X-100).

In an embodiment of the invention, the reagent layer is disposed on the circuit layer by coating, and the coating method comprises an air brush, a doctor blade or a screen printing. ). Accordingly, the present invention provides an electrochemical biosensor test piece incorporating a ferricyanide stabilizer and a light shielding agent to improve the optical and thermal stability of an electron transport medium such as ferricyanide. Therefore, the electrochemical biosensor test piece of the present invention is suitable for a commercial one year or longer shelf life, resistant to extreme heat or humidity, and resistant to light damage.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

The invention provides a high stability electrochemical biosensor test piece with a ferricyanide stabilizer and a light shielding agent component, which can effectively overcome the conversion of ferricyanide to ferrocyanide by using iron cyanide as an electron transport medium. The compound, which causes the test piece to deteriorate, and can maintain the activity of the enzyme for a long time. The present invention demonstrates that the electrochemical biosensor test piece has good high temperature stability, light stability, and measurement stability using the following examples. Embodiment 1 Method for manufacturing electrochemical biosensor test piece with high stability of the present invention

As shown in FIG. 2, the present invention provides a method for manufacturing a test piece for an electrochemical biosensor having light and thermal stability, the method comprising: providing a substrate 110; and disposing a circuit layer 120 on the substrate 110, wherein the circuit layer 120 includes at least a first electrode 121 and a second electrode 122; a reagent layer 130 is coated on the circuit layer 120, wherein the first electrode 121 and the second electrode 122 are formed. An electrode reaction zone 131 is formed by overlapping with the reagent layer 130; an adhesive layer 140 is coated on the reagent layer 130, and the bottom electrode reaction zone 131 is partially exposed to facilitate detection of a sample to be tested; The cover layer 150 is on the adhesive layer 140, and partially exposes the underlying reagent layer 130 and the electrode reaction zone 131, wherein the coating comprises an air brush, a doctor blade or a screen printing the way. Thereafter, it was dried at 40 ° C for 30 minutes, and then placed in a dry tank in the dark to complete the test piece.

The reagent layer 130 of the present invention comprises: a ferricyanide, a oxidoreductase, a ferricyanide stabilizer, and a light shielding agent.

In one embodiment of the invention, the ferricyanide stabilizer is selected from the group consisting of polyvinyl pyrrolidone, polyacrylamides, gelatin, dextrin, cellulose. A group consisting of alkali metal salts of cellulose, hydrolyzed polyvinyl alcohol, and any combination thereof.

In an embodiment of the invention, the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), aluminum oxide (Al ₂ O ₃ ), a group consisting of cerium oxide (SiO ₂ ), kaolin, calcined kaolin, clay and china clay, wherein barium sulfate has a refractive index of 1.64, which has good refractive power, can play an optimal light diffusing agent, and has special The erect, stylistic shape, can enhance the reflection of light, to achieve the role of light transmission opacity, is preferred.

In one embodiment of the invention, the ferricyanide is potassium ferricyanide or sodium ferricyanide.

In an embodiment of the invention, wherein the test piece is to measure glucose, the oxidoreductase is glucose oxidase or glucose dehydrogenase; and the test piece is uric acid, the oxidoreductase is uric acid Enzyme; when the test piece is to measure cholesterol, the oxidoreductase is cholesterol esterase or cholesterol oxidase; when the test piece is to measure triglyceride, the oxidoreductase esterase, glycerol kinase or glycerol phosphate An oxidase; and when the test piece is to measure creatinine, the oxidoreductase is the creatinine, creatase or sarcosine oxidase.

The reagent layer of the present invention further comprises: a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, a polymerase structural stabilizer, a cellulose thickener, and a buffer, wherein the enzyme The stabilizer is selected from the group consisting of trehalse, sodium gluconate, mannose, cyclodextrin, sucrose, glycine, alanine. ), serine, arabinose, sorbitan, 2-deoxy-D-glucose, xylose, fructose, and glutamic acid ( a group consisting of glutamic acid); and the polymerase structural stabilizer is selected from the group consisting of polyvinyl alcohol (PVA), polyvinyl pyrrolidone, polyethylene oxide, and poly a group consisting of copolymers of polyvinyl pyrrolidone and polyvinyl acetate and copolymers of polyvinyl alcohol and polyvinyl acetate; The cellulose thickener is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC); and the buffer is 0.1 M phosphate buffer; the nonionic surfactant is Ketone (Triron X-100).

In an embodiment of the invention, the substrate is an electrically insulating substrate, and the material thereof may include, but not limited to, polyvinyl chloride, polystyrene, polyester, polycarbonate, polyether, polyethylene, polypropylene, polyethylene. Phthalate or polyethylene terephthalate.

In an embodiment of the invention, the first electrode and the second electrode of the circuit layer are respectively a working electrode and an auxiliary electrode, and the relative positions thereof are not limited. In still another embodiment of the present invention, the first electrode and the second electrode structure may print a desired pattern via screen printing, and the working electrode and the auxiliary electrode may include, but are not limited to, carbon, a single metal, an alloy, and/or Other conductive materials. Further, the present invention is not limited to the relative position, shape and size of the working electrode and the auxiliary electrode.

In an embodiment of the invention, the adhesive layer may be a double-sided tape having an insulating interlayer.

In an embodiment of the invention, the cover layer is a transparent electrical insulating film having hydrophilic properties, which may include, but is not limited to, polyvinyl chloride, polystyrene, polyester, polycarbonate, polyether, polyethylene, polypropylene. , polyethylene terephthalate, or polyethylene terephthalate. Example 2 Heat resistance stability of a test piece for an electrochemical biosensor of the present invention

In one embodiment of the present invention, a test piece for an electrochemical biosensor uses potassium ferricyanide as an electron transport medium, but potassium ferricyanide is converted to potassium ferrocyanide by being susceptible to heat. The disadvantage of causing the test piece to deteriorate. Especially when potassium ferricyanide is mixed with an enzyme to form a reagent, even if it is stored in a dry environment, this transformation will still proceed. Therefore, the present invention tests for the heat resistance of the test piece.

This example is exemplified by glucose oxidase. First, glucose oxidase was dissolved in 0.1 M phosphate buffer, and a mixture of all the ferricyanide stabilizers in the above Example 1 was added, which contained: polyvinyl pyrrolidone, polyacrylamides. Gelatin, dextrin, alkali metal salts of cellulose, and hydrolyzed polyvinyl alcohol were dried under vacuum at 40 °C. Another person who did not add ferricyanide stabilizer was used as a control group. The two were placed in a 40 ° C box, and the activity was measured weekly and compared with those placed on the first day, and the percentage of residual activity was calculated.

The results are shown in Fig. 3. Compared with the control group without the addition of ferricyanide stabilizer, the activity of glucose oxidase has been reduced to near no residual activity for 10 weeks at 40 °C; the addition of ferricyanide stabilizer to the present invention The electrochemical biosensor test strips were maintained at 90% for 10 weeks at 40 °C. It was confirmed that the electrochemical biosensor test piece of the present invention showed good heat resistance stability by the addition of the ferricyanide stabilizer, and retained about 90% of the enzyme activity after 10 weeks of storage.

Further, in this embodiment, glucose oxidase is taken as an example, and an electrochemical biosensor test piece is manufactured according to the procedure of Example 1. After completion, it is vacuum dried at 40 ° C, placed in a drying tank, and placed in an oven at 50 ° C. In the middle, every seven days, the current value of different concentrations of glucose solution (50, 100, 250 mg / dL) was measured with a potentiostat (Logic SP-150).

The results are shown in Figure 4. After storage in a high temperature environment of 50 °C, a glucose test strip containing a ferricyanide stabilizer was added to test 50, 100, 250 mg/dL glucose solutions of different concentrations, and stable measurements were still displayed after 28 days. . It was confirmed that the electrochemical biosensor test piece of the present invention showed good high temperature stability due to the addition of the ferricyanide stabilizer. Example 3 Photostability of a test piece for an electrochemical biosensor of the present invention

The test piece for the electrochemical biosensor of the present invention uses potassium ferricyanide as an electron transport medium, but an electrochemical test piece using potassium ferricyanide as an electron transporter is exposed to a light environment, iron Potassium cyanide is converted to potassium ferrocyanide. This conversion often occurs during the test strip process from reagent coating to dry canning or long-term storage of the test piece, which will cause an increase in the background value of the test piece. , resulting in a high detection value. Therefore, the present invention tests the light resistance of the test piece.

In this example, an example of the glucose oxidase was used to prepare an electrochemical biosensor test piece with a light shielding agent as an experimental group according to the procedure of Example 1, wherein the reagent layer contained: 0.75 mg/mL glucose oxidase (250 U/ Mg), 100 mg/mL potassium ferricyanide, 1.5 g/mL light screening agent barium sulfate (BaSO ₄ ), 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X- 100) and a mixture of all the above-mentioned ferricyanide stabilizers; another electrochemical biosensor test piece without added light shielding agent was used as a control group, wherein the reagent layer contained: 0.75 mg/mL glucose oxidase (250 U/mg) ), 100 mg/mL potassium ferricyanide, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X-100), and a mixture of all the above-mentioned ferricyanide stabilizers. The two test pieces were coated on the reaction zone with an air brush, placed in a 40 ° C dry box, dried for 30 minutes, and exposed to a fluorescent UV lamp for 0 hours, 0.5 hours, 1 hour, 6 hours, respectively. 12 hours, 24 hours, and then placed in a dry can without light, stored at room temperature, and taken out every other day to measure the background current value with a potentiostat.

The results are shown in Table 1. The electrochemical biosensor test piece without the added light shielding agent showed a gradually increasing background current value as the exposure time increased, and the test piece of the electrochemical biosensor with the light shielding agent added. A significant increase in background current value occurs only after exposure for more than 12 hours. It was confirmed that the electrochemical biosensor test piece of the present invention showed good photostability due to the addition of a light shielding agent. Table 1. Effect of adding light shielding agent on background current of electrochemical biosensor <TABLE border="1"borderColor="#000000"width="85%"><TBODY><tr><td></td><td> background current (uA) </td></tr><tr><td> exposure time (hours) </td><td> no added BaSO₄ ( -) </td><td> Add BaSO<sub>4</+> (+) </td></tr><tr><td></td><td></td><td></td></tr><tr><td> 0 </td><td> 2.55 </td><td> 2.55 </td></tr><tr><td> 0.5 </td><td> 4.27 </td><td> 2.57 </td></tr><tr><td> 1 </td><td> 7.76 </td><td> 2.71 </td></tr ><tr><td> 6 </td><td> 11.42 </td><td> 2.82 </td></tr><tr><td> 12 </td><td> 12.92 </td ><td> 4.50 </td></tr><tr><td> 24 </td><td> 13.30 </td><td> 7.73 </td></tr></TBODY></ TABLE> Example 4 Enzyme stability of the test piece for electrochemical biosensor of the present invention

In the past, it has been known that macromolecular polymers can be used to protect heme detecting substances, such as ferricyanide, from being unstable. This protective effect is presumed to be a high molecular polymer coating to avoid direct contact with light. The present invention utilizes such a coating to stabilize the current, so as to avoid long-term direct contact between the electron-transporting material, ferricyanide and the enzyme, in the electrochemical test piece, and the instability of the background value of the detected current is increased.

In this embodiment, an electrochemical biosensor test piece according to the procedure of Example 1 is taken as an experimental group, wherein the reagent layer further comprises: 0.75 mg/mL glucose oxidase (250 U/mg), 100. Mg/mL potassium ferricyanide, 5 mg/mL polymer enzyme structural stabilizer polyvinyl pyrrolidone, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X -100) and adding a mixture of all the above-mentioned ferricyanide stabilizers; and preparing an electrochemical biosensor test piece without adding a polymerase structural stabilizer as a control group, wherein the reagent layer comprises: 0.75 mg/mL glucose oxidase (250 U/mg), 100 mg/mL potassium ferricyanide, 0.5 mg/mL carboxymethyl cellulose, 0.1 mg/mL ketol (Triron X-100), and all the ferricyanide added above a mixture of agents. Both test pieces were coated on the reaction zone with an air brush, placed in a 40 ° C dry box, dried for 30 minutes, and placed in a dry tank protected from light. The test pieces of the two sets of test pieces were placed in an oven at 40 °C and taken out every three days. The current values of different concentrations of glucose solution were measured by a potentiostat (Logic SP-150) to test the stability.

The results are shown in Figures 5 and 6. Electrochemical biosensor test strips without added ferricyanide stabilizer showed a measured background value that gradually increased with time (Fig. 5). Electrochemistry with addition of a ferricyanide stabilizer This is not the case with biosensor test strips (Figure 6). It was confirmed that the electrochemical biosensor test piece of the present invention showed measurement stability by the addition of a ferricyanide stabilizer.

In summary, the present invention provides a high stability electrochemical biosensor test piece having a ferricyanide stabilizer and a light shielding agent component, wherein the ferricyanide stabilizer is preferably a high molecular polymer, light. The masking agent is preferably barium sulfate (BaSO ₄ ). The electrochemical biosensor test piece overcomes the shortcomings of using the reaction of the ferricyanide as an electron transport medium with the enzyme during the storage or the auto-oxidation-reduction reaction, resulting in a wrong diagnosis result; and overcoming the electron transfer medium to the light. Sensitive, thus causing the disadvantage of inaccuracies in measurement. Therefore, the electrochemical biosensor test piece of the invention has the advantages of stable manufacturing process, long shelf life, high temperature resistance and light resistance, is suitable for commercial use, achieves the purpose of self-health management of the patient, and helps prevent and slow down. The occurrence of related disease complications and the enormous burden of medical resources.

110‧‧‧Substrate

120‧‧‧ circuit layer

121‧‧‧First electrode

122‧‧‧second electrode

130‧‧‧Reagent layer

131‧‧‧Electrode reaction zone

140‧‧‧Adhesive layer

150‧‧‧ Coverage

Figure 1 is a schematic illustration of the reaction process of an electrochemical biosensor using electron transport media to assist electron transport. 2 is a structural view of a test piece of an electrochemical biosensor having high stability according to the present invention. Fig. 3 is a graph showing the heat resistance stability of a composition in which a ferricyanide stabilizer and glucose oxidation are added. Fig. 4 is a graph showing the heat resistance stability of an electrochemical biosensor test piece to which a ferricyanide stabilizer is added according to the present invention. Figure 5 is a graph showing the reaction current of an electrochemical biosensor test piece without the addition of a polymerase structural stabilizer. Fig. 6 is a graph showing the reaction current of an electrochemical biosensor test piece incorporating a polymerase structural stabilizer according to the present invention.

Claims (22)

An electrochemical biosensor test piece for detecting a sample to be tested in a sample, comprising: a substrate; a circuit layer disposed on the substrate, wherein the circuit layer comprises at least a first electrode and a second electrode; a reagent layer disposed on the circuit layer, wherein the first electrode interacts with the second electrode to overlap with the reagent layer to form an electrode reaction zone; an adhesive layer is disposed on the reagent layer, and a portion Exposing the electrode reaction zone underneath to facilitate detection of a test object; and a cover layer disposed over the adhesive layer and partially exposing the underlying reagent layer and the electrode reaction zone; wherein the reagent layer comprises a reagent layer composition, and the reagent layer composition comprises: a ferricyanide, a oxidoreductase, a ferricyanide stabilizer, and a light shielding agent; and the oxidoreductase is specific to the analyte . The electrochemical biosensor test piece according to claim 1, wherein the ferricyanide stabilizer is selected from the group consisting of polyacrylamides and alkali metal salts of Group of cellulose), hydrolyzed polyvinyl alcohol, and any combination thereof. The electrochemical biosensor test piece according to claim 1, wherein the ferricyanide is potassium ferricyanide or sodium ferricyanide. The electrochemical biosensor test piece according to claim 1, wherein the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), and the like. A group consisting of aluminum oxide (Al ₂ O ₃ ), calcined kaolin, clay, and china clay. The electrochemical biosensor test piece according to claim 1, wherein the oxidoreductase is glucose oxidase or glucose dehydrogenase when the test substance is glucose. The electrochemical biosensor test piece according to claim 1, wherein the oxidoreductase is uricase when the test substance is uric acid. The electrochemical biosensor test piece according to claim 1, wherein the oxidoreductase is cholesterol esterase or cholesterol oxidase when the test substance is cholesterol. The electrochemical biosensor test piece according to claim 1, wherein the test substance is triglyceride, the oxidoreductase esterase, glycerol kinase or phosphoglycerol oxidase. The electrochemical biosensor test piece according to claim 1, wherein the oxidoreductase is the creatinine, creatase or sarcosine oxidase when the test substance is creatinine. The electrochemical biosensor test piece according to claim 1, wherein the reagent layer composition further comprises: a nonionic surfactant, an antifoaming agent, an enzyme stabilizer, and a polymer enzyme structure. Stabilizer, a cellulose thickener and a buffer. The electrochemical biosensor test piece according to claim 10, wherein the buffer is 0.1 M phosphate buffer; and the nonionic surfactant is ketol (Triron X-100). The electrochemical biosensor test piece according to claim 10, wherein the enzyme stabilizer is selected from the group consisting of trehalse, sodium gluconate, and mannose. Cyclodextrin, sucrose, glycine, alanine, serine, arabinose, sorbitan, 2-deoxygen A group consisting of 2-deoxy-D-glucose, xylose, fructose, and glutamic acid. The electrochemical biosensor test piece according to claim 10, wherein the polymer enzyme structural stabilizer is selected from the group consisting of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone. , polyethylene oxide, polyvinyl pyrrolidone and polyvinyl acetate and polyvinyl alcohol and polyvinyl acetate (polyvinyl acetate and polyvinyl acetate copolymer) Group. The electrochemical biosensor test piece according to claim 10, wherein the cellulose thickening agent is carboxymethyl cellulose (CMC) or hydroxymethyl cellulose (HEC). . The electrochemical biosensor test piece according to claim 1, wherein the reagent layer is disposed on the circuit layer by coating, and the coating method comprises an air brush and a scraper ( Doctor blade) or screen printing. A method for manufacturing an electrochemical biosensor test strip, comprising: providing a substrate; and disposing a circuit layer on the substrate, wherein the circuit layer comprises at least a first electrode and a second electrode; On the circuit layer, the first electrode and the second electrode are alternately formed to overlap with the reagent layer to form an electrode reaction zone, and the reagent layer comprises a ferricyanide, a oxidoreductase, and a ferricyanide stabilized. And a light shielding agent; coating an adhesive layer on the reagent layer, and partially exposing the electrode reaction zone underneath to facilitate detection of a test object; A coating layer is coated on the adhesive layer, and the underlying reagent layer and the electrode reaction zone are partially exposed. The manufacturing method of claim 16, wherein the coating comprises an air brush, a doctor blade or a screen printing method. The manufacturing method according to claim 16, wherein the ferricyanide stabilizer is selected from the group consisting of polyacrylamides, alkali metal salts of cellulose, and hydrolyzed polyethylene. A group consisting of hydrolyzed polyvinyl alcohol and combinations thereof. The manufacturing method according to claim 16, wherein the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium oxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), and aluminum oxide (Al _{2 ).} O ₃ ), a group of calcined kaolin, clay, and china clay. A reagent layer for detecting a sample in a sample, comprising: a ferricyanide; a oxidoreductase; a ferricyanide stabilizer; and a light shielding agent. The reagent layer according to claim 20, wherein the ferricyanide stabilizer is selected from the group consisting of polyacrylamides, alkali metal salts of cellulose, and hydrolyzed polyethylene. A group consisting of hydrolyzed polyvinyl alcohol and combinations thereof. The reagent layer according to claim 20, wherein the light shielding agent is selected from the group consisting of barium sulfate (BaSO ₄ ), titanium dioxide (TiO ₂ ), calcium carbonate (CaCO ₃ ), and aluminum oxide (Al _{2 ).} O ₃ ), a group of calcined kaolin, clay, and china clay.