Enzyme solution, electrochemical biosensor for measuring blood glucose and preparation method of electrochemical biosensor

Abstract

The invention relates to the technical field of biosensing, in particular to enzyme liquid, an electrochemical biosensor for blood glucose measurement and a preparation method of the electrochemical biosensor. The enzyme liquid is used for preparing the electrochemical biosensor for blood glucose determination and comprises the following components in percentage by mass: 60%-80% of a buffer system, 10%-20% of a disintegrating agent, 0.1%-5% of a stabilizer, 0.01%-2% of a surfactant, 0.1%-8% of an electron transfer agent and 0.1%-5% of glucose dehydrogenase, the electron transfer agent is selected from a phenanthrenequinone compound or a phenanthroline compound containing diketone. The current amplification effect of the electrochemical biosensor can be improved, and the detection sensitivity can be further improved.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, in particular to an enzyme solution and an electrochemical biosensor for blood sugar determination and a preparation method thereof. Background Art

[0002] Biosensors based on electrochemical principles have been widely used in POCT rapid diagnosis. This type of biosensor uses an insulating substrate (PET substrate) as a carrier, prints the biosensor electrode on the insulating substrate, and then sets an enzyme layer in the reaction area of ​​the substrate. The electrochemical redox reaction between the enzyme and the analyte generates current, and the analyte is detected by the current strength. This biosensor has been widely used in auxiliary diagnosis and treatment of diabetes, such as blood glucose test strips.

[0003] Electron transfer agents, such as potassium ferrocyanide, potassium ferrocyanide and hexaammonium ruthenium trichloride, need to be added to the enzyme solution that forms the enzyme layer. The main reason for adding electron transfer agents is that the electroactive center of the enzyme molecule used in the biosensor is deeply buried inside the molecule and is easily deformed after adsorption on the electrode surface, making it difficult for the enzyme to transfer electrons directly to the electrode, so electron transfer agents are needed to transmit the signal from the enzyme active center.

[0004] However, the existing biosensors prepared from enzyme solutions formed by electron transfer agents have problems such as weak current amplification and low detection sensitivity.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide an enzyme solution and an electrochemical biosensor for blood sugar determination and a preparation method thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides an enzyme solution, which is used to prepare an electrochemical biosensor for blood glucose measurement. The enzyme solution has the following composition, measured by mass percentage:

[0009] The buffer system comprises 60%-80%, a disintegrant 10%-20%, a stabilizer 0.1%-5%, a surfactant 0.01%-2%, an electron transfer agent 0.1%-8%, and a glucose dehydrogenase 0.1%-5%; the electron transfer agent is selected from phenanthroquinone compounds or diketone-containing phenanthroline compounds.

[0010] In an optional embodiment, the enzyme solution is composed of: 65%-75% of the buffer system, 15%-20% of the disintegrant, 0.5%-4.5% of the stabilizer, 0.05%-0.5% of the surfactant, 1%-5% of the electron transfer agent, and 0.5%-5% of the glucose dehydrogenase;

[0011] Preferably, the enzyme solution is composed of: 74% of the buffer system, 18% of the disintegrant, 1% of the stabilizer, 0.5% of the surfactant, 3% of the electron transfer agent, and 3.5% of the glucose dehydrogenase.

[0012] In an alternative embodiment, the electron transfer agent is selected from 9,10-phenanthrolinequinone or 1,10-phenanthroline-5,6-dione.

[0013] In an optional embodiment, the buffer system is selected from any one or at least two of sodium citrate buffer, sodium succinate buffer, bellate buffer and sodium fumarate buffer;

[0014] The disintegrant is selected from polyvinyl compounds; preferably polyvinyl pyrrolidone;

[0015] The stabilizer is selected from disaccharide compounds or sugar alcohol compounds, preferably any one of maltose, trehalose, sucrose and maltitol;

[0016] The surfactant is selected from nonionic surfactants, preferably Tween or Triton;

[0017] The glucose dehydrogenase is selected from flavin adenine dinucleotide glucose dehydrogenase.

[0018] In a second aspect, the present invention provides an electrochemical biosensor for blood glucose measurement, comprising an enzyme layer, wherein the enzyme layer is prepared by the enzyme solution described in any one of the aforementioned embodiments.

[0019] In an optional embodiment, the electrochemical biosensor comprises a substrate, an electrode group, an insulating layer, an enzyme layer and a siphon guide layer arranged in sequence;

[0020] Preferably, the electrode group includes an impedance detection working electrode, a working electrode, a counter electrode and an impedance detection counter electrode; wherein the impedance detection working electrode and the impedance detection counter electrode constitute an impedance loop, and the working electrode and the counter electrode constitute a sample test loop.

[0021] In a third aspect, an embodiment of the present invention provides a method for preparing an electrochemical biosensor for blood glucose measurement, comprising: forming an enzyme layer using the above enzyme solution.

[0022] In an optional embodiment, the method comprises: pre-treating the electrode group;

[0023] Preferably, the pretreatment is an electrochemical pretreatment;

[0024] More preferably, the pre-treatment comprises treatment using cyclic voltammetry or current-time curve method.

[0025] In an optional embodiment, the conditions of cyclic voltammetry treatment include: an applied voltage range of -1.5V-1.5V, a scan rate of 20mv / s-300mv / s, a pretreatment buffer including any one of acetate buffer solution, phosphate buffer solution and carbonate buffer solution, the molar concentration of the pretreatment buffer is 0.01-0.2mmol / L, and the pH range of the pretreatment buffer is 4-9.

[0026] In an optional embodiment, the current time curve method is used: the applied voltage range is 1.0V-5.0V, the voltage application time is 20-300s, the pretreatment buffer can be any one of acetate buffer solution, phosphate buffer solution and carbonate buffer solution, the molar concentration of the pretreatment buffer is 0.01-0.2mmol / L, and the pH range of the pretreatment buffer is 4-9.

[0027] The present invention has the following beneficial effects: The embodiment of the present invention provides a new enzyme solution system, which can be used for the preparation of an electrochemical biosensor, which can enhance the current amplification effect of the electrochemical biosensor and further enhance the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 A schematic diagram of the structure of an electrochemical biosensor provided in an embodiment of the present invention;

[0030] Figure 2 Cyclic voltammetry curves of potassium ferrocyanide before and after electrochemical pretreatment provided by an embodiment of the present invention;

[0031] Figure 3 A diagram showing the difference between the current signal generated by the electron transfer agent used in the embodiment of the present invention and the current signal generated by other electron transfer agents;

[0032] Figure 4 Linear fitting diagram of the measurement results of the glucose electrochemical biosensor prepared in the embodiment of the present invention and the reference instrument.

[0033] Icon: 100-electrochemical biosensor; 110-substrate; 120-electrode group; 121-impedance detection working electrode; 122-working electrode; 123-counter electrode; 124-impedance detection counter electrode; 130-insulating layer; 140-enzyme layer; 150-siphon guide channel layer; 160-logo membrane strip. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] The present invention provides an enzyme solution, which has the following composition in terms of mass percentage:

[0036] The buffer system comprises 60%-80%, a disintegrant 10%-20%, a stabilizer 0.1%-5%, a surfactant 0.01%-2%, an electron transfer agent 0.1%-8%, and a glucose dehydrogenase 0.1%-5%; the electron transfer agent is selected from phenanthroquinone compounds or diketone-containing phenanthroline compounds.

[0037] The enzyme solution can be used to prepare an electrochemical biosensor for blood glucose measurement, which can improve the amplification effect of the current of the electrochemical biosensor and further improve the detection sensitivity. Specifically, the buffer solution can provide a stable environment for the reaction to occur, the disintegrant can quickly turn the powder into a liquid state, so as to quickly participate in the reaction, the stabilizer can protect the enzyme activity from being lost, the surfactant can make the enzyme solution more stable, the electrode electronic agent can improve the reaction signal, and the glucose dehydrogenase can specifically recognize glucose, thereby enabling the enzyme solution to be used for blood glucose detection.

[0038] Specifically, the amount of the buffer system is 60%, 65%, 70%, 75% and 80%, etc., any value between 60-80%, or a range value between any two values. For example, preferably 65-75%. The buffer system includes, but is not limited to, any one or at least two of sodium citrate buffer, sodium succinate buffer, bellate buffer and sodium fumarate buffer.

[0039] The amount of the disintegrant is 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20%, etc., any value between 15-20%, or a range between any two values. For example, preferably the disintegrant is 15%-20%. The disintegrant is selected from polyvinyl compounds; for example, including but not limited to polyvinyl pyrrolidone.

[0040] The amount of the stabilizer is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%, etc., any value between 0.1% and 5%, or a range between any two values. For example, the stabilizer is 0.5-4.5%. The disintegrant is selected from polyethylene compounds. The stabilizer is selected from disaccharide compounds or sugar alcohol compounds, for example, including but not limited to any one of maltose, trehalose, sucrose and maltitol.

[0041] The amount of the surfactant is any value between 0.01% and 2%, or a range between any two values, such as 0.01%, 0.5%, 1%, 1.5%, and 2%. For example, preferably 0.05-0.5%. The surfactant is selected from nonionic surfactants, such as Tween 20, Tween 80, and other Tweens or Triton.

[0042] The amount of glucose dehydrogenase is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%, etc., any value between 0.1-5% or a range between any two values. For example, preferably 0.5-5%. Glucose dehydrogenase is selected from flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH).

[0043] The amount of the electron transfer agent is 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8%, etc., any value between 0.1% and 8%, or a range between any two values. For example, preferably 1-5%. The electron transfer agent is selected from 9,10-phenanthroquinone or 1,10-phenanthroline-5,6-dione.

[0044] Quinol compounds in the prior art have been used as redox mediators for electrocatalysis and preparation of enzymatic biofuel cells due to their chemical stability, good equilibrium potential, electrochemical reversibility and high reactivity to redox-active enzymes. The oxidation-catalytic reduction of quinones was found to be carried out by a two-electron (hydride) transfer reaction, which conforms to the "outer sphere" ET mechanism, and its reactivity is mainly controlled by the electron accepting ability of the compound, and is not substantially affected by its structural characteristics. And the high self-exchange rate constant of quinones makes quinones used as signal transduction media in enzyme-catalyzed biosensor systems. Because of its efficient self-exchange rate, it can amplify the current of electrochemical biosensors during the reaction process. Therefore, the embodiment of the present invention adopts the phenanthroline quinone compounds such as 9,10-phenanthroline or 1,10f-phenanthroline-5,6-diketone or phenanthroline compounds containing diketone as electron transfer agents. Compared with potassium ferrocyanide and hexachlororuthenium trichloride, which are commonly used electron transfer agents in products on the market, the current intensity generated by the use of 9,10-phenanthroquinone or 1,10-phenanthroline-5,6-dione (PD) as an electron transfer agent in the present invention is higher, which helps to improve the sensitivity and linear range of the reaction, and has better stability and electrochemical activity.

[0045] More preferably, the enzyme solution is composed of: 74% of the buffer system, 18% of the disintegrant, 1% of the stabilizer, 0.5% of the surfactant, 3% of the electron transfer agent, and 3.5% of the glucose dehydrogenase.

[0046] In a second aspect, an embodiment of the present invention provides an electrochemical biosensor for blood glucose measurement, which includes a substrate, an electrode group, an insulating layer, an enzyme layer and a siphon guide channel layer arranged in sequence; the enzyme layer is prepared by the above enzyme solution.

[0047] Specifically, the electrode group includes an impedance detection working electrode, a working electrode, a counter electrode and an impedance detection counter electrode. The impedance detection working electrode and the impedance detection counter electrode form an impedance loop, and then the impedance value of the blood sample can be detected. Specifically, the hematocrit (HCT) of the sample can be measured by the above two electrodes, and then the measurement result can be compensated according to the corresponding relationship between the hematocrit (HCT) and the current, thereby reducing the influence of the hematocrit (HCT) on the measurement result, making the glucose measurement result in the blood sample more accurate.

[0048] The working electrode and the counter electrode form a sample test circuit, which then realizes the determination of glucose in the sample. Specifically, when the blood sample passes through the working electrode and the counter electrode, a rated voltage is applied, and a current is generated. The current intensity is positively correlated with the glucose concentration, and the glucose concentration can be calculated based on the collected current intensity.

[0049] In summary, the embodiment of the present invention can measure the impedance of a blood sample during measurement through the impedance detection working electrode with impedance measurement and the impedance detection counter electrode, and then convert it into the hematocrit of the sample, provide compensation for the hematocrit of the blood sample, and make the measurement result more accurate.

[0050] The electrochemical biosensor has an enzyme layer formed by an enzyme solution, and the glucose dehydrogenase in the enzyme layer reacts with glucose in the blood. When a fixed voltage is applied, a current that is positively correlated with the glucose concentration is generated. Therefore, the concentration of glucose in the blood sample can be obtained based on the current intensity. Using the above enzyme solution to form an enzyme layer can amplify the electrical signal generated by the electrochemical sensor and increase the sensitivity of the reaction.

[0051] In a third aspect, an embodiment of the present invention provides a method for preparing the electrochemical biosensor, comprising:

[0052] The preparation is carried out by screen printing technology. Specifically, carbon paste, silver paste and insulating material are printed on a substrate (such as a polyethylene terephthalate (PET) substrate) to form an electrode group and an insulating layer. The conditions and processes of the screen printing technology can adopt existing conditions and processes, and the embodiments of the present invention will not be described in detail.

[0053] The use of screen printing technology to prepare electrochemical biosensors makes it easy to miniaturize and integrate the prepared electrodes, consume less reagents, and have lower costs. However, in the process of preparing screen-printed electrodes, the carbon paste used for printing is composed of graphite powder, organic binder, organic solvent and additives. These organic insulating substances have a great influence on the performance of the electrode, such as the decrease in conductivity and electron transfer rate, which will lead to poor precision and low detection sensitivity of the test paper.

[0054] Therefore, in the preparation process of electrochemical biosensors, after forming the electrode group and before forming the enzyme layer, the electrode needs to be pretreated and activated to remove surface organic matter, polymers and other impurities, so as to expose the carbon active points of the screen-printed electrode and enhance the conductivity, hydrophilicity and consistency of the electrode.

[0055] There are several methods for pre-treatment and activation of electrodes in the prior art, including mechanical polishing, laser treatment and acid-base washing. The first two methods are expensive, require high professional knowledge of the operator, and are easy to damage the electrode structure. Acid-base washing may cause changes in the chemical properties of the electrode surface and the treatment time is long.

[0056] The embodiment of the present invention uses electrochemical treatment to treat the electrode to achieve activation. Specifically, the embodiment of the present invention uses electrochemical treatment to not only remove surface organic matter, polymers and other impurities, but also increase its conductivity and electron transfer rate, which is conducive to the occurrence of electrochemical reactions. At the same time, the electrochemical treatment belongs to anodizing treatment, and an oxidation reaction occurs on the electrode surface, which will cause many hydrophilic groups on the electrode surface, such as hydroxyl (-OH) and carboxyl (-COOH), thereby increasing the wettability of the electrode surface, which is conducive to the diffusion of the enzyme solution and makes the dot liquid more uniform.

[0057] Specifically, the embodiment of the present invention adopts cyclic voltammetry (CV method) or current-time curve method (i-t method) for pre-treatment.

[0058] The conditions for cyclic voltammetry treatment include: the applied voltage range is -1.5V-1.5V, such as -1.5V, -1V, 1V and 1.5V, etc., any value between -1.5V-1.5V. The scanning speed is 20mv / s-300mv / s, such as 20mv / s, 50mv / s, 100mv / s, 150mv / s, 200mv / s, 250mv / s and 300mv / s, etc., any value between 20-300mv / s. The pretreatment buffer includes any one of acetate buffer solution, phosphate buffer solution and carbonate buffer solution, and the molar concentration of the pretreatment buffer is 0.01-0.2mmol / L, such as 0.01mmol / L, 0.05mmol / L, 0.1mmol / L, 0.15mmol / L and 0.2mmol / L, etc., any value between 0.01-0.2mmol / L. The pH of the pretreatment buffer is in the range of 4-9, for example, any value between 4, 5, 6, 7, 8 and 9.

[0059] Current time curve method treatment: the applied voltage range is 1.0V-5.0V, for example, any value between 1.0V-5.0V such as 1.0V, 2.0V, 3.0V, 4.0V and 5.0V. The voltage application time is 20-300s, for example, any value between 20s, 50s, 100s, 150s, 200s, 250s and 300s. The pretreatment buffer can be any one of acetate buffer solution, phosphate buffer solution and carbonate buffer solution, and the molar concentration of the pretreatment buffer is 0.01-0.2mmol / L, for example, any value between 0.01mmol / L, 0.05mmol / L, 0.1mmol / L, 0.15mmol / L and 0.2mmol / L such as 0.01-0.2mmol / L. The pH of the pretreatment buffer is in the range of 4-9, for example, any value between 4, 5, 6, 7, 8 and 9.

[0060] In summary, the electrodes formed by screen printing in the embodiments of the present invention can be treated by cyclic voltammetry (CV) or current-time curve method (i-t method) to remove impurities on the electrode surface, improve electrode conductivity and electrode transfer rate, and thus improve the detection sensitivity of the test paper. At the same time, the area of ​​the electrode active site can be increased, the consistency of the electrode surface can be improved, and the precision of the test paper can be improved, so that the product quality is better.

[0061] After the pretreatment is completed, the enzyme solution is applied to form an enzyme layer, and then the siphon guide layer and logo film strip are set.

[0062] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0063] Example 1

[0064] The present invention provides an enzyme solution, the components of which are as follows:

[0065] The buffer system is 74%, the disintegrant is 18%, the stabilizer is 1%, the surfactant is 0.5%, the electron transfer agent is 3%, and the glucose dehydrogenase is 3.5%. The buffer system is sodium citrate, the disintegrant is polyvinyl pyrrolidone PVP, the stabilizer is maltitol, the surfactant is Triton, the glucose dehydrogenase is flavin adenine dinucleotide glucose dehydrogenase (FAD-GDH), and the electron transfer agent is selected from 1,10-phenanthroline-5,6-dione.

[0066] Example 2

[0067] The present invention provides an electrochemical biosensor 100 for measuring blood glucose. Figure 1 It includes a substrate 110, which is provided with an electrode group 120, and the electrode group 120 includes an impedance detection working electrode 121, a working electrode 122, a counter electrode 123 and an impedance detection counter electrode 124; wherein the impedance detection working electrode 121 and the impedance detection counter electrode 124 constitute an impedance loop, and the working electrode 122 and the counter electrode 123 constitute a sample test loop.

[0068] The electrode group 120 is provided with an insulating layer 130, and one side of the insulating layer 130 has a hole, and the hole is provided with an enzyme layer 140, and the enzyme layer 140 is formed by the enzyme solution. The insulating layer 130 is provided with a siphon guide layer 150 and a logo film strip 160 in sequence.

[0069] Example 3

[0070] This embodiment provides a method for preparing the electrochemical biosensor for blood glucose measurement of the above embodiment 2, comprising:

[0071] Carbon paste, silver paste, and insulating material are printed on the substrate, respectively, to form an electrode group and an insulating layer 130 .

[0072] The electrode group is then pretreated using cyclic voltammetry, the conditions of which include: an applied voltage range of -1.0V-1.0V, a scan rate of 100mv / s, an acetate buffer solution as the pretreatment buffer, a molar concentration of 0.1mmol / L, and a pH range of 5.6.

[0073] Then, the enzyme solution of Example 1 was added dropwise to form an enzyme layer, and then a siphon guide groove layer and a logo film strip were arranged.

[0074] Example 4

[0075] This example prepares an electrochemical biosensor for blood glucose measurement by referring to the preparation method provided in Example 3, the only difference being that the method for pre-treating the electrode group is different, and the other conditions are the same as those in Example 3. Specifically, this example adopts the current time curve method (i-t method) for treatment, and the conditions are as follows: the applied voltage is 1.5V, the voltage application time is 120s, the molar concentration of the phosphate buffer solution used is 0.1mmol / L, and the pH range of the solution is 7.4.

[0076] Example 5-Example 7

[0077] Examples 5 to 7 provide enzyme solutions respectively, as follows:

[0078] Example 5: 60% buffer system, 20% disintegrant, 5% stabilizer, 2% surfactant, 8% electron transfer agent, and 5% glucose dehydrogenase.

[0079] Example 6: 80% buffer system, 10% disintegrant, 0.1% stabilizer, 1.4% surfactant, 8% electron transfer agent, and 0.5% glucose dehydrogenase.

[0080] Example 7: Buffer system 74.79%, disintegrant 20%, stabilizer 5%, surfactant 0.01%, electron transfer agent 0.1%, glucose dehydrogenase 0.1%.

[0081] Embodiment 8-9

[0082] Embodiment 8-9 provides a method for preparing an electrochemical biosensor for blood glucose measurement, which is similar to that of Embodiment 3, except that the conditions of cyclic voltammetry are different, and the other conditions are the same as those of Embodiment 3. Specifically,

[0083] Example 8: The scanning rate is 20 mv / s; the buffer solution is a phosphate buffer solution; the buffer solution concentration is 0.01 mmol / L; and the pH is 9.

[0084] Example 9: The scanning rate is 300 mv / s; the buffer solution is a carbonate buffer solution; the buffer solution concentration is 0.2 mmol / L; and the pH is 4.

[0085] Embodiment 10-11

[0086] Embodiments 10-11 provide a method for preparing an electrochemical biosensor for blood glucose measurement, which is similar to that of Embodiment 4, except that the conditions of the current-time curve method are different, and the other conditions are the same as those of Embodiment 4. Specifically,

[0087] Example 10: The applied voltage is 1.0 V; the buffer solution is a phosphate buffer solution, the voltage application time is 20 s, the buffer solution concentration is 0.01 mmol / L; and the pH is 9.

[0088] In Example 11, the applied voltage is 5.0 V, the buffer solution is a carbonate buffer solution, the voltage application time is 300 s, the buffer solution concentration is 0.2 mmol / L; and the pH is 4.

[0089] Test Example 1

[0090] The electrode group before and after the pretreatment provided in Example 3 was tested, and the test method was: cyclic voltammetry (CV), test voltage -0.8V-0.8V, scan rate 100mV / S;

[0091] Waiting time: 5s; Test substance: potassium ferrocyanide solution (10mmol / L).

[0092] Results Figure 2 ,according to Figure 2 It can be seen that in the cyclic voltammetry curve, there is a clear difference between the potassium ferrocyanide solution curve before and after the electrochemical pretreatment of the screen-printed electrode. Compared with the screen-printed electrode without electrochemical pretreatment, the oxidation peak current intensity of the potassium ferrocyanide curve of the screen-printed electrode after electrochemical pretreatment is stronger, and the current intensity increases by about 50%. It further shows that the electrochemical pretreatment removes impurities on the electrode surface, and the generated hydrophilic groups, such as hydroxyl (-OH) and carboxyl (-COOH), are more conducive to the transfer of electrons, thereby enhancing the electron transfer rate and transfer rate of the screen-printed electrode.

[0093] Test Example 2

[0094] A comparative electrochemical biosensor was prepared by referring to the preparation method provided in Example 3, except that the electrode group was not pre-treated by cyclic voltammetry.

[0095] Then, the electrochemical biosensor of Example 3 and the comparative example was tested. Specifically, samples with blood glucose concentrations of 50 mg / dL, 110 mg / dL, 275 mg / dL, 360 mg / dL, and 480 mg / dL were configured. Each sample was tested 20 times, and then the average value, SD, and CV were calculated. The coefficient of variation (CV) is an effective indicator for evaluating the precision of the test paper. The smaller the value, the higher the precision of the test paper.

[0096] The results are shown in Tables 1 and 2.

[0097] Table 1 Test results of pre-treated electrodes

[0098] Concentration / mg / dL 50 110 275 360 480 Test 1 46 107 258 380 510 Test 2 47 113 276 348 488 Test 3 49 101 290 354 521 Test 4 49 115 263 394 493 Test 5 47 119 260 376 535 Test 6 52 110 276 390 506 Test 7 49 100 256 354 461 Test 8 54 101 290 359 483 Test 9 50 106 275 392 507 Test 10 53 105 281 382 470 Test 11 54 106 264 348 474 Test 12 51 117 270 370 473 Test 13 53 103 278 380 526 Test 14 46 107 260 351 485 Test 15 52 118 284 353 532 Test 16 47 101 256 356 511 Test 17 53 114 268 374 491 Test 18 50 119 289 347 470 Test 19 47 101 282 387 505 55 114 285 390 530 average value 50.2 108.9 273.1 369.3 498.6 SD 2.86 6.57 11.50 16.60 22.44 CV 5.7％ 6.0％ 4.2％ 4.5％ 4.5％

[0099] Table 2 Test results of electrodes without pretreatment

[0100] Concentration / mg / dL 50 110 275 360 480 Test 1 45 100 259 365 480 Test 2 49 102 264 345 468 Test 3 50 101 273 363 457 Test 4 47 103 259 366 466 Test 5 46 109 264 348 482 Test 6 49 104 267 359 481 Test 7 47 104 268 361 480 Test 8 48 107 274 351 490 Test 9 50 110 264 347 470 Test 10 45 103 265 352 456 Test 11 51 108 269 362 470 Test 12 46 107 264 374 452 Test 13 50 103 258 358 478 Test 14 51 101 257 368 480 Test 15 51 102 256 368 458 Test 16 50 103 267 374 457 Test 17 49 112 275 357 461 Test 18 46 106 272 355 467 Test 19 48 110 267 356 456 Test 20 46 102 269 368 484 average value 48.2 104.9 265.6 359.9 469.7 SD 2.01 3.42 5.53 8.36 11.25 CV 4.2％ 3.3％ 2.1％ 2.3％ 2.4％

[0101] According to Table 1 and Table 2, before electrochemical pretreatment, the precision of the blood glucose test strips was in the range of 4.2%-6.0%, while after electrochemical pretreatment, the precision of the blood glucose test strips was in the range of 2.1%-4.2%. It can be seen that after electrochemical pretreatment, the precision of the test strips can be significantly improved, thereby making the product quality better. It further explains that the electrochemical treatment produces an oxidation reaction on the electrode surface, which will cause many hydrophilic groups on the electrode surface, such as hydroxyl (-OH) and carboxyl (-COOH), thereby increasing the wettability of the electrode surface, which is beneficial to the diffusion of the enzyme solution and makes the dot liquid more uniform.

[0102] Test Example 3

[0103] The potassium ferrocyanide enzyme solution is prepared, and its ingredients are as follows: 74% of the buffer system, 18% of the disintegrant, 1% of the stabilizer, 0.5% of the surfactant, 3% of the electron transfer agent, and 3.5% of the glucose dehydrogenase.

[0104] The hexachlororuthenium enzyme solution is prepared, and its ingredients are as follows: 74% of the buffer system, 18% of the disintegrant, 1% of the stabilizer, 0.5% of the surfactant, 3% of the electron transfer agent, and 3.5% of the glucose dehydrogenase.

[0105] Then, the electrochemical biosensor 100 was prepared by using the enzyme solution according to the method of Example 3. The preparation of the electrochemical biosensor 100 was only to change the enzyme solution, and the rest was the same as that of Example 3. The enzyme solution used in Example 1 was denoted as PD enzyme solution.

[0106] Then the electrochemical biosensor was used for detection. Specifically, seven samples with target blood glucose concentrations of 50 mg / dL, 110 mg / dL, 275 mg / dL, 360 mg / dL, 480 mg / dL, 525 mg / dL, and 610 mg / dL were prepared, and then the concentration values ​​were tested by YSI-2500 and electrochemical biosensor respectively. The YSI-2500 test value was used as a reference value. Then the YSI-2500 and the test value of the electrochemical biosensor were linearly fitted to calculate the linear correlation.

[0107] Results Figure 3 and Figure 4 ,according to Figure 3 It can be seen that the signal enhancement intensity is significantly different when different electron transfer agents are used in the formula. The best signal enhancement effect is 1,10-phenanthroline-5,6-dione (PD) as an electron transfer agent, followed by hexaammonium ruthenium trichloride, and the weakest signal enhancement is potassium ferrocyanide. 1,10-phenanthroline-5,6-dione (PD) has good chemical stability and electrochemical activity, and its efficient self-exchange rate constant enables it to amplify the electrical signal generated by the electrochemical sensor and increase the sensitivity of the reaction.

[0108] according to Figure 4 It can be seen that the glucose concentration in the blood sample measured by the electrochemical biosensor provided in the embodiment of the present invention has a good linear correlation with the reference instrument YSI-2500, R 2 =0.998. Therefore, the prepared electrochemical biosensor can be applied to the determination of glucose in blood samples.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An enzyme solution, characterized in that The enzyme solution is used to prepare an electrochemical biosensor for blood glucose measurement. The enzyme solution has the following composition in terms of mass percentage: The buffer system comprises 60%-80%, a disintegrant 10%-20%, a stabilizer 0.1%-5%, a surfactant 0.01%-2%, an electron transfer agent 0.1%-8%, and a glucose dehydrogenase 0.1%-5%; the electron transfer agent is selected from phenanthroquinone compounds or diketone-containing phenanthroline compounds.

2. The enzyme solution according to claim 1, characterized in that The enzyme solution is composed of: 65%-75% of the buffer system, 15%-20% of the disintegrant, 0.5%-4.5% of the stabilizer, 0.05%-0.5% of the surfactant, 1%-5% of the electron transfer agent, and 0.5%-5% of the glucose dehydrogenase; Preferably, the enzyme solution is composed of: 74% of the buffer system, 18% of the disintegrant, 1% of the stabilizer, 0.5% of the surfactant, 3% of the electron transfer agent, and 3.5% of the glucose dehydrogenase.

3. The enzyme solution according to claim 1, characterized in that The electron transfer agent is selected from 9,10-phenanthrolinequinone or 1,10f-phenanthroline-5,6-dione.

4. The enzyme solution according to any one of claims 1 to 3, characterized in that The buffer system is selected from any one or at least two of sodium citrate buffer, sodium succinate buffer, bellate buffer and sodium fumarate buffer; The disintegrant is selected from polyvinyl compounds; preferably polyvinyl pyrrolidone; The stabilizer is selected from disaccharide compounds or sugar alcohol compounds, preferably any one of maltose, trehalose, sucrose and maltitol; The surfactant is selected from nonionic surfactants, preferably Tween or Triton; The glucose dehydrogenase is selected from flavin adenine dinucleotide glucose dehydrogenase.

5. An electrochemical biosensor for blood glucose measurement, characterized in that: The invention comprises an enzyme layer, wherein the enzyme layer is prepared by the enzyme solution according to any one of claims 1 to 4.

6. The electrochemical biosensor for blood glucose measurement according to claim 5, characterized in that: The electrochemical biosensor comprises a substrate, an electrode group, an insulating layer, an enzyme layer and a siphon guide channel layer which are arranged in sequence; Preferably, the electrode group includes an impedance detection working electrode, a working electrode, a counter electrode and an impedance detection counter electrode; wherein the impedance detection working electrode and the impedance detection counter electrode constitute an impedance loop, and the working electrode and the counter electrode constitute a sample test loop.

7. A method for preparing an electrochemical biosensor for blood glucose measurement according to claim 5, characterized in that: include: The enzyme layer is formed by using the enzyme solution described in any one of claims 1 to 4.

8. The method for preparing an electrochemical biosensor for blood glucose measurement according to claim 7, characterized in that: include: Pre-treating the electrode group; Preferably, the pretreatment is an electrochemical pretreatment; More preferably, the pre-treatment comprises treatment using cyclic voltammetry or current-time curve method.

9. The method for preparing an electrochemical biosensor for blood glucose measurement according to claim 8, characterized in that: The conditions for cyclic voltammetry treatment include: an applied voltage range of -1.5V-1.5V, a scan rate of 20mv / s-300mv / s, a pretreatment buffer solution including any one of an acetate buffer solution, a phosphate buffer solution and a carbonate buffer solution, a molar concentration of the pretreatment buffer solution of 0.01-0.2mmol / L, and a pH range of 4-9.

10. The method for preparing an electrochemical biosensor for blood glucose measurement according to claim 8, characterized in that: Current time curve method treatment: the applied voltage range is 1.0V-5.0V, the voltage application time is 20-300s, the pretreatment buffer can be any one of acetate buffer solution, phosphate buffer solution and carbonate buffer solution, the molar concentration of the pretreatment buffer is 0.01-0.2mmol / L, and the pH range of the pretreatment buffer is 4-9.

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