WO2023246298A1 - 电化学生物传感器及其制备方法 - Google Patents

电化学生物传感器及其制备方法 Download PDF

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WO2023246298A1
WO2023246298A1 PCT/CN2023/091081 CN2023091081W WO2023246298A1 WO 2023246298 A1 WO2023246298 A1 WO 2023246298A1 CN 2023091081 W CN2023091081 W CN 2023091081W WO 2023246298 A1 WO2023246298 A1 WO 2023246298A1
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electrode
film
insulating film
substrate
auxiliary electrode
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PCT/CN2023/091081
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English (en)
French (fr)
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崔刚
康泰荣
俞在炫
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爱森斯(江苏)生物科技有限公司
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Publication of WO2023246298A1 publication Critical patent/WO2023246298A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/333—Ion-selective electrodes or membranes

Definitions

  • the invention belongs to the technical field of electrochemical biosensors, and specifically relates to an electrochemical biosensor that uses electrochemical methods to analyze the concentration of analyte contained in a sample in more detail and a preparation method thereof.
  • Diagnosing and preventing diabetes requires periodic testing of blood glucose levels. Measuring blood glucose levels is very important for diabetic patients to regulate sugar absorption, and is especially necessary for early detection and treatment of diabetic patients.
  • Blood glucose can be easily measured using biosensors in the form of test strips.
  • Biosensors that measure blood glucose work based on colorimetric or electrochemical methods.
  • the colorimetric method uses a mixture of indicators o-toluidine and benzidine to measure blood glucose concentration by observing the color change of the indicator after the reaction.
  • the disadvantage of this method is that the measurement accuracy is insufficient.
  • the electrochemical method can improve the accuracy of measuring blood glucose concentration, shorten the measurement time, and facilitate measurement. It has been widely used in recent years.
  • electron transfer mediators include ferrocene, ferrocene derivatives, quinones, quinone derivatives, and transition metal-containing Organic or inorganic compounds (ruthenium hexaammonium chloride, osmium-containing polymers, potassium ferricyanide, etc.), organic conducting salt, viologen, etc.
  • Electrochemical biosensors are based on the following reactions:
  • GOx represents glucose oxidase (Glucose oxidase);
  • GOx-FAD and GOx-FADH 2 represent the active site FAD (flavin adenine dinucleotide) of the associated glucose oxidase in the oxidized and reduced states respectively.
  • Reaction formula (1) Glucose in the blood is first oxidized to gluconic acid under the catalysis of glucose oxidase. At this time, FAD at the active site of glucose oxidase is reduced to FADH 2 ;
  • the transfer mediator is oxidized to FAD through the redox reaction FADH2 , and the electron transfer mediator is reduced.
  • the reduced electron transfer mediator generated by the reaction diffuses to the electrode surface, and the potential for oxidation of the reduced electron transfer mediator on the electrode surface is applied through the working electrode, and the current generated at this time is measured to measure the blood glucose concentration.
  • an insulating lower substrate S11
  • the lower substrate is formed by an integrated electrode 10, an insulating film 20, and a sensing film 30. It is then made by pasting it with the middle substrate and the upper substrate in sequence.
  • the integrated electrode 10 includes a first working electrode 11, a first auxiliary electrode 12, a second working electrode 13, and a second auxiliary electrode 14.
  • the sensing film 30 includes an oxidase and an electrode transfer mediator.
  • the step of making integrated electrodes (S12) The step of making the insulating film (S13) is to screen-print the insulating slurry on the lower substrate that has completed the integrated electrode 10 and then heat it to make it harden.
  • the step of making the sensing film (S14) is to apply the sensing film composition material on the insulating film 20 and then dry it to form;
  • the intermediate substrate pasting step (S15) is to put the intermediate substrate with a double-sided tape structure with fine channels ( (not shown) is pasted on the lower substrate to cover the insulating film (20);
  • the upper substrate pasting step (S16) is to paste the upper substrate (not shown) with air holes on the middle substrate.
  • the measurement accuracy and precision of this type of electrochemical biosensor are affected by the uniformity of the sensing film 30. Furthermore, exposure of the sensing film 30 to moisture not only affects the accuracy and precision, but also may cause abnormal operation. However, in the past, during the process of forming the sensing film 30 after making the insulating film 20 of the biosensor, the problem that the sensing film composition cannot be evenly applied to the insulating film frequently occurs. As shown in Figures 3(a) and (b), during the process of applying the sensing film composition to the insulating film 20, the sensing film composition irregularly shifts to one side, or is prone to excessive diffusion as shown in Figure 3(c). .
  • the sensing film 30 formed in a state where the sensing film composition is unevenly applied in this way comes into contact with the blood sample sucked in through the fine flow channel between the upper and lower substrates, the contact area or shape size of the blood sample and the sensing film 30 will be different. At this time, the blood The reaction amount between the sample and the oxidase in the sensing membrane 30 will be different, which will lead to a decrease in measurement accuracy and precision.
  • the non-uniformity of the sensing film 30 also causes the sensing film 30 and the blood sample to easily flow to the second working electrode 13 during the dissolution process.
  • the electrochemical reaction between the oxidase and the electron transfer mediator in the sensing film 30 is based on the electron flow between the first working electrode 11 and the first auxiliary electrode to measure blood glucose concentration.
  • the oxidase and the electron transfer mediator If the composition flows too much on the second working electrode 13, it will affect the measurement of the second working electrode 13, thereby affecting the accuracy and precision.
  • an inspection process is required to eliminate defects in the sensing film, which will lead to an increase in production time.
  • moisture easily penetrates into the sensing film from the fine flow channel between the lower substrate and the upper substrate, making it easy to react and affecting measurement accuracy and precision.
  • the present invention provides an electrochemical biosensor and a preparation method thereof, which can ensure that the contact area between the sensing film and the measurement sample remains uniform, thereby improving measurement accuracy and precision, and can also reduce moisture content. Penetration effects, maintaining good measurement accuracy even in high humidity environments.
  • the invention provides an electrochemical biosensor, which includes a lower substrate, a middle substrate and an upper substrate.
  • the lower substrate is provided with an integrated electrode.
  • the integrated electrode includes a first working electrode and a first auxiliary electrode spaced apart from each other.
  • the second working electrode and the second auxiliary electrode, the first working electrode and the first auxiliary electrode form a first electrode part, the second working electrode and the second auxiliary electrode form a second electrode part;
  • the intermediate substrate is sandwiched between the lower substrate and the upper substrate, and a fine flow channel is provided on the middle substrate.
  • the fine flow channel is used to continuously introduce the sample into the first electrode part and the second electrode part.
  • An insulating film is also disposed between the upper substrate and the middle substrate.
  • the insulating film covers part of the integrated electrode and limits the inflowing sample from reaching the first electrode part and the second electrode part. ;
  • a sensing film is exposed between the lower substrate and the insulating film.
  • the sensing film is exposed relative to the part of the fine flow channel, and the remaining part is covered by the insulating film.
  • the sensing film is disposed on the On the first working electrode and the first auxiliary electrode, the insulating film is provided with an insulating film opening that opens from the inside to the outside, and the insulating film opening corresponds to the fine flow channel.
  • the upper substrate is provided with a confirmation window corresponding to the fine flow channel, and the confirmation window is provided with air holes.
  • the insulating film further includes at least one barrier film, the barrier film is disposed transversely to the opening of the insulating film, and the barrier film is located between the first working electrode and the first auxiliary electrode. /or between the air hole and the first auxiliary electrode.
  • the insulating film further includes at least one blocking film, the blocking film is disposed transversely to the opening of the insulating film, and the blocking film is located between the first electrode part and the second electrode part, The blocking film can inhibit the flow of dissolved substances dissolved by the reaction between the sensing film and the sample.
  • the blocking film and the barrier film are arranged in sequence.
  • the second auxiliary electrode, the second working electrode, the first working electrode and the first auxiliary electrode are arranged in sequence along the direction of sample inflow in the fine flow channel.
  • the first auxiliary electrode and the second auxiliary electrode are connected through a connecting electrode to form an auxiliary electrode body; a biosensor confirmation electrode is provided at the end of the auxiliary electrode body.
  • the lower substrate is provided with a production barcode information confirmation electrode.
  • the volume of the fine flow channel is 0.3-1.0 ⁇ L, preferably 0.3-0.7 ⁇ L.
  • the insulating film covers part of the first electrode part and part of the second electrode part.
  • the insulating film covers part of the first working electrode, part of the first auxiliary electrode, part of the second working electrode and part of the second electrode part. Two auxiliary electrodes.
  • the invention also provides a preparation method for the above-mentioned electrochemical biosensor, which includes the following steps:
  • Lower substrate preparation First prepare the lower substrate that can support integrated electrodes;
  • Make an integrated electrode make a first electrode part composed of a mutually spaced first working electrode and a first auxiliary electrode, and a second electrode part composed of a mutually spaced second working electrode and a second auxiliary electrode on the lower substrate;
  • Paste the intermediate substrate Paste the intermediate substrate on the insulating film
  • Paste the upper substrate Paste the upper substrate on the middle substrate.
  • the sensing film contains an electron transfer mediator and an oxidase
  • the sensing film composition containing the electron transfer mediator and oxidase is applied to the first working electrode and the first auxiliary electrode, and heated and dried. , forming a sensing film
  • step S4 the insulating film uses an insulating material, and a mixture of the insulating material is printed on the lower substrate provided with the integrated electrode and the sensing film, and is heated and dried to cover the integrated electrode and the sensing film to form insulation. film.
  • the electron transfer mediator is selected from the group consisting of ferrocene, benzoquinone, benzoquinone derivatives, organic conductive salts, paraquat, hexaammine ruthenium trichloride, potassium ferricyanide, potassium ferrocyanide, Methylferrocene, ferrocene ion, ferrocenecarboxylic acid, 7,7,8,8,-tetracyanoquinodimethane, nickelrocene, ammonium N-methylate, tetrathiotetraene, tetrasulfide Fulvalene, N-methylphenazine, hydroquinone, 3-dimethylaminobenzoic acid, 3-methyl-2-benzothiazolinonehydrazone, 2-methoxy-4-allyl Phenol, 4-aminoantipyrine, dimethylaniline, 4-aminothipyrene, 4-methoxynaphthol, 3,3',5,5'-tetramethyl
  • the oxidase is selected from the group consisting of glucose oxidase, lactate oxidase, cholesterol oxidase, glutamate oxidase, horseradish peroxidase, alcohol oxidase, glucose dehydrogenase, cholesterol esterase, ascorbic acid oxidase, alcohol dehydrogenase. At least one of hydrogenase and bilirubin oxidase.
  • the electrochemical biosensor of the present invention arranges an insulating film on the sensing film. Only the part of the sensing film corresponding to the fine flow channel is exposed, and the rest is covered by the insulating film. Regardless of the shape of the sensing film, the fine flow channel is The exposed sensing membrane area remains consistent, and the sensing membrane area in contact with the blood sample when the blood sample flows in through the fine channel is always consistent. In addition, the reaction amount between the blood sample and the enzyme in the sensing membrane remains constant, thus continuously ensuring the accuracy and precision of the measurement.
  • the electrochemical biosensor of the present invention transversely prints the fine flow channel between the first working electrode and the first auxiliary electrode to form a barrier film to prevent moisture from easily penetrating, thereby limiting the reaction between moisture and the sensing film, preventing measurement accuracy and The precision decreases, so that the electrochemical biosensor can maintain moisture resistance stability in high humidity environments;
  • the electrochemical biosensor of the present invention only needs to use a small amount of blood sample, does not require pre-processing of the blood sample, and can be introduced quickly and stably.
  • the micro-volume blood sample collected by the user can quickly output accurate blood glucose measurement results within a few seconds, and Reproducibility is good;
  • the present invention helps to avoid the defective phenomenon of applying the sensing film during the production process of general electrochemical biosensors, thereby helping to eliminate the detection process of confirming the defective sensing film, thereby shortening the production time and increasing the output.
  • Figure 1 is a process diagram for the preparation of electrochemical biosensors in the prior art
  • Figure 2 is a partial block diagram of a prior art electrochemical biosensor
  • FIG. 3 shows various forms of sensing films in electrochemical biosensors in the prior art
  • Figure 4 is an exploded view of the electrochemical biosensor in Embodiment 1 of the present invention.
  • Figure 5 is a perspective view of the electrochemical biosensor according to Embodiment 1 of the present invention.
  • Figure 6 is a perspective view of the electrochemical biosensor in Embodiment 1 of the present invention from another angle;
  • FIG. 7 is a partial block diagram of the electrochemical biosensor according to Embodiment 1 of the present invention.
  • Figure 8 is a preparation process diagram of the electrochemical biosensor in Embodiment 1 of the present invention.
  • Figure 9 is an exploded view of the electrochemical biosensor of Embodiment 2 of the present invention.
  • FIG. 10 is a partial block diagram of the electrochemical biosensor in Embodiment 2 of the present invention.
  • Figure 11 shows various forms of sensing films in the electrochemical biosensor according to Embodiment 2 of the present invention.
  • Figure 12 is a consensus error grid (Consensus Error Grid) curve diagram of the results of modulated blood measurement by the electrochemical biosensor in Embodiment 2 of the present invention.
  • Figure 13 is an accuracy curve chart of the electrochemical biosensor measuring blood modulation results in Embodiment 2 of the present invention.
  • FIG. 1 to 3 The marks in Figures 1 to 3 are: 10: integrated electrode, 11: first working electrode, 12: first auxiliary electrode, 13: second working electrode, 14: second auxiliary electrode, 20: insulating film, 30: induction membrane.
  • the marks in Figures 4 to 11 are: 100, 200: electrochemical biosensor, 110: lower substrate, 120: integrated electrode, 121: first electrode part, 122: first working electrode, 123: first auxiliary electrode, 125: third Two electrode parts, 126: second working electrode, 127: second auxiliary electrode, 129: connection electrode, 130: auxiliary electrode body, 131: biosensor confirmation electrode, 133: production barcode information confirmation electrode, 140: sensing film, 150,210 : Insulating film, 151, 211: Insulating film opening, 160: Intermediate substrate, 161: Fine flow channel, 170: Upper substrate, 171: Confirmation window, 172: Air hole, 212: Barrier film, 213: Blocking film.
  • the electrochemical biosensor 100 of this embodiment includes a lower substrate 110 and an upper substrate 110 .
  • the integrated electrode 120 is provided, the sensing film 140 is provided on the integrated electrode 120, the insulating film 150 is provided on the lower substrate 110 to cover part of the integrated electrode 120 and part of the sensing film 140, and the intermediate substrate 160 is pasted on the insulating film 150.
  • the upper substrate 170 is pasted on the middle substrate 160 .
  • the upper substrate 170 is used in conjunction with the lower substrate 110.
  • the upper substrate 170 covers the integrated electrode 120, the lower substrate 110 supports the integrated electrode 120, and the lower substrate 110 and the upper substrate 170 together protect the integrated electrode 120.
  • the shape and material of the lower substrate 110 There is no particular limitation.
  • a rectangular lower substrate 110 is preferred.
  • the intermediate substrate 160 is sandwiched between the lower substrate 110 and the upper substrate 170.
  • the intermediate substrate 160 is provided with a fine flow channel 161.
  • the electrochemical biosensor 100 allows the sample to flow into the integrated electrode 120 through the fine flow channel 161, and is utilized through an electrochemical reaction.
  • the current generated on the integrated electrode 120 measures the concentration of the analyte contained in the sample.
  • the electrochemical biosensor 100 is used to measure the blood sample, and this is used as an example for explanation.
  • the integrated electrode 120 includes a first working electrode 122 and a first auxiliary electrode 123 spaced apart from each other and a second working electrode 126 and a second auxiliary electrode 127 spaced apart from each other.
  • the first working electrode 122 and the first auxiliary electrode 123 form a first working electrode 122 and a first auxiliary electrode 123 .
  • the electrode part 121, the second working electrode 126 and the second auxiliary electrode 127 constitute the second electrode part 125, and the fine flow channel 161 is used to continuously introduce the sample into the first electrode part 121 and the second electrode part 125.
  • the first auxiliary electrode 123 and the second auxiliary electrode 127 are connected as a whole on the lower substrate 110 , that is, the first auxiliary electrode 123 and the second auxiliary electrode 127 are connected through the connecting electrode 129 to form the auxiliary electrode body 130 .
  • the end of the auxiliary electrode body 130 A biosensor confirmation electrode 131 is provided. If necessary, a production barcode information confirmation electrode 133 can also be provided on the lower substrate 110 .
  • the second electrode part 125 is arranged at the upstream end of the first electrode part 121.
  • the auxiliary electrodes 123 are arranged in sequence to form the first electrode part 121 and the second electrode part 125 .
  • the materials of the first working electrode 122, the first auxiliary electrode 123, the second working electrode 126, and the second auxiliary electrode 127 that make up the integrated electrode 120 are conductive substances and are not particularly limited, and their manufacturing methods are also not particularly limited.
  • the potential difference between the first working electrode 122 and the first auxiliary electrode 123 constituting the first electrode part 121 can be induced by applying direct current, low frequency or high frequency AC, high impedance or various forms of impulse. Furthermore, changes in the electrical signal of the first electrode part 121 may be caused by changes in voltage, current, impedance, capacitance, etc., and the blood glucose concentration in the blood sample can be measured through the above changes in the electrical signal of the first electrode part 121
  • the potential difference between the second working electrode 126 and the second auxiliary electrode 127 constituting the second electrode part 125 can be induced by applying low-frequency or high-frequency alternating current, high impedance, or various forms of impulses. Furthermore, changes in the electrical signal of the second electrode part 125 may be caused by changes in voltage, current, impedance, capacitance, etc., and the hematocrit in the blood sample is measured through the changes in the electrical signal of the second electrode part 125 .
  • the sensing film 140 contains electron transfer mediators and oxidases.
  • the sensing film 140 is the integrated electrode 120 printed on the lower substrate 110
  • the sensing film 140 is then coated on the integrated electrode 120 to make the sensing film 140 exposed relative to the fine flow channel 161 , and the remaining portion is covered by the insulating film 150 .
  • the sensing film 140 is disposed on the first working electrode 122 and the first auxiliary electrode 123, and is made by applying a sensing film composition containing an electron transfer mediator and an oxidase to the first working electrode 122 and the first auxiliary electrode 123. on and made by heating and drying.
  • the sensing film 140 may also contain an appropriate concentration of potassium ferricyanide (III) and an appropriate concentration of water-soluble polymer.
  • the sensing film 140 and the blood sample are mixed and dissolved to perform a thin-layer electrochemical cell reaction.
  • the oxidase reacts with the glucose in the blood sample and is reduced.
  • a potential difference occurs in the first electrode part 121, and the blood glucose concentration in the blood sample is measured by measuring the current of the first electrode part 121.
  • the electron transfer mediator in the sensing film 140 reacts with the enzyme that reduces glucose in the blood to perform a redox reaction and is reduced.
  • the reduced electron transfer mediator diffuses to the electrode surface to generate an oxidation potential on the electrode surface to form a current.
  • the electron transfer mediator reacts with metabolic substances and is reduced after an oxidation-reduction reaction with the reduced enzyme. When the reduced electron transfer mediator diffuses to the electrode surface, it will apply an oxidation potential to the electrode surface to form a current.
  • the electron transfer mediator can also be hexaammineruthenium(III)chloride, potassium ferricyanide, potassium ferrocyanide, dimethylferrocene ( dimethylferrocene (DMF)), ferricinium, ferocene monocarboxylic acid (FCOOH), 7,7,8,8-tetracyanoquinodimethane (7,7,8,8 -tetracyanoquino-dimethane(TCNQ)), nickelocene(Nc)), N-methyl acidinium(NMA + ), tetrathiatetracene(TTT), tetrathiaphenyl Tetrathia fulvalene (TTF), N-methylphenazinium (NMP + ), hydroquinone, 3-dimethylaminobenzoic acid (MB
  • the sensing film 140 may also be mixed with polyvinylpyrrolidone (polyvinyl pyrrolidone; PVP), polyvinyl alcohol (PVA), perfluoro sulfonate (perfluoro sulfonate), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl cellulose (HPC), At least one of macromolecules such as methyl cellulose (carboxy methyl cellulose; CMC), cellulose acetate (cellulose acetate) or polyamide (polyamide).
  • PVP polyvinyl pyrrolidone
  • PVA polyvinyl alcohol
  • HEC hydroxyethyl cellulose
  • HPC hydroxypropyl cellulose
  • HPC hydroxypropyl cellulose
  • HPC hydroxypropyl cellulose
  • HPC hydroxypropyl cellulose
  • At least one of macromolecules such as methyl cellulose (carboxy methyl cellulose; CMC), cellulose a
  • the insulating film 150 is provided with an insulating film opening 151 that is open from the inside to the outside.
  • the insulating film opening 151 corresponds to the fine flow channel 161 of the intermediate substrate 160.
  • the insulating film 150 covers part of the first working electrode 122, Part of the first auxiliary electrode 123 , part of the second working electrode 126 and part of the second auxiliary electrode 127 have the insulating film 150 covering part of the first electrode part 121 and part of the second electrode part 125 , which helps to limit the flow from the fine flow channel 161
  • the inflowing blood sample reaches the area of the first electrode part 121 and the second electrode part 125 covered by the insulating film 150, thereby preventing the first working electrode 122 and the second working electrode 126 from being connected in the fine flow channel 161 area, and also preventing The first auxiliary electrode 123 and the second auxiliary electrode 127 are connected in the fine flow channel 161 area.
  • the insulating film 150 is an insulating mixture with an insulating material printed on the lower substrate 110 provided with the integrated electrode 120 and the sensing film 140, and is heated and dried to cover the integrated electrode 120 and the sensing film 140. Since the insulating film 150 is printed on the sensing film 140, only the part of the sensing film 140 corresponding to the fine flow channel 161 is exposed, and the rest is covered by the insulating film 150. Therefore, regardless of the shape or size of the sensing film 140 applied on the integrated electrode 120, the blood sample When flowing in through the fine flow channel 161, the area of the sensing film 140 in contact with the blood sample remains consistent.
  • the middle substrate 160 is sandwiched between the lower substrate 110 and the upper substrate 170 .
  • the middle substrate 160 can be made of double-sided tape with adhesive force on both sides for pasting the lower substrate 110 and the upper substrate 170 .
  • the intermediate substrate 160 has a fine flow channel 161 that allows the blood sample to flow into the first electrode part 121 and the second electrode part 125.
  • the fine flow channel 161 is open from the inside to the outside on the intermediate substrate 160, and the blood sample flows into the fine flow channel 161. Can be retained.
  • the fine flow channel 161 can accommodate microliter unit-level blood samples.
  • the internal volume of the fine flow channel 161 can be limited to less than 0.7 ⁇ L.
  • the volume of the fine flow channel 161 is less than 0.3 ⁇ L, it will affect the error range of the electrochemical biosensor. The measurement accuracy cannot be ensured.
  • the volume of the fine flow channel 161 is greater than 1.0 ⁇ L, a relatively large number of user blood samples need to be collected, which is also undesirable.
  • the upper substrate 170 corresponds to the lower substrate 110, and both can be made of the same material.
  • the upper substrate 170 is stacked on the lower substrate 110 and covers the first electrode part 121 and the second electrode part 125.
  • the upper substrate 170 is provided with a corresponding fine flow channel 161.
  • the confirmation window 171 can be made of transparent or translucent material so that the fine flow channel 161 can be confirmed from the outside. This product can confirm the filling status of the blood sample flowing into the fine flow channel 161 through the confirmation window 171.
  • the confirmation window 171 is also provided with an air hole 172. When the blood sample flows into the fine flow channel 161, the air in the fine flow channel 161 can be discharged through the air hole 172.
  • the arrangement of the air holes 172 facilitates the blood sample to flow into the fine flow channel 161 more smoothly through the siphon phenomenon.
  • the distance between the first working electrode 122 and the first auxiliary electrode 123 is within several hundred ⁇ m to form a thin-layer electrochemical cell. (thin layer electrochemical cell) structure, after the blood sample flows into the fine channel 161, the analytes in the collected sample are formed by enzymes and electron transfer mediators to form a continuous cycle of oxidation/reduction reaction effects, and it takes several seconds for the current to reach a stable state ( steady state), in order to meet the above conditions, the sensing film 140 needs to quickly dissolve in the sample flowing into the fine flow channel 161.
  • the produced electrochemical biosensor 100 can reach a steady state within a few seconds.
  • the insulating film 150 is disposed on the sensing film 140. Only the portion of the sensing film 140 corresponding to the fine flow channel 161 is exposed, and the remaining portions are covered by the insulating film 150.
  • the sensing film, 140 shape and size the area of the exposed sensing film 140 in the fine flow channel 161 remains consistent, and the area of the sensing membrane 140 that contacts the blood sample when the blood sample flows in through the fine flow channel 161 remains consistent, plus the amount of reaction between the blood sample and the enzyme in the sensing membrane 140 Maintain constant, thereby continuously ensuring the accuracy and precision of measurement.
  • this embodiment can also eliminate the detection process that has traditionally been used to confirm defects in the sensing film 140, thereby helping to shorten production time and increase output.
  • the electrochemical biosensor 100 of this embodiment can introduce specific enzymes and appropriate electron transfer mediators based on the same principle as blood glucose monitoring, and can thereby measure a variety of metabolic substances, such as: cholesterol, lactic acid, creatinine, protein, hydrogen peroxide, Concentrations of various organic or inorganic substances in living specimens, environmental specimens, agricultural specimens, industrial specimens or food specimens such as alcohol, amino acids, GPT (glutamate pyruvate transaminase), GOT (glutamate oxaloacetate transmianse) and other enzymes.
  • metabolic substances such as: cholesterol, lactic acid, creatinine, protein, hydrogen peroxide, Concentrations of various organic or inorganic substances in living specimens, environmental specimens, agricultural specimens, industrial specimens or food specimens such as alcohol, amino acids, GPT (glutamate pyruvate transaminase), GOT (glutamate oxaloacetate transmianse) and other enzymes.
  • the electrochemical biosensor 100 can quantitatively measure a variety of metabolic substances by adjusting the types of enzymes contained in the sensing film 140, for example, using glucose oxidase, lactate oxidase, cholesterol oxidase, glutamate oxidase, horseradish ( Horseradish) peroxidase and alcohol oxidase quantitatively measure cholesterol, lactate, glutamate, hydrogen peroxide and alcohol.
  • glucose dehydrogenase (glucose dehydrogenase; GDH), glucose oxidase (glucose oxidase; GOx), cholesterol oxidase, cholesterol esterase, lactate oxidase, ascorbic acid oxidase (ascorbic acid oxidase), alcohol oxidase,
  • GDH glucose dehydrogenase
  • Gx glucose oxidase
  • cholesterol oxidase cholesterol esterase
  • lactate oxidase ascorbic acid oxidase (ascorbic acid oxidase)
  • alcohol oxidase The selected oxidase from the group consisting of alcohol dehydrogenase and bilirubin oxidase can also be placed in the sensing membrane 140.
  • the preparation process of the electrochemical biosensor 100 of this embodiment is shown in Figure 8.
  • the preparation process includes S1, preparing the lower substrate; S2, making the integrated electrode; S3, making the sensing film; S4, making the insulating film; S5, pasting the intermediate substrate ;S6. Paste the upper substrate.
  • step S1 the lower substrate 110 that can support the integrated electrode 120 is first prepared.
  • a first electrode part 121 consisting of a first working electrode 122 and a first auxiliary electrode 123 spaced apart from each other, and a third electrode part 121 consisting of a spaced apart second working electrode 126 and a second auxiliary electrode 127 are formed on the lower substrate 110.
  • the two electrode parts 125 in which the first auxiliary electrode 123 and the second auxiliary electrode 127 are connected on the lower substrate 110, are more advantageous than the first auxiliary electrode 123 and the second auxiliary electrode 127 being produced independently; the integrated electrode 120 can be made in various ways. , for example, the method of screen printing the conductive mixture with conductive substances and then curing it, the method of pasting the charged film, the conductive Substance precipitation methods, etc.
  • a sensing film 140 containing an electron transfer mediator and an oxidase is produced on the first working electrode 122 and the first auxiliary electrode 123.
  • the specific production process is to combine the composition of the sensing film containing the electron transfer mediator and the oxidase. Apply on the first working electrode 122 and the first auxiliary electrode 123, and form the sensing film 140 by heating and drying.
  • the heating temperature and heating time of the dry sensing film composition may vary depending on the types of electron transfer mediators and oxidases contained in the sensing film 140. Make adjustments.
  • an insulating film 150 is formed on the lower substrate 110 provided with the integrated electrode 120 and the sensing film 140.
  • the insulating film 150 covers part of the first working electrode 122, part of the first auxiliary electrode 123, and part of the second working electrode 126. and part of the second auxiliary electrode 127, and restrict the inflowing sample from reaching the area of the first electrode part 121 and the second electrode part 125 covered by the insulating film 150, and the insulating film 150 excludes the corresponding fine flow channel 161 in the sensing film 140.
  • the part of the sensing film 140 corresponding to the fine flow channel 161 is exposed in the fine flow channel 161;
  • the insulating film 150 is made of insulating material.
  • the specific preparation process is as follows: printing a mixture of insulating materials on the lower substrate 110 provided with the integrated electrode 120 and the sensing film 140, and heating and drying to cover the integrated electrode 120 and the sensing film. 140.
  • the heating and drying temperature of the insulating film 150 should be selected at an appropriate temperature that does not damage the oxidase in the sensing film 140.
  • step S5 in order to form the fine flow channel 161 through which the first electrode part 121 and the second electrode part 125 flow into the blood sample, the intermediate substrate 160 is pasted on the insulating film 150.
  • the intermediate substrate 160 matches the lower substrate 110, and double-sided can be used. Adhesive double-sided tape material;
  • step S6 the upper substrate 170 is pasted on the middle substrate 160.
  • the upper substrate 170 is used in conjunction with the lower substrate 110.
  • the upper substrate 170 can arrange the first electrode part 121 and the first electrode part 121 in the fine flow channel 161 where a certain amount of blood sample flows.
  • the two electrode parts 125 are formed to complete the electrochemical biosensor 100.
  • an electrochemical biosensor 200 includes a lower substrate 110, an integrated electrode 120 provided on the lower substrate 110, a sensing film 140 provided on the integrated electrode 120, a covering part of the integrated electrode 120 and Part of the sensing film 140 is provided on the insulating film 210 on the lower substrate 110, the intermediate substrate 160 pasted on the insulating film 210, and the upper substrate 170 pasted on the intermediate substrate 160.
  • the insulating film 210 of this embodiment is the same as the insulating film of Embodiment 1.
  • 150, the rest of the structure is the same as Embodiment 1.
  • the insulating film 210 is disposed on the lower substrate 110.
  • the insulating film 210 covers part of the first working electrode 122, part of the first auxiliary electrode 123, part of the second working electrode 126 and part of the second auxiliary electrode 127.
  • the insulating film 210 is provided with a The insulating film opening 211 is open from the inside out, and the insulating film opening 211 corresponds to the fine flow channel 161 of the intermediate substrate 160 .
  • the insulating film 210 of this embodiment also includes a barrier film 212 and a blocking film 213, which are arranged in sequence along the sample inflow direction in the fine flow channel 161 in the order of the blocking film 213 and the barrier film 212.
  • the number of barrier films 212 is one.
  • the barrier film 212 is disposed transversely on the opening 211 of the insulating film.
  • the barrier film 212 is transversely inserted between the first working electrode 122 and the first auxiliary electrode 123.
  • the barrier film 212 prevents The moisture in the outside air easily penetrates into the sensing film 140. If the moisture penetrates into the sensing film 140 through the fine flow channel 161, the sensing film 140 will react with the moisture, resulting in a reduction in the accuracy and precision of blood sample measurement.
  • the arrangement of the barrier film 212 can inhibit moisture from penetrating into the sensing film 140, thereby effectively preventing the above problems from occurring.
  • the barrier film 212 can also be inserted horizontally between the air holes 172 and the first auxiliary electrode 123 to prevent moisture in the external air entering through the air holes 172 from easily penetrating into the sensing film 140.
  • the number of barrier films 212 is 2 or more, corresponding barrier films 212 can be provided between the air holes 172 and the first auxiliary electrode 123 and between the first working electrode 122 and the first auxiliary electrode 123 .
  • the number of blocking films 213 is one.
  • the blocking film 213 is disposed transversely on the opening 211 of the insulating film.
  • the blocking film 213 is located between the first electrode part 121 and the second electrode part 125 .
  • the blocking film 213 can suppress the induction film. 140The flow of dissolved substances that react with the sample. If the solution dissolved by the reaction between the sensing film 140 and the blood sample does not flow to the second electrode part 125, it is beneficial to the measurement to stay at the first electrode part 121.
  • the blocking film 213 in this embodiment is between the first electrode part 121 and the second electrode part 125 and plays a role in preventing the dissolved matter that reacts between the sensing film 140 and the blood sample from flowing to the second electrode part 125 easily.
  • the blocking film 213 The settings can also effectively improve measurement accuracy and precision.
  • the blocking film 213 of this embodiment also plays a role in inhibiting the penetration of moisture through the fine flow channel 161, which can reduce the measurement accuracy and precision of the electrochemical biosensor due to the influence of moisture in a high-humidity environment. Descent problem.
  • the blocking film 213 can also prevent the area of the sensing film 140 from being exposed in the fine flow channel 161 , that is, the blocking film 213 covers the portion of the sensing film 140 close to the second electrode part 125 , limiting the sensing close to the second electrode part 125 Reaction of membrane 140 with blood sample.
  • the barrier film 212 has an irregular smearing phenomenon on the sensing film 140 , and the sensing film 140 reacts with the blood sample to dissolve even near the second electrode portion 125 . material, it will also effectively prevent the dissolved matter from reaching the second electrode part 125. Furthermore, as shown in FIG. 11(c) , the barrier film 212 over-diffuses the sensing film 140 , and even if the sensing film 140 reacts with the blood sample and dissolves dissolved substances near the second electrode part 125 , the arrival of the dissolved substances will be effectively suppressed. second electrode part 125.
  • the preparation method of the electrochemical biosensor 200 of this embodiment is the same as that of Embodiment 1.
  • Example 1 Taking the electrochemical biosensor 100 of Example 1 as an example, specifically, a corresponding sensing film composition is prepared.
  • the preparation process is as follows: adding 10.0 g of potassium ferricyanide and hydroxyethyl fiber. A mixture of hydroxyethyl cellulose (HEC) 2.0g, Triton X-100 0.3g and glucose dehydrogenase 2.0g was dissolved in 100mL PBS buffer (pH 6.4, concentration 0.1mol/L), and mixed evenly , and then remove the particles in the solution to obtain the sensing membrane composition.
  • HEC hydroxyethyl cellulose
  • the structure of the electrochemical biosensor 100 is shown in Figures 4 to 7.
  • the detailed manufacturing process is: S1. First prepare the lower substrate 110 that can support the integrated electrode 120; S2. Use the carbon stir material as raw material and screen-print it. In a curing manner, the first working electrode 122, the second working electrode 126, the auxiliary electrode body 130 composed of the first auxiliary electrode 123 and the second auxiliary electrode 127, the biosensor confirmation electrode 131, and the production barcode information confirmation electrode are printed on the lower substrate. 133, and then dry in an environment of 130°C for 5 minutes; S3. Select the above-mentioned sensing film composition, apply the sensing film composition on the first working electrode 122 and the first auxiliary electrode 123, and then dry in an environment of 70°C for 5 minutes.
  • the middle substrate 160 is pasted on the insulating film 150; f.
  • the upper substrate 170 is pasted On the middle substrate 160, the upper substrate 170 is processed with air holes 172 and corresponding tester connector insertion parts, thereby completing the production of the electrochemical biosensor 100.
  • Comparative Example 1 The electrochemical biosensor of Comparative Example 1 was prepared according to the above production process. The only difference between Comparative Example 1 and the electrochemical biosensor 100 of Example 1 is that: Comparative Example 1 first made an insulating film and then made a sensing film, and then implemented Example 1: Make the sensing film first and then the insulating film.
  • Example 1 The measurement precision of the electrochemical biosensors of Example 1 and Comparative Example 1 was compared.
  • venous blood was collected from blood collection tubes containing heparin and centrifuged to separate plasma and blood cells.
  • the separated plasma and blood cells were mixed to prepare prepared blood with a hematocrit of 42% and a blood glucose concentration as shown in Table 1.
  • Five blood glucose testers were used. Measure 5 times each.
  • the electrochemical biosensors of Example 1 and Comparative Example 1 were respectively used for measurement. The specific results are shown in Table 2. The average CV% of the measurement results of the electrochemical biosensor of Example 1 was 2.85. On the contrary, the electrochemical biosensor of Comparative Example 1 The average CV% of the biosensor measurements was 5.00. Based on the above results, it can be confirmed that the process of making the sensing film first and then the insulating film can help improve the measurement precision of the electrochemical biosensor.
  • Example 2 Preparing the electrochemical biosensor of Example 2 with reference to the preparation process of Example 1 in the above precision measurement.
  • the insulating film 210 of Embodiment 2 is provided with a barrier film 212 and a blocking film 213.
  • the barrier film 212 is inserted horizontally between the first working electrode 122 and the Between the first auxiliary electrodes 123, the blocking film 213 is located between the first electrode part 121 and the second electrode part 125.
  • the insulating film 150 of Embodiment 1 is not provided with a blocking film and a blocking film.
  • the electrodes of Embodiments 1 and 2 are The chemical and biological sensors were exposed to a temperature of 35°C and a humidity of 85% for performance testing and comparison.
  • the temperature of the constant temperature and humidity laboratory is adjusted to 35°C and the humidity is adjusted to 85%.
  • the tester and electrochemical biosensor are placed in the constant temperature and humidity laboratory for more than 30 minutes to stabilize.
  • the separated plasma and blood cells are mixed to prepare prepared blood with a hematocrit of 42% and a blood glucose concentration as shown in Table 3 below. Five units are used for testing.
  • the instrument measures at 30min, 60min, 90min, 120min, 150min and 180min respectively.
  • Example 2 The specific measurement results are shown in Table 4 below, compared with the measurement data of test strips not exposed to humidity at different corresponding times.
  • Example 1 there is no barrier film between the first working electrode 122 and the first auxiliary electrode 123.
  • the measurement result of the electrochemical biosensor 100 without blocking film between the electrode part 121 and the second electrode part 125 is more than 2 times higher than that of the electrochemical biosensor 200 with the blocking film 212 and the blocking film 213 printed, and the measurement accuracy is reduced. Based on the results, it can be determined that printing barrier films and blocking films can improve the accuracy of electrochemical biosensors.
  • the electrochemical biosensor 200 produced according to Embodiment 2 was tested for accuracy in modulating blood.
  • the centrifuged plasma and blood cells are mixed to adjust the hematocrit to 42% and the partial pressure of oxygen to adjust the capillary oxygen partial pressure to 60 to 80 mmHg.
  • the reference equipment YSI 2300STAT Plus was used to modulate and measure the concentrations listed in Table 5.
  • the electrochemical biosensor produced according to the embodiment was measured a total of 50 times.

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Abstract

一种电化学生物传感器（100,200）及其制备方法，其中传感器（100,200）包括下基板（110）、中间基板（160）和上基板（170），下基板（110）设有集成电极（120），集成电极（120）包含相互间隔的第一工作电极（122）和第一辅助电极（123）以及相互间隔的第二工作电极（126）和第二辅助电极（127）；中间基板（160）夹在下基板（110）和上基板（170）之间，中间基板（160）上设有微细流道（161）；下基板（110）和中间基板（160）之间还设有绝缘薄膜（150,210），绝缘薄膜（150,210）盖住部分集成电极（120）；下基板（110）和绝缘薄膜（150,210）之间裸露设置有感应膜（140），感应膜（140）相对于微细流道（161）的部分进行裸露，其余部分被绝缘薄膜（150,210）盖住，感应膜（140）设置在第一工作电极（122）和第一辅助电极（123）上，且绝缘薄膜（150,210）设有从里向外开放形态的绝缘薄膜开口部（151,211），绝缘薄膜开口部（151,211）与微细流道（161）相对应。该传感器有助于提高测量准确度及精密度，还能够降低水分渗透影响。

Description

电化学生物传感器及其制备方法 技术领域
本发明属于电化学生物传感器技术领域，具体涉及一种利用电化学方法更详细地分析样本中所含分析物质浓度的电化学生物传感器及其制备方法。
背景技术
诊断和预防糖尿病需要对血液葡萄糖水平进行周期性检测，而测量血液中葡萄糖水平对糖尿病患者调节糖吸收量非常重要，尤其对早期发现糖尿病患者及治疗过程是必要的。
血糖可通过试纸形态的生物传感器轻松测量，测量血糖的生物传感器的工作原理以比色法或电化学方法为基础。比色法是用指示剂邻甲苯胺和联苯胺混合物，通过观察反应后的指示剂颜色变化测量血糖浓度，但是此方法的弊端是测量准确度不足。电化学的方法相比比色法可提高测量血糖浓度的准确度，缩短测量时间，方便测量，近几年得到了广泛应用。
利用电化学方法测量血糖的生物传感器最大的特点就是使用电子转移介体，一般用于电子转移介体的有二茂铁、二茂铁衍生物、醌类、醌类衍生物、含过渡金属的有机或无机化合物(三氯化六铵合钌、含锇高分子、铁氰化钾等)，有机导电盐(organic conducting salt)，紫罗碱(viologen)等。电化学生物传感器基于如下反应：
(1)葡萄糖+GOx-FAD→葡萄糖酸+GOx-FADH2
(2)GOx-FADH2+电子转移介体(氧化)→GOx-FAD+电子转移介体(还原)
上式中，GOx表示葡萄糖氧化酶(Glucose oxidase)；GOx-FAD和GOx-FADH2分别表示氧化和还原态的关联葡萄糖氧化酶的活性部位FAD(flavin adenine dinucleotide)。反应式(1)：血液内葡萄糖先与葡萄糖氧化酶催化作用下被氧化成葡萄糖酸，这时葡萄糖氧化酶活性部位的FAD还原成FADH2；反应式(2)：还原后的FADH2与电子转移介体通过氧化还原反应FADH2氧化为FAD，电子转移介体被还原。反应生成的还原态电子转移介体扩散至电极表面，通过工作电极施加电极表面还原态电子转移介体氧化的电势，测量此时产生的电流来测量血糖浓度。
如图1和图2所示，一般电化学生物传感器采用绝缘下基板(S11)，下基板上由集成电极10和绝缘薄膜20及感应膜30形成，之后与中间基板及上基板依次粘贴方式制作而成。在此，集成电极10包含第一工作电极11、第一辅助电极12、第二工作电极13、第二辅助电极14，感应膜30包含氧化酶及电极转移介体。在制备方法中，制作集成电极(S12)步 骤是将带导电性搅拌物采用网版印刷在下基板上，加热使其硬化而成；制作绝缘薄膜(S13)步骤是把绝缘浆料丝网印刷在完成集成电极10的下基板上后加热使其硬化而成；制作感应膜(S14)步骤是把感应膜组成物质涂抹在绝缘薄膜20后干燥后形成；中间基板粘贴步骤(S15)是将把带微细通道的双面胶结构的中间基板(未图示)为盖住绝缘薄膜(20)而粘贴在下基板；上基板粘贴步骤(S16)是将把带有气孔的上基板(未图示)粘贴在中间基板上面。
此类电化学生物传感器的测量准确度及精密度受感应膜30的均匀程度影响，再有，感应膜30暴露于水分当中不仅影响准确度及精密度，而且也会发生工作异常。但是，历来生物传感器制作绝缘薄膜20后形成感应膜30的过程中，频繁发生感应膜组成物无法均匀涂抹在绝缘薄膜的问题。如图3(a)及(b)所示，感应膜组成物涂抹在绝缘薄膜20过程中感应膜组成物不规则的向一侧偏移，或者如图3(c)所示容易过度往外扩散。如此在感应膜组成物不均匀涂抹状态下形成的感应膜30，通过上下基板之间微细流道吸入的血液样品接触时，血液样品与感应膜30接触面积或形态大小会出现差异，这时血液样品与感应膜30内氧化酶之间的反应量会不同，进而导致测量准确度及精密度下降。
此外，感应膜30不均匀还导致感应膜30与血样溶解过程中容易流动至第二工作电极13。感应膜30内氧化酶与电子转移介体之间的电化学反应是基于在第一工作电极11与第一辅助电极之间的电子流动才能用于血糖浓度的测量，氧化酶与电子转移介体等组成物多在第二工作电极13上流动则影响第二工作电极13测量，随之影响准确度及精密度。当电化学生物传感器的感应膜制作完成后，为剔除感应膜不良需通过检查工艺流程，则会导致生产时间的增加。再有，历来生物传感器在高温高湿环境下，水分容易从下基板与上基板之间的微细流道渗透到感应膜，易进行反应，也会导致影响测量准确度及精密度。
发明内容
针对上述存在的技术问题，本发明提供了一种电化学生物传感器及其制备方法，能够保证感应膜与测量样本之间的接触面积保持均匀，进而提高测量准确度及精密度，还能够降低水分渗透影响，即使在高湿度环境下也能维持良好的测量准确度。
本发明的技术方案为：
本发明提供了一种电化学生物传感器，包括下基板、中间基板和上基板，所述下基板设有集成电极，所述集成电极包含相互间隔的第一工作电极和第一辅助电极以及相互间隔的第二工作电极和第二辅助电极，所述第一工作电极和所述第一辅助电极组成第一电极部，所述第二工作电极和所述第二辅助电极组成第二电极部；
所述中间基板夹在所述下基板和所述上基板之间，所述中间基板上设有微细流道，所述微细流道用于将样本连续导入第一电极部和第二电极部中；
所述上基板和所述中间基板之间还设有绝缘薄膜，所述绝缘薄膜盖住部分所述集成电极，同时限制流入的样本到达所述第一电极部及所述第二电极部的区域；
所述下基板和所述绝缘薄膜之间裸露设置有感应膜，所述感应膜相对于所述微细流道的部分进行裸露，其余部分被所述绝缘薄膜盖住，所述感应膜设置在所述第一工作电极和所述第一辅助电极上，且所述绝缘薄膜设有从里向外开放形态的绝缘薄膜开口部，所述绝缘薄膜开口部与所述微细流道相对应。
优选地，所述上基板设有与所述细微流道对应的确认窗，所述确认窗设有气孔。
优选地，所述绝缘薄膜还包括至少一个阻隔膜，所述阻隔膜横向设于所述绝缘薄膜开口部，且所述阻隔膜位于所述第一工作电极和所述第一辅助电极之间和/或所述气孔和所述第一辅助电极之间。
优选地，所述绝缘薄膜还包括至少一个阻塞膜，所述阻塞膜横向设于所述绝缘薄膜开口部，且所述阻塞膜位于所述第一电极部和所述第二电极部之间，该阻塞膜能够抑制感应膜与样本反应溶解的溶解物的流动。
优选地，沿着所述微细流道内样本流入方向，按照所述阻塞膜、所述阻隔膜的顺序进行依次排列。
优选地，沿着所述微细流道内样本流入方向，按照所述第二辅助电极、所述第二工作电极、所述第一工作电极和所述第一辅助电极的顺序进行依次排列。
优选地，所述第一辅助电极和所述第二辅助电极通过连接电极相连形成辅助电极体；所述辅助电极体的末端设有生物传感器确认电极。
优选地，所述下基板设有生产条码信息确认电极。
优选地，所述微细流道的容积为0.3-1.0μL，优选0.3-0.7μL。
优选地，所述绝缘薄膜盖住部分第一电极部和部分第二电极部，优选地，所述绝缘薄膜盖住部分第一工作电极、部分第一辅助电极、部分第二工作电极和部分第二辅助电极。
本发明还提供了上述电化学生物传感器的制备方法，包括如下步骤：
S1、下基板准备：先准备可支持集成电极的下基板；
S2、制作集成电极：在下基板上制作相互间隔的第一工作电极及第一辅助电极组成的第一电极部，以及相互间隔的第二工作电极及第二辅助电极组成的第二电极部；
S3、制作感应膜：在第一工作电极及第一辅助电极上制作感应膜；
S4、制作绝缘薄膜：在设有集成电极和感应膜的下基板上制作绝缘薄膜；
S5、中间基板粘贴：把中间基板粘贴在绝缘薄膜上；
S6、上基板粘贴：将上基板粘贴在中间基板上。
优选地，在步骤S3中，所述感应膜含有电子转移介体和氧化酶，将含有电子转移介体和氧化酶的感应膜组成物涂抹在第一工作电极及第一辅助电极上，加热干燥，形成感应膜；
在步骤S4中，所述绝缘薄膜采用绝缘性材料，将绝缘性材料组成的搅拌物印刷在设有集成电极和感应膜的下基板，并加热干燥使其盖住集成电极和感应膜，形成绝缘薄膜。
优选地，所述电子转移介体选自二茂铁、苯醌、苯醌衍生物、有机导电盐、百草枯、三氯化六氨合钌、铁氰化钾、亚铁氰化钾、二甲基二茂铁、二茂铁离子、二茂铁甲酸、7,7,8,8,-四氰基醌二甲烷、二茂镍、N-甲基酸铵、四硫四烯、四硫富瓦烯、N-甲基吩嗪、对苯二酚、3-二甲基氨基安息香酸、3-甲基-2-苯并噻唑啉酮腙、2-甲氧基-4-烯丙基苯酚、4-氨基安替比林、二甲基苯胺、4-氨基噻芘、4-甲氧基萘酚、3,3',5,5'-四甲基联苯胺、2,2'-联氮-双-3-乙基苯并噻唑啉-磺酸、邻联茴香胺、邻甲苯胺、2,4二氯苯酚、4-氨基安替比林、联苯胺及普鲁士蓝中至少一种；
所述氧化酶选自葡萄糖氧化酶、乳酸氧化酶、胆固醇氧化酶、谷氨酸氧化酶、辣根过氧化物酶、酒精氧化酶、葡萄糖脱氢酶、胆固醇酯酶、抗坏血酸氧化酶、酒精脱氢酶、胆红素氧化酶中至少一种。
本发明的有益效果是：
(1)本发明的电化学生物传感器将绝缘薄膜设置在感应膜之上，感应膜只裸露与微细流道对应部分，其余部分都被绝缘薄膜盖住，进而不管感应膜形态大小，微细流道中裸露的感应膜区域保持一致，血样通过微细流道流入时与血样接触的感应膜面积始终保持一致，加上血样与感应膜内酶的反应量维持恒定，进而可持续保证测量的准确度及精密度；
(2)本发明的电化学生物传感器把第一工作电极和第一辅助电极之间的微细流道横向印刷形成阻隔膜使水分不容易渗透，进而限制水分与感应膜反应，避免测量准确度及精密度下降，从而使得该电化学生物传感器在高湿环境也能保持抗湿稳定性；
(3)本发明的电化学生物传感器只需利用少量血样，无需对血样进行预处理，并能够快速、稳定导入，使用者采集的微量血样在数秒内就能快速输出准确的血糖测量结果，且再现性较好；
(4)本发明有助于避免一般电化学生物传感器生产过程中涂抹感应膜的不良现象，从而有助于去除确认感应膜不良的检测工艺流程，进而缩短生产时间、提高产量。
附图说明
下面结合附图及实施例对本发明作进一步描述：
图1是现有技术的电化学生物传感器制备工艺图；
图2是现有技术的电化学生物传感器的部分框图；
图3是现有技术的电化学生物传感器中感应膜的多种形态；
图4是本发明实施例1的电化学生物传感器分解图；
图5是本发明实施例1的电化学生物传感器的立体图；
图6是本发明实施例1的电化学生物传感器另一角度的立体图；
图7是本发明实施例1的电化学生物传感器的部分框图；
图8是本发明实施例1的电化学生物传感器制备工艺图；
图9是本发明实施例2的电化学生物传感器的分解图；
图10是本发明实施例2的电化学生物传感器的部分框图；
图11是本发明实施例2的电化学生物传感器中感应膜的多种形态；
图12是本发明实施例2的电化学生物传感器测量调制血结果的共识误差网络(Consensus Error Grid)曲线图；
图13是本发明实施例2的电化学生物传感器测量调制血结果的准确性(Accuracy)曲线图。
图1至3中的标记为：10：集成电极、11：第一工作电极、12：第一辅助电极、13：第二工作电极、14：第二辅助电极、20：绝缘薄膜、30：感应膜。
图4至11中的标记为：100,200:电化学生物传感器、110:下基板、120:集成电极、121:第一电极部、122:第一工作电极、123:第一辅助电极、125:第二电极部、126:第二工作电极、127:第二辅助电极、129:连接电极、130:辅助电极体、131:生物传感器确认电极、133：生产条码信息确认电极、140:感应膜、150,210:绝缘薄膜、151,211:绝缘薄膜开口部、160:中间基板、161:微细流道、170:上基板、171:确认窗、172:气孔、212:阻隔膜、213:阻塞膜。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
实施例1
如图4至图7所示，本实施例的电化学生物传感器100包括下基板110、下基板110上 设有的集成电极120、集成电极120上设有的感应膜140、盖住部分集成电极120和部分感应膜140而设置在下基板110上的绝缘薄膜150、粘贴在绝缘薄膜150上的中间基板160、中间基板160上面粘贴的上基板170。上基板170与下基板110配套使用，上基板170盖住集成电极120，下基板110支撑集成电极120，且下基板110与上基板170一起对集成电极120进行保护，下基板110的形状及材质没有特别限制，本实施例优选长方形的下基板110。中间基板160夹在下基板110和上基板170之间，中间基板160上设有微细流道161，此电化学生物传感器100通过微细流道161使样本流入到集成电极120上，通过电化学反应利用集成电极120上产生的电流测量样本中含有的待分析物浓度，本实施例通过电化学生物传感器100测量血液样本，并以此为例进行说明。
其中，集成电极120包含相互间隔的第一工作电极122和第一辅助电极123以及相互间隔的第二工作电极126和第二辅助电极127，第一工作电极122和第一辅助电极123组成第一电极部121，第二工作电极126和第二辅助电极127组成第二电极部125，微细流道161用于将样本连续导入第一电极部121和第二电极部125中。第一辅助电极123和第二辅助电极127在下基板110上连接为一体，即第一辅助电极123和第二辅助电极127通过连接电极129相连形成辅助电极体130，此外，辅助电极体130的末端设有生物传感器确认电极131，根据需要，下基板110上还可以设置有生产条码信息确认电极133。
沿着微细流道161内样本流入方向，第二电极部125排在第一电极部121的上流端，具体地，按照第二辅助电极127、第二工作电极126、第一工作电极122和第一辅助电极123的顺序进行依次排列，并形成第一电极部121和第二电极部125。组成集成电极120的第一工作电极122、第一辅助电极123、第二工作电极126、第二辅助电极127的材料为导电性物质，无特别限制，其制作方法也不特别限制。
组成第一电极部121的第一工作电极122及第一辅助电极123之间的电位差可选用施加直流、低频过高频交流、高阻抗或多种形态的迈冲来引发。再有，第一电极部121的电信号变化有可能是电压、电流、阻抗、电容等变化引起的，可以通过第一电极部121的上述电信号变化测量血样中的血糖浓度
组成第二电极部125的第二工作电极126和第二辅助电极127之间的电位差可选用施加低频或高频交流、高阻抗或多种形态的迈冲来引发。再有，第二电极部125的电信号变化有可能是电压、电流、阻抗、电容等变化引起的，通过第二电极部125上述电信号变化测量血样中的红细胞比容(hematocrit)。
感应膜140含有电子转移介体和氧化酶，感应膜140是下基板110印刷集成电极120 后在其集成电极120上进行涂抹制成，该感应膜140相对于微细流道161的部分进行裸露，其余部分被则绝缘薄膜150盖住。该感应膜140设置在第一工作电极122和第一辅助电极123上，其制作方式是通过含有电子转移介体和氧化酶的感应膜组成物涂抹在第一工作电极122及第一辅助电极123上，并通过加热干燥制作。
感应膜140还可含有适当浓度的铁氰化钾(Ⅲ)和适当浓度的水溶性高分子，血样通过微细流道161流入时，感应膜140与血样混合溶解进行薄层电化学池反应，这时氧化酶与血样中的葡萄糖进行反应并被还原，此电化学反应过程中第一电极部121发生电位差，通过测量第一电极部121电流来测量血样中血糖浓度。
感应膜140中的电子转移介体与血液中葡萄糖反应还原的酶进行氧化还原反应被还原，还原态的电子转移介体扩散至电极表面使电极表面产生氧化电位形成电流。电子转移介体与代谢物质反应，与还原态酶氧化还原反应后被还原，上述还原态电子转移介体扩散至电极表面时将对电极表面施加氧化电势形成电流作用。
历来用于电子转移介体的有二茂铁(ferrocene)、二茂铁衍生物、苯醌(quinones)、苯醌衍生物、有机导电盐(organic conducting salt)或百草枯(viologen)，进一步地，电子转移介体还可选用三氯化六氨合钌(hexaammineruthenium(Ⅲ)chloride)、铁氰化钾(potassium ferricyanide)、亚铁氰化钾((potassium ferrocyanide)、二甲基二茂铁(dimethylferrocene(DMF))、二茂铁离子(ferricinium)、二茂铁甲酸(ferocene monocarboxylic acid(FCOOH))、7,7,8,8,-四氰基醌二甲烷(7,7,8,8-tetracyanoquino-dimethane(TCNQ))、二茂镍(nickelocene(Nc))、N-甲基酸铵(N-methyl acidinium(NMA+))、四硫四烯(tetrathiatetracene(TTT))、四硫富瓦烯(tetrathia fulvalene(TTF))、N-甲基吩嗪(N-methylphenazinium(NMP+))、对苯二酚(hydroquinone)、3-二甲基氨基安息香酸(3-dimethylaminobenzoic acid(MBTHDMAB))、3-甲基-2-苯并噻唑啉酮腙(3-methyl-2-benzothiozolinone hydrazone)、2-甲氧基-4-烯丙基苯酚(2-methoxy-4-allylphenol)、4-氨基安替比林(4-aminoantipyrin(AAP))、二甲基苯胺(dimethylaniline)、4-氨基噻芘(4-aminoantipyrene)、4-甲氧基萘酚(4-methoxynaphthol)、3,3',5,5'-四甲基联苯胺(3,3',5,5'-tetramethyl benzidine(TMB))、2,2'-联氮-双-3-乙基苯并噻唑啉-磺酸(2,2-azino-di-[3-ethyl-benzthiazoline sulfonate])邻联茴香胺(o-dianisidine))、邻甲苯胺(o-toluidine)、2,4二氯苯酚(2,4-dichlorophenol)、4-氨基安替比林(4-amino phenazone)、联苯胺(benzidine)及普鲁士蓝(prussian blue)中至少一种。
进一步地，为有效体现酶的特性，感应膜140中还可混合有聚乙烯吡咯烷酮(polyvinyl  pyrrolidone；PVP)、聚乙烯醇(polyvinyl alcohol；PVA)、全氟磺酸盐(perfluoro sulfonate)、羟乙基纤维素(hydroxyethyl cellulose；HEC)、羟丙基纤维素(hydroxypropyl cellulose；HPC)，羟甲基纤维素(carboxy methyl cellulose；CMC)、醋酸纤维素(cellulose acetate)或聚酰胺(polyamide)等高分子中的至少一种。
具体地，绝缘薄膜150设有从里向外开放形态的绝缘薄膜开口部151，绝缘薄膜开口部151与中间基板160的微细流道161相对应，绝缘薄膜150盖住部分第一工作电极122、部分第一辅助电极123、部分第二工作电极126和部分第二辅助电极127，将绝缘薄膜150盖住部分第一电极部121和部分第二电极部125，有助于限制从微细流道161流入的血样到达绝缘薄膜150所盖住的第一电极部121及第二电极部125的区域，从而防止第一工作电极122和第二工作电极126在微细流道161区域内相连，还可以防止第一辅助电极123和第二辅助电极127在微细流道161区域内相连。绝缘薄膜150是带有绝缘性物质的绝缘搅拌物印刷在设有集成电极120和感应膜140的下基板110上，并加热干燥使其盖住集成电极120和感应膜140。由于绝缘薄膜150印刷在感应膜140上面，使感应膜140只裸露与微细流道161对应的部分，其余部分被绝缘薄膜150盖住，进而集成电极120上涂抹的感应膜140不管形状大小，血样通过微细流道161流入时与血样接触的感应膜140面积始终保持一致。
中间基板160夹在下基板110和上基板170之间，中间基板160可以选择双面附有黏合力的双面胶，用于粘贴下基板110与上基板170。
中间基板160拥有使血样流入到第一电极部121及第二电极部125的微细流道161，微细流道161在中间基板160上呈从里向外开放状态，微细流道161内流入血样并可以保留。微细流道161为可容纳微升单位级的血样，优选地，微细流道161内容积可限制为0.7μL以下，当微细流道161容积小于0.3μL时，因影响电化学生物传感器的误差范围无法确保测量准确度，另外，微细流道161的容积大于1.0μL时需采集比较多的使用者血样，因而也不可取。
上基板170对应下基板110，两者可以为同一个材质，上基板170叠在下基板110上并盖住第一电极部121及第二电极部125，上基板170设有与微细流道161对应的确认窗171，确认窗171材质可选用透明或半透明材质，以便从外部确认微细流道161。本产品通过确认窗171可确认微细流道161流入的血样装满状态，该确认窗171还设有气孔172，血样流入到微细流道161时，微细流道161的空气可通过气孔172排出，气孔172的设置便于血样通过虹吸现象更顺畅的流入到微细流道161内。
第一工作电极122和第一辅助电极123之间的间隔在数百μm以内形成薄层电化学池 (thin layer electrochemical cell)结构，血样流入到微细流道161后，采集的样本内分析物被酶和电子转移介体形成的连续循环的氧化/还原反应效果，需数秒内电流要达到稳定状态(steady state)，为满足上述条件感应膜140需快速溶解于微细流道161流入的样本当中，所制得的电化学生物传感器100可满足数秒内达到稳定状态(steady state)。
而且，本实施例的电化学生物传感器100将绝缘薄膜150设置在感应膜140之上，感应膜140只裸露与微细流道161对应部分，其余部分都被绝缘薄膜150盖住，进而不管感应膜140形态大小，微细流道161中裸露的感应膜140区域保持一致，血样通过微细流道161流入时与血样接触的感应膜140面积始终保持一致，加上血样与感应膜140内酶的反应量维持恒定，进而可持续保证测量的准确度及精密度。此外，本实施例还能去除历来用于确认感应膜140不良的检测工艺流程，进而有助于缩短生产时间、提高产量。
本实施例的电化学生物传感器100可导入与血糖监测相同原理的特定酶及合适的电子转移介体，进而可测量多种代谢物质，例如：胆固醇、乳酸、肌酸酐、蛋白质、过氧化氢、酒精、氨基酸、GPT(glutamate pyruvate transaminase)、GOT(glutamate oxaloacetate transmianse)等酶的活体标本、环境标本、农业标本、工业标本或食品标本中的多种有机物或无机物浓度。而且，该电化学生物传感器100调整感应膜140所含的酶的种类可定量测量多种代谢物质，例如：使用葡萄糖氧化酶、乳酸氧化酶、胆固醇氧化酶、谷氨酸氧化酶、辣根(horseradish)过氧化物酶、酒精氧化酶定量测量胆固醇、乳酸、谷氨酸、过氧化氢及酒精。进一步地，将葡萄糖脱氢酶(glucose dehydrogenase；GDH)、葡萄糖氧化酶(glucose oxidase；GOx)、胆固醇氧化酶、胆固醇酯酶、乳酸氧化酶、抗坏血酸氧化酶(ascorbic acid oxidase)、酒精氧化酶、酒精脱氢酶、胆红素氧化酶(bilirubin oxidase)组成的群中被选中的氧化酶也可放入感应膜140内。
本实施例的电化学生物传感器100制备工艺如图8所示，该制备工艺包括S1、下基板准备；S2、制作集成电极；S3、制作感应膜；S4、制作绝缘薄膜；S5、中间基板粘贴；S6、上基板粘贴。
在S1步骤中，先准备可支持集成电极120的下基板110。
在S2步骤中，在下基板110上制作相互间隔的第一工作电极122及第一辅助电极123组成的第一电极部121，以及相互间隔的第二工作电极126及第二辅助电极127组成的第二电极部125，其中第一辅助电极123和第二辅助电极127在下基板110上相连，相比第一辅助电极123和第二辅助电极127独立分开生产工艺更有利；集成电极120的制作方式多样，例如将带导电性物质的导电性搅拌物丝网印刷后固化方式、粘贴带电性薄膜方式、导电性 物质沉淀方式等。
在S3步骤中，在第一工作电极122及第一辅助电极123上制作含电子转移介体和氧化酶的感应膜140,具体制作过程是将含电子转移介体和氧化酶的感应膜组成物涂抹在第一工作电极122及第一辅助电极123上，通过加热干燥方式形成感应膜140，干燥感应膜组成物的加热温度及加热时间随感应膜140包含的电子转移介体及氧化酶种类可进行调整。
在S4步骤中，在设有集成电极120和感应膜140的下基板110上制作绝缘薄膜150，绝缘薄膜150盖住部分第一工作电极122、部分第一辅助电极123、部分第二工作电极126和部分第二辅助电极127，并限制流入的样本到达绝缘薄膜150所盖住的第一电极部121及第二电极部125的区域，且绝缘薄膜150将感应膜140中对应微细流道161以外的部分盖住，感应膜140对应微细流道161部分裸露于微细流道161中；
绝缘薄膜150采用绝缘性材料，具体制备过程为：将绝缘性材料组成的搅拌物印刷在设有集成电极120和感应膜140的下基板110，并加热干燥使其盖住集成电极120和感应膜140，绝缘薄膜150加热干燥过程中感应膜140受热过高会导致感应膜140内氧化酶损伤，因此绝缘薄膜150加热干燥温度应选择不损伤感应膜140内氧化酶的适当温度。
在S5步骤中，为形成第一电极部121及第二电极部125流入血样的微细流道161，把中间基板160粘贴在绝缘薄膜150上，中间基板160对应匹配下基板110，可选用双面带粘性的双面胶材质；
在S6步骤中，将上基板170粘贴在中间基板160上，上基板170与下基板110配套使用，粘贴上基板170可在一定量血样流入的微细流道161中排列第一电极部121和第二电极部125，从而完成制作电化学生物传感器100。
实施例2
如图9和10所示，一种电化学生物传感器200，包括下基板110、下基板110上设有的集成电极120、集成电极120上设有的感应膜140、盖住部分集成电极120和部分感应膜140而设置在下基板110上的绝缘薄膜210、粘贴在绝缘薄膜210上的中间基板160、中间基板160上面粘贴的上基板170，本实施例的绝缘薄膜210与实施例1的绝缘薄膜150存在区别，其余结构与实施例1相同。
绝缘薄膜210设置在下基板110上，该绝缘薄膜210盖住部分第一工作电极122、部分第一辅助电极123、部分第二工作电极126和部分第二辅助电极127，该绝缘薄膜210设有从里向外开放形态的绝缘薄膜开口部211，绝缘薄膜开口部211与中间基板160的微细流道161相对应。
本实施例的绝缘薄膜210还包括阻隔膜212和阻塞膜213，沿着微细流道161内样本流入方向，按照阻塞膜213、阻隔膜212的顺序进行依次排列。
具体地，阻隔膜212数量为1个，阻隔膜212横向设于绝缘薄膜开口部211，且阻隔膜212横插在第一工作电极122和第一辅助电极123之间，阻隔膜212起到防止外部空气当中的水分轻易渗透到感应膜140上的作用，若水分通过微细流道161渗透到感应膜140，则感应膜140与水分进行反应，导致降低了对血样测量的准确度及精密度，而阻隔膜212的设置能抑制水分渗透到感应膜140，从而可有效阻止上述问题发生。此外，当阻隔膜212数量为1个时，阻隔膜212还可以横插在气孔172和第一辅助电极123之间，防止经气孔172进入的外部空气当中的水分轻易渗透到感应膜140上，当阻隔膜212数量为2个或者大于2个时，气孔172和第一辅助电极123之间以及第一工作电极122和第一辅助电极123之间均可以设置相应的阻隔膜212。
具体地，阻塞膜213数量为1个，阻塞膜213横向设于绝缘薄膜开口部211，且阻塞膜213位于第一电极部121和第二电极部125之间，该阻塞膜213能够抑制感应膜140与样本反应溶解的溶解物的流动。若感应膜140与血样反应溶解的溶解物不向第二电极部125流动，保持待在第一电极部121对测量有利。本实施例的阻塞膜213在第一电极部121和第二电极部125之间，起到不轻易让感应膜140与血样反应溶解的溶解物向第二电极部125流动的作用，阻塞膜213的设置也可有效地提高测量准确度及精密度。
本实施例的阻塞膜213也与阻隔膜212一样也起到抑制水分通过微细流道161渗透的作用，可降低在高湿环境下因受水分影响导致电化学生物传感器的测量准确度和精密度下降问题。此外，阻塞膜213还可抑制感应膜140裸露于微细流道161中的区域，即阻塞膜213把感应膜140中靠近第二电极部125的部分盖住，限制靠近第二电极部125的感应膜140与血样的反应。
进一步地，阻隔膜212如图11的(a)及(b)所示，感应膜140存在不规则的涂抹现象，即使在靠近第二电极部125的地方发生感应膜140与血样反应溶解的溶解物，也会有效抑制溶解物到达第二电极部125。而且，阻隔膜212如图11(c)所示，感应膜140过度扩散涂抹，即使在靠近第二电极部125的地方发生感应膜140与血样反应溶解的溶解物，也会有效抑制溶解物到达第二电极部125。
本实施例的电化学生物传感器200的制备方法与实施例1相同。
对实施例1和实施例2的电化学生物传感器进行性能测试，具体如下。
(1)感应膜设置顺序不同的电化学生物传感器的精密度测量
实施例1：以实施例1的电化学生物传感器100为例，具体地，制备对应的感应膜组成物，其制备过程为：将含铁氰化钾(potassium ferricyanide)10.0g、羟乙基纤维素(hydroxyethyl cellulose；HEC)2.0g、Triton X-100 0.3g和葡萄糖脱氢酶2.0g所组成的混合物溶解于100mL的PBS缓冲液(pH为6.4，浓度为0.1mol/L)中，混合均匀，然后去除溶液内微粒子，得到感应膜组成物。
电化学生物传感器100的结构如图4至图7所示，详细的制作过程为：S1、先准备可支持集成电极120的下基板110；S2、以碳搅拌物为原料，利用丝网印刷后固化的方式，在下基板上印刷第一工作电极122、第二工作电极126、第一辅助电极123与第二辅助电极127相连组成的辅助电极体130、生物传感器确认电极131、生产条码信息确认电极133，然后在130℃环境下干燥5min；S3、选择上述的感应膜组成物，将感应膜组成物涂抹在第一工作电极122及第一辅助电极123上，然后在70℃环境下干燥5min，形成感应膜140；S4、将绝缘性材料组成的搅拌物印刷在设有集成电极120和感应膜140的下基板110，并于70℃环境下干燥5min，形成绝缘薄膜150；S5、用聚酯纤维材质的双面胶模具加工后形成具有微细流道161的中间基板160，此时微细流道的容积为0.5μL，把中间基板160粘贴在绝缘薄膜150上；f、然后将上基板170粘贴在中间基板160上，上基板170上加工有气孔172以及对应的测试仪连接器插入部分，从而完成制作电化学生物传感器100。
对比例1：按照上述制作过程制备对比例1的电化学生物传感器，对比例1与实施例1的电化学生物传感器100区别仅在于：对比例1先制作绝缘薄膜，后制作感应膜，而实施例1先制作感应膜，后制作绝缘薄膜。
对实施例1和对比例1的电化学生物传感器进行测量精密度比较。
具体分析，利用含肝素的采血管中采集静脉血并离心分离血浆和血细胞，分离的血浆和血细胞混合制作红细胞比容为42％、血糖浓度如下表表1的调制血，并用5台血糖测试仪分别测量5次。
表1

分别利用实施例1和对比例1的电化学生物传感器进行测量，具体结果如表2所示，实施例1的电化学生物传感器的测量结果平均CV％为2.85，相反，对比例1的电化学生物传感器的测量结果平均CV％是5.00。依据上述结果可以确认，先制作感应膜、后制作绝缘薄膜的工艺有助于提高电化学生物传感器的测量精密度。
表2
(2)有无阻隔膜、阻塞膜的电化学生物传感器的湿度测量
实施例2：参照上述精密度测量中实施例1的制备过程制备实施例2的电化学生物传感 器200，实施例2的电化学生物传感器200结构如图9至11所示，实施例2的绝缘薄膜210设有阻隔膜212、阻塞膜213，阻隔膜212横插在第一工作电极122和第一辅助电极123之间，阻塞膜213位于第一电极部121和第二电极部125之间，实施例1的绝缘薄膜150不设有阻隔膜和阻塞膜，将实施例1和2的电化学生物传感器裸露于温度35℃，湿度85％环境下进行性能测试比较。
具体而言，恒温恒湿实验室温度调整为35℃，湿度调整为85％，设定温度及湿度达到稳定水平后，测试仪及电化学生物传感器放入恒温恒湿实验室内稳定30min以上后进行测量，将含肝素的采血管中采集静脉血并离心分离血浆和血细胞，分离的血浆和血细胞混合制备红细胞比容为42％，血糖浓度如下表表3所示的调制血，使用5台测试仪分别在30min、60min、90min、120min、150min、180min进行测量。
表3
具体测量结果如下表表4所示，与不同对应时间的不暴露于湿度的试纸的测量数据进行对比，实施例1这种第一工作电极122和第一辅助电极123之间无阻隔膜、第一电极部121和第二电极部125之间也无阻塞膜的电化学生物传感器100比印刷阻隔膜212及阻塞膜213的电化学生物传感器200测量结果高2倍以上，测量准确度下降。依据结果可以确定，印刷阻隔膜、阻塞膜可提高电化学生物传感器的准确度。
表4

(3)电化学生物传感器的准确度测量
根据实施例2制作的电化学生物传感器200对调制血的准确度测试。
具体而言，将含肝素的采血管采集的静脉血离心分离血浆和血细胞后，利用离心的血浆和血细胞混合调制红细胞比容为42％、氧分压调制毛细血管氧分压为60～80mmHg，利用基准设备YSI 2300STAT Plus按下列表表5浓度进行调制并测量，根据实施例制作的电化学生物传感器共计测量50次。
表5
测量结果如图12和图13所示，基准设备YSI 2300 STAT Plus对比斜率为1.0086、截距为-0.026、线性为0.9984，100％满足公示网络误差(Consensus Error Grid)A领域，并100％满足Accuracy±15％基准。
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化和修改 例。

Claims (13)

  1. 一种电化学生物传感器，其特征在于，包括下基板、中间基板和上基板，所述下基板设有集成电极，所述集成电极包含相互间隔的第一工作电极和第一辅助电极以及相互间隔的第二工作电极和第二辅助电极，所述第一工作电极和所述第一辅助电极组成第一电极部，所述第二工作电极和所述第二辅助电极组成第二电极部；
    所述中间基板夹在所述下基板和所述上基板之间，所述中间基板上设有微细流道，所述微细流道用于将样本连续导入第一电极部和第二电极部中；
    所述上基板和所述中间基板之间还设有绝缘薄膜，且所述绝缘薄膜设有从里向外开放形态的绝缘薄膜开口部，所述绝缘薄膜开口部与所述微细流道相对应，所述绝缘薄膜盖住部分第一电极部和第二电极部，同时限制从所述微细流道流入的样本到达所述绝缘薄膜所盖住的第一电极部及第二电极部的区域；
    所述下基板和所述绝缘薄膜之间裸露设置有感应膜，所述感应膜相对于所述微细流道的部分进行裸露，其余部分被所述绝缘薄膜盖住，所述感应膜设置在所述第一工作电极和所述第一辅助电极上。
  2. 根据权利要求1所述的电化学生物传感器，其特征在于，所述上基板设有与所述细微流道对应的确认窗，所述确认窗设有气孔。
  3. 根据权利要求2所述的电化学生物传感器，其特征在于，所述绝缘薄膜还包括至少一个阻隔膜，所述阻隔膜横向设于所述绝缘薄膜开口部，且所述阻隔膜位于所述第一工作电极和所述第一辅助电极之间和/或所述气孔和所述第一辅助电极之间。
  4. 根据权利要求3所述的电化学生物传感器，其特征在于，所述绝缘薄膜还包括至少一个阻塞膜，所述阻塞膜横向设于所述绝缘薄膜开口部，且所述阻塞膜位于所述第一电极部和所述第二电极部之间。
  5. 根据权利要求4所述的电化学生物传感器，其特征在于，沿着所述微细流道内样本流入方向，按照所述阻塞膜、所述阻隔膜的顺序进行依次排列。
  6. 根据权利要求1所述的电化学生物传感器，其特征在于，沿着所述微细流道内样本流入方向，按照所述第二辅助电极、所述第二工作电极、所述第一工作电极和所述第一辅助电极的顺序进行依次排列。
  7. 根据权利要求1所述的电化学生物传感器，其特征在于，所述第一辅助电极和所述第二辅助电极通过连接电极相连形成辅助电极体；所述辅助电极体的末端设有生物传感器确认电极。
  8. 根据权利要求1所述的电化学生物传感器，其特征在于，所述下基板设有生产条码信 息确认电极。
  9. 根据权利要求1所述的电化学生物传感器，其特征在于，所述微细流道的容积为0.3-1.0μL，优选0.3-0.7μL。
  10. 根据权利要求1所述的电化学生物传感器，其特征在于，所述绝缘薄膜盖住部分第一工作电极、部分第一辅助电极、部分第二工作电极和部分第二辅助电极。
  11. 一种如权利要求1-10任一项所述的电化学生物传感器的制备方法，其特征在于，包括如下步骤：
    S1、下基板准备：先准备可支持集成电极的下基板；
    S2、制作集成电极：在下基板上制作相互间隔的第一工作电极及第一辅助电极组成的第一电极部，以及相互间隔的第二工作电极及第二辅助电极组成的第二电极部；
    S3、制作感应膜：在第一工作电极及第一辅助电极上制作感应膜；
    S4、制作绝缘薄膜：在设有集成电极和感应膜的下基板上制作绝缘薄膜；
    S5、中间基板粘贴：把中间基板粘贴在绝缘薄膜上；
    S6、上基板粘贴：将上基板粘贴在中间基板上。
  12. 根据权利要求11所述的电化学生物传感器的制备方法，其特征在于，在步骤S3中，所述感应膜含有电子转移介体和氧化酶，将含有电子转移介体和氧化酶的感应膜组成物涂抹在第一工作电极及第一辅助电极上，加热干燥，形成感应膜；
    在步骤S4中，所述绝缘薄膜采用绝缘性材料，将绝缘性材料组成的搅拌物印刷在设有集成电极和感应膜的下基板，并加热干燥使其盖住集成电极和感应膜，形成绝缘薄膜。
  13. 根据权利要求12所述的电化学生物传感器的制备方法，其特征在于，所述电子转移介体选自二茂铁、苯醌、苯醌衍生物、有机导电盐、百草枯、三氯化六氨合钌、铁氰化钾、亚铁氰化钾、二甲基二茂铁、二茂铁离子、二茂铁甲酸、7,7,8,8,-四氰基醌二甲烷、二茂镍、N-甲基酸铵、四硫四烯、四硫富瓦烯、N-甲基吩嗪、对苯二酚、3-二甲基氨基安息香酸、3-甲基-2-苯并噻唑啉酮腙、2-甲氧基-4-烯丙基苯酚、4-氨基安替比林、二甲基苯胺、4-氨基噻芘、4-甲氧基萘酚、3,3',5,5'-四甲基联苯胺、2,2'-联氮-双-3-乙基苯并噻唑啉-磺酸、邻联茴香胺、邻甲苯胺、2,4二氯苯酚、4-氨基安替比林、联苯胺及普鲁士蓝中至少一种；
    所述氧化酶选自葡萄糖氧化酶、乳酸氧化酶、胆固醇氧化酶、谷氨酸氧化酶、辣根过氧化物酶、酒精氧化酶、葡萄糖脱氢酶、胆固醇酯酶、抗坏血酸氧化酶、酒精脱氢酶、胆红素氧化酶中至少一种。
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