CN111220667B - Light-addressable electrochemical sensor and preparation method and detection method thereof - Google Patents

Light-addressable electrochemical sensor and preparation method and detection method thereof Download PDF

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CN111220667B
CN111220667B CN202010124420.6A CN202010124420A CN111220667B CN 111220667 B CN111220667 B CN 111220667B CN 202010124420 A CN202010124420 A CN 202010124420A CN 111220667 B CN111220667 B CN 111220667B
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glucose
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laser diode
electrochemical sensor
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CN111220667A (en
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张文
陈芷羽
刘超
魏晓鸥
张俊俊
邹小波
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Jiangsu University
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    • 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
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    • G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3273—Devices therefor, e.g. test element readers, circuitry
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    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3274—Corrective measures, e.g. error detection, compensation for temperature or hematocrit, calibration
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    • 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
    • G—PHYSICS
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    • 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
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Abstract

The invention provides a light-addressable electrochemical sensor and a preparation method and a detection method thereof, the invention combines a potential sensor with photoelectric effect to detect glucose, the preparation of the light-addressable electrochemical sensor comprises the construction of a system topological structure, the manufacture and the assembly of a LAPS chip, the modification of the sensor function, the manufacture of a reference electrode and the assembly of the sensor, and the glucose concentration detection is carried out by utilizing the prepared sensor. Because the sensing surface of the LAPS chip reacts with a specific substance in a solution to be detected, a layer of ions are adsorbed on the surface to generate a membrane potential, so that SiO of an insulating layer2And the voltage between the silicon substrate is deviated, so that the standard curve of the volt-ampere characteristic is deviated, and the concentration of the ions to be measured is obtained through the deviation. The invention designs the micro optical addressing electrochemical sensor according to the principle to realize the field detection of the glucose, has the advantages of low sensor preparation cost, simple and convenient preparation process, convenient carrying and the like, and well solves the problems existing in the detection of the traditional electrochemical sensor.

Description

Light-addressable electrochemical sensor and preparation method and detection method thereof
Technical Field
The invention belongs to the field of biological sample electrochemical sensor detection, and relates to a light-addressable electrochemical sensor and a preparation method and a detection method thereof.
Background
Diabetes is a group of diseases which cause chronic metabolic disorders and serious complications, and in which blood sugar and urine sugar contents are important criteria for diabetes, so that whether the detection of blood sugar and urine sugar can be effectively performed is very important for early screening of diabetes. In recent years, electrochemical analysis and detection of glucose concentration have been studied. The traditional method for detecting glucose by using the electrochemical sensor has the advantages of sensitive reaction, large detection range, strong stability and the like, but the conditions of long sensor manufacturing period, overlarge equipment, high cost and the like generally exist.
The light addressing potential sensor LAPS is a sensitive device based on the photoelectric effect in a semiconductor. It includes a reference electrode, a sensitive surface and a silicon substrate, and forms a closed loop through an electric lead. When semiconductor silicon is irradiated by light of a certain wavelength, photons are absorbed and transition is completed, and electron-hole pairs appear. A bias voltage is applied to produce a detectable photocurrent in the external circuit.
At present, no formed equipment for realizing sensitive detection of glucose by using a sensor prepared by using a LAPS photoelectric effect exists in the market. Among the existing electrochemical sensors applied to glucose detection, the patent "preparation method of electrochemical glucose biosensor and detection method thereof for glucose test" CN105548317A discloses a preparation method of electrochemical glucose biosensor modified by calcium titanate nanoparticles and a detection method using the electrochemical glucose biosensor, which can detect glucose by using glucose oxidase fixed by calcium titanate nanoparticles. However, the preparation period of the electrode is long, and in-situ detection cannot be realized easily by preparing the sensor. However, the electrochemical sensor combining the photoelectric effect of the present invention can effectively solve the above-mentioned problems.
Disclosure of Invention
The invention discloses a light-addressable electrochemical sensor and a preparation method and a detection method thereof, aiming at the problems that the existing electrochemical glucose detection sensor is difficult to realize rapid on-site detection, high in cost, complex in preparation and the like. As the sensing surface of the LAPS chip reacts with a specific substance in a solution to be detected, a layer of ions is adsorbed on the surface to generate a membrane potential, so that the SiO of the insulating layer2And the voltage between the silicon substrate, thereby causing the standard curve of the volt-ampere characteristic to deviateAnd moving to obtain the concentration of the ions to be measured through the offset. The invention designs the micro optical addressing electrochemical sensor according to the principle to realize the field detection of the glucose, has the advantages of low sensor preparation cost, simple and convenient preparation process, convenient carrying and the like, and well solves the problems existing in the detection of the traditional electrochemical sensor.
The technical scheme adopted by the invention for solving the technical problem is as follows: a photo-addressable electrochemical sensor comprises an integrated active driver, a laser component, an optical fiber, a LAPS chip, a reference electrode and a trans-impedance amplifier;
the laser assembly comprises a laser diode and a micro lens; the laser diode, the semi-reflecting and semi-transmitting lens and the micro lens are arranged on the same light path, the laser diode is connected with the integrated active driver, a light source of the laser diode reaches one end of the optical fiber through the micro lens, the other end of the optical fiber is provided with a LAPS chip, and the LAPS chip is used for measuring the concentration of glucose;
the LAPS chip and the reference electrode are respectively connected with one end of the transimpedance amplifier, and the other end of the transimpedance amplifier is used for connecting external equipment.
In the scheme, the LAPS chip comprises a first gold layer, a silicon dioxide layer, an N-type silicon substrate and a second gold layer which are sequentially connected; diluting the isolation epoxy glue with acetone, coating the isolation epoxy glue on other areas except the surface of the first gold layer, and dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer in sequence to serve as a sensing surface.
In the above scheme, the laser assembly further includes a photodiode, a half-reflecting and half-transmitting mirror and a reflective mirror;
the photoelectric diode is connected with the integrated active driver, part of light source of the laser diode reaches one end of the optical fiber through the semi-reflecting and semi-transparent mirror and the micro lens, and the other part of light source of the laser diode is reflected to the photoelectric diode through the reflecting mirror.
A method for manufacturing an electrochemical sensor based on the optical addressing, comprising the following steps:
and (3) constructing a topological structure: the optical addressing electrochemical sensor comprises an integrated active driver, a laser assembly, an optical fiber, a LAPS chip, a reference electrode and a trans-impedance amplifier;
the laser assembly comprises a laser diode and a micro lens; the laser diode, the semi-reflecting and semi-transmitting lens and the micro lens are arranged on the same light path, the laser diode is connected with the integrated active driver, a light source of the laser diode reaches one end of the optical fiber through the micro lens, the other end of the optical fiber is provided with a LAPS chip, and the LAPS chip is used for measuring the concentration of glucose;
the LAPS chip and the reference electrode are respectively connected with one end of a transimpedance amplifier, and the other end of the transimpedance amplifier is used for connecting external equipment;
manufacturing and assembling the LAPS chip: taking an N-type silicon wafer as a substrate, growing a layer of silicon dioxide on each side of the N-type silicon wafer under the thermal dry oxidation, completely removing the layer of silicon dioxide on the N-type silicon wafer by hydrofluoric acid (HF), depositing a first gold layer and a second gold layer on each side of the N-type silicon wafer by a thermal evaporation method, photoetching a micro-ring on the second gold layer by photolithography, directly contacting the second gold layer with the surface of the N-type silicon wafer, and simultaneously obtaining a solid gold surface, namely a first gold layer on the opposite side; cutting the chip into round chips, placing the optical fibers in the centers of gold rings of the round chips, assembling the LAPS chip and the optical fibers by using silver glue, connecting the LAPS chip and the transimpedance amplifier by using the silver glue through silver paint wires, and diluting the isolating epoxy glue by using acetone to coat the isolating epoxy glue on other areas except the surfaces of the first gold layers;
functional modification of a sensor: sequentially dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer to serve as a sensing surface;
manufacturing a reference electrode: placing the bare silver wire in hydrochloric acid to generate an Ag-AgCl thin layer, and assembling the Ag-AgCl thin layer near the LAPS chip 7 by using epoxy resin as a reference electrode;
assembling the sensor: and assembling the integrated active driver, the laser assembly and the transimpedance amplifier on a printed circuit board.
In the above scheme, the laser component in the step of constructing the topological structure further comprises a photodiode, a semi-reflecting and semi-transmitting mirror and a reflective mirror;
the photodiode is connected with the integrated active driver, part of light sources of the laser diode reach one end of the optical fiber through the semi-reflecting and semi-transparent mirror and the micro lens, and the other part of light sources of the laser diode are reflected to the photodiode through the reflecting mirror.
In the scheme, the concentration of the glucose oxidase GOx solution in the sensor function modification step is 10-50g/L, and the using amount is 0.5-1.0 mu mL.
In the scheme, the perfluorosulfonic acid-polytetrafluoroethylene copolymer in the step of sensor function modification is a diluent, the diluent is prepared by diluting the perfluorosulfonic acid-polytetrafluoroethylene copolymer to 1% by mass with 95% ethanol, and the dosage of the diluent is 0.4 μmL.
A glucose detection method using the optical addressing electrochemical sensor comprises the following steps:
establishing an I/V curve of the photocurrent: the sensing surface of the LAPS chip is directly contacted with a sample to be detected, and under the action of glucose oxidase GOx, along with the change of the glucose concentration of the sample, the oxidation-reduction potential is changed, so that an I/V standard curve of photocurrent is established;
and measuring the concentration of the glucose to be measured according to the established I/V curve of the photocurrent: the sensing areas of the LAPS chip and the reference electrode are contacted with a sample to be tested, a depletion layer is generated after a direct current bias voltage is applied to an interface of an N-type silicon crystal substrate and a silicon dioxide layer, when the LAPS chip is irradiated by modulated light, a photocurrent signal is amplified by a trans-impedance amplifier and transmitted to a computer for data analysis, the photocurrent is determined by the direct current bias voltage and the oxidation-reduction potential of a metal layer, and when glucose oxidase GOx exists on the sensing surface, an enzyme catalytic reaction is carried out and H is induced to be generated2O2Due to H2O2The glucose concentration can be measured by the displacement of the standard I/V curve established on the photocurrent and the bias current.
In the above scheme, the standard curve of I/V of the photocurrent is: y958-101.1 lgX, R20.992, X is the concentration of glucose in the sampleAnd the degree mM, Y is the voltage mV obtained after the sample with the concentration is detected by the optical addressing electrochemical sensor.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, the glucose concentration is measured by the reaction of the glucose oxidase and the glucose in the sample and the output of the detection result by combining the photoelectric effect, so that the portable sensitive design of the electrochemical sensor is realized, and the possibility is provided for the online real-time detection of the glucose. The novel modification method of the light-addressable electrochemical sensor sensing surface makes up the defects of complex modification, long manufacturing period and high cost of a reference electrode of a common electrochemical sensor, can realize in-situ detection of the concentration of glucose in a sample, and can be applied to blood sugar detection of a diabetic patient.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1: the invention relates to a circuit arrangement of a micro optical addressing potential sensor on the end face of an optical fiber;
FIG. 2: modifying the sensitive surface of the light addressable potentiometric sensor;
FIG. 3: a glucose concentration measurement curve;
FIG. 4: a standard curve plot of glucose concentration versus sensor signal;
FIG. 5: the method of the invention detects the blood sugar concentration of the sample and detects the contrast chart with the standard method;
FIG. 6: the method of the invention detects the concentration of the urine glucose in the sample and the standard method detects the contrast graph.
In the figure, 1, an active driver is integrated; 2. a laser assembly; 3. a laser diode; 4. a photodiode; 5. a microlens; 6. an optical fiber; 7. a LAPS chip; 8. a reference electrode; 9. a transimpedance amplifier; 10. a potentiostat interface; 11. a half-reflecting and half-transmitting mirror; 12. a reflector.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the present invention, unless otherwise explicitly stated or limited, the terms "mounted," "connected," "fixed," and the like are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Example 1
A manufacturing method of a light-addressable electrochemical sensor comprises the following steps:
and (3) constructing a topological structure: as shown in fig. 1, the photo-addressable electrochemical sensor comprises an integrated active driver 1, a laser component 2, an optical fiber 6, a LAPS chip 7, a reference electrode 8 and a trans-impedance amplifier 9;
the laser component 2 comprises a laser diode 3, a photodiode 4, a half-reflecting and half-transmitting mirror 11, a reflecting mirror 12 and a micro lens 5; the laser diode 3, the semi-reflecting and semi-transmitting mirror 11 and the micro lens 5 are arranged on the same light path, the laser diode 3 and the photodiode 4 are respectively connected with the integrated active driver 1, part of light source of the laser diode 3 reaches one end of the optical fiber 6 through the semi-reflecting and semi-transmitting mirror 11 and the micro lens 5, the other end of the optical fiber 6 is provided with the LAPS chip 7, and the LAPS chip 7 is used for measuring the glucose concentration; another part of the light source of the laser diode 3 is reflected to the photodiode 4 through a reflector 12;
the LAPS chip 7 and the reference electrode 8 are respectively connected with one end of a transimpedance amplifier 9, and the other end of the transimpedance amplifier 9 is used for connecting external equipment.
Preferably, most of the light generated by the laser diode 3, which is greater than or equal to 70%, is focused by the micro lens 5 and then output by the beam tail optical fiber 6, and about 5-10% of the light generated by the laser diode 3 is coupled to the photodiode 4 and excites the light current to be transmitted to the integrated active driver 1 as a detection and feedback signal. When the LAPS chip 7 and the solid-state reference electrode 8 extend into a sample to be detected for detection, the optical fiber 6 is used for modulating light to irradiate the LAPS chip 7 and then detectable photocurrent is generated.
The transimpedance amplifier 9 is respectively connected with the LAPS chip 7 and the reference electrode 8, so that the signal length can be shortened, and the electromagnetic interference can be reduced.
The integrated active driver 1 receives and amplifies an external sinusoidal signal to drive the laser diode 3, the laser diode 3 does not have a linear V-I characteristic, and the integrated active driver 1 needs to be designed into a current amplification mode, wherein the output current in the current amplification mode is in direct proportion to the voltage amplitude of an input signal. Meanwhile, the resistance of the sample to be tested and the error signal generated by the photodiode 4 serve as the negative feedback factor of the integrated active driver 1.
The circuit shown in fig. 1, specifically, in the integrated active driver 1, the operational amplifier OP1a receives an excitation sine wave signal provided from an external device, such as a potentiostat, and amplifies the excitation sine wave signal to drive the laser diode 3; the two ends of the sampling resistor R1a are respectively connected with the inverted input end of the OP1a and the ground, the driving current flowing through the laser diode 3 is grounded through the sampling resistor R1a, and a current negative feedback signal is generated on the R1 a. Laser generated by the laser diode 3 is divided into two parts in the module by the half-reflecting and half-transmitting mirror 11, the reflective mirror 12 and the micro lens 5, wherein most energy is excited and irradiated to the LAPS chip 7 through the optical fiber 6; other energy excites and irradiates the photodiode 4 inside the module. The photo signal generated by the photodiode 4 is buffered and amplified by the OP1b, and then output to the reverse input terminal of the OP1a through the R1b as the light intensity negative feedback signal. The LAPS chip 7 and the reference electrode 8 form a two-electrode system in a solution environment, and are respectively connected with the OP10 and the transimpedance amplifier 9. The transimpedance amplifier 9 is connected with a reference electrode interface of an external potentiostat, and a constant potential signal is output to the solid reference electrode 8 through a circuit formed by an operational amplifier OP9 and a resistor R9; the photocurrent signal generated by the LAPS chip 7 is subjected to I/V conversion by the OP10 and then is output to an external potentiostat.
As shown in fig. 2, fabrication and assembly of the LAPS chip 7: taking an N-type silicon wafer with the thickness of 500 mu m and the crystal face orientation of 100 as a substrate, carrying out hot dry oxidation for 45 minutes at the temperature of 1100 ℃, growing a layer of silicon dioxide on each side of the N-type silicon wafer, completely removing the layer of silicon dioxide on the upper side by hydrofluoric acid HF, depositing a first gold layer and a second gold layer with the thickness of 90-100nm on each side of the N-type silicon wafer by using a thermal evaporation method, namely AutoE306, Edwards and UK, and photoetching a micro-ring on the second gold layer by using photolithography, wherein the second gold layer is directly contacted with the surface of the N-type silicon wafer, and meanwhile, obtaining a solid gold surface, namely the first gold layer on the opposite side; the chip is cut into round chips, preferably, the inner diameter of the round chip is 900 microns, the outer diameter of the round chip is 950 microns, and the diameter of the cut chip is larger than that of the optical fiber, so that better passing rate can be achieved. The optical fiber is placed in the center of the gold ring by using a precise motion platform, and the effective irradiation can be met only by keeping close contact. Placing the optical fiber 6 in the center of the gold ring of the round chip, assembling the LAPS chip 7 and the optical fiber 6 by using silver adhesive, connecting the LAPS chip 7 and the transimpedance amplifier 9 by using the silver adhesive through a silver paint line, and diluting the isolating epoxy adhesive by using acetone to coat the isolating epoxy adhesive on other areas except the surface of the first gold layer;
functional modification of the sensor: glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer are sequentially dripped on the surface of the first gold layer to serve as a sensing surface, specifically, 500mg of glucose oxidase GOx is accurately measured and dissolved in 0.01M Phosphate Buffered Saline (PBS), the concentration of glucose oxidase GOx is 10-50g/L, the value of corresponding PBS is 10-50mL, the obtained solution is purified for 15 minutes by nitrogen flow to remove dissolved oxygen, before functional modification, a first gold layer of a sensing surface of the LAPS chip 7 is washed by ethanol and distilled water, 0.5-1.0 mu mL of glucose oxidase GOx solution is dripped on the first gold layer, natural drying is carried out at room temperature, 95% ethanol is used for diluting perfluorosulfonic acid-polytetrafluoroethylene copolymer to the mass fraction of 1%, then 0.4 mu mL of diluted solution is dripped on the first gold layer and dried for 12 hours, and the modified sensing surface can detect the glucose concentration;
manufacturing a reference electrode: treating a bare silver wire by using an ultraviolet/ozone method for more than 20 minutes, preferably, the diameter of the bare silver wire is 100 mu m, the bare silver wire is placed in hydrochloric acid to generate an Ag-AgCl thin layer, and the Ag-AgCl thin layer is assembled near a LAPS chip 7 which is smaller than 1mm by using epoxy resin and is used as a reference electrode;
assembling the sensor: the integrated active driver 1, the laser assembly 2, and the transimpedance amplifier 9 are assembled on a printed circuit board. The on-board small signal circuit at the bottom layer of the printed circuit board is shielded by adopting a grounding or buffering synchronous signal loop so as to avoid unnecessary electromagnetic interference.
According to the present embodiment, preferably, the amplifiers are low noise and ultra-low noise operational amplifiers, wherein the models of the OP1a and OP1b are OPA134AU, the model of the OP9 amplifier is OPA627AU, and the model of the OP10 amplifier is OPA129 AU.
According to the present embodiment, preferably, the integrated active driver 1 uses a domestic external power supply ± 7.5V,45mA and a sinusoidal modulation signal emitted by a potentiostat. In addition, the integrated active driver 1 employs 2 rechargeable batteries 7.4V,100mAh as an independent power source to obtain better signal-to-noise ratio.
According to this embodiment, preferably, a potentiostat is used for all potential measurements. Collecting the photocurrent on the LAPS chip 7 by a transimpedance amplifier 9, and converting the photocurrent into a photocurrent with a fixed coefficient of 5 × 105V/A voltage signal. The voltage signal is connected to a potentiostat and amplified with an optional gain of 0-60 db. And the analog-to-digital converter on the potentiostat obtains digital data and transmits the digital data to a computer for data analysis.
Example 2
A photo-addressable electrochemical sensor, in this embodiment 2, is prepared by the method described in embodiment 1, and thus has the beneficial effects described in embodiment 1, and details are not repeated here.
As shown in fig. 1, the optical addressing electrochemical sensor comprises an integrated active driver 1, a laser component 2, an optical fiber 6, a LAPS chip 7, a reference electrode 8 and a transimpedance amplifier 9;
the laser component 2 comprises a laser diode 3, a photodiode 4, a half-reflecting and half-transmitting mirror 11, a reflecting mirror 12 and a micro lens 5; the laser diode 3, the semi-reflecting and semi-transparent mirror 11 and the micro lens 5 are arranged on the same light path, the laser diode 3 and the photodiode 4 are respectively connected with the integrated active driver 1, part of light source of the laser diode 3 reaches one end of the optical fiber 6 through the semi-reflecting and semi-transparent mirror 11 and the micro lens 5, the other end of the optical fiber 6 is provided with the LAPS chip 7, and the LAPS chip 7 is used for measuring the concentration of glucose; another part of the light source of the laser diode 3 is reflected to the photodiode 4 through a reflector 12;
the LAPS chip 7 and the reference electrode 8 are respectively connected with one end of a transimpedance amplifier 9, and the other end of the transimpedance amplifier 9 is used for connecting external equipment.
According to this embodiment, preferably, the LAPS chip 7 includes a first gold layer, a silicon dioxide layer, an N-type silicon crystal substrate, and a second gold layer, which are connected in sequence; diluting the isolation epoxy glue with acetone, coating the isolation epoxy glue on other areas except the surface of the first gold layer, and dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer in sequence to serve as a sensing surface.
According to the present embodiment, preferably, the laser assembly 2 further includes a photodiode 4, a half-reflecting and half-transmitting mirror 11 and a reflective mirror 12;
the photodiode 4 is connected with the integrated active driver 1, part of light source of the laser diode 3 reaches one end of the optical fiber 6 through the half-reflecting and half-transmitting mirror 11 and the micro lens 5, and the other part of light source of the laser diode 3 is reflected to the photodiode 4 through the reflecting mirror 12.
Example 3
As shown in fig. 3, a method for detecting glucose using the photoaddressable electrochemical sensor according to embodiment 2 includes the following steps:
establishing an I/V curve of the photocurrent: the sensing surface of the LAPS chip 7 is directly contacted with a sample to be detected, and under the action of glucose oxidase GOx, the redox potential is changed along with the change of the glucose concentration of the sample, so that an I/V standard curve of photocurrent is established;
and measuring the concentration of the glucose to be measured according to the established I/V curve of the photocurrent: the sensing areas of the LAPS chip 7 and the reference electrode 8 are contacted with a sample to be tested, and after a direct current bias is applied to the interface of the N-type silicon substrate and the silicon dioxide layerGenerating depletion layer, amplifying the light signal by transimpedance amplifier 9 and capturing the signal by external circuit such as potentiostat or phase-locked amplifier (also called phase detector) via potentiostat interface 10 after LAPS chip 7 is irradiated by modulated light, transmitting the signal to computer for data analysis, wherein the photocurrent is determined by DC bias voltage and oxidation-reduction potential of metal layer, and when glucose oxidase GOx exists on the sensing surface, enzyme catalyzing reaction and inducing generation of H2O2，
Figure GDA0003623282860000071
Due to H2O2Corresponding to different concentrations of glucose, different oxidation-reduction potentials are generated,
Figure GDA0003623282860000081
since an I/V standard curve established on the photocurrent and the bias current is obtained, the glucose concentration can be measured by the displacement thereof. The light-addressable electrochemical sensor has the advantages of simple structure and convenient operation, and can be well used for on-site detection and analysis.
According to this embodiment, preferably, the standard curve of I/V of the photocurrent is: y958-101.1 lgX, R2＝0.992。
The invention utilizes the light-addressable potentiometric sensor to realize the detection of the glucose concentration, so that the sensor which combines the photoelectric effect to realize the rapid on-site detection has the characteristics which other sensors do not have, and the light-addressable electrochemical sensor is firstly applied on the micron size.
According to the embodiment, preferably, the optical addressing electrochemical sensor is set to have the light source power set to be 1.0mW, the optical fiber modulation ratio is 0.8, the glucose oxidase GOx concentration is 30g/L, the pH value is about 7.0, and the optimal working pH value is very close to the human blood pH value of 7.35, which is beneficial to simplifying the sample processing procedure.
Measurement range: within the range of 0.01-100mm, there is a good log linear relationship between the glucose concentration and the signal of the photo-addressable electrochemical sensor, defined as Y958-101.1 lgX, R2And when the concentration is 0.992, X is the concentration mM of glucose in the sample, and Y is the voltage mV obtained after the sample with the concentration is detected by the photoaddress electrochemical sensor. The slope was 101.1 mV/decade, and when the glucose concentration exceeded 100mm, the photo-addressable electrochemical sensor began to lose sensitivity due to the saturation behavior of the enzyme reaction. From the predetermined glucose concentration and the sensor reading, a calibration chart is obtained, as shown in fig. 4.
And (3) sample analysis: the diabetic patients in the subsidiary hospital of Jiangsu university are taken as test objects, and the patients voluntarily participate. The collected blood and urine samples were subjected to clinical analysis and classified according to patient age into 30-45 years, 45-60 years and >60 years. Each sample was measured 3 times over a short time period of <180s, with the average of 3 readings as the final record. Meanwhile, glucose (NIST SRM 965b) in the serum of a frozen human is selected AS a standard sample, and an authenticated clinical analyzer (Labospect 008AS, Hitachi, Japan) is selected for comparison, and then the analysis is carried out by using a statistical method of a t test. The data obtained by the method and standard method of the present invention on the sample are shown in fig. 5 and 6, and the data of the two methods are not statistically different when the predetermined significance level is 0.01. The micro optical addressing electrochemical sensor based on the photoelectric effect has good sensitivity on glucose detection, can effectively realize the detection of the concentration of the glucose in real time, has the characteristics of quick detection response, accurate detection result, simple operation, wide measurable range, convenient carrying and the like, and can realize the field detection of the glucose and be applied to micro analysis and the like.
It should be understood that although the present description has been described in terms of various embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and those skilled in the art will recognize that the embodiments described herein may be combined as suitable to form other embodiments, as will be appreciated by those skilled in the art.
The above-listed detailed description is only a specific description of possible embodiments of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (9)

1. A photo-addressable electrochemical sensor is characterized by comprising an integrated active driver (1), a laser component (2), an optical fiber (6), a LAPS chip (7), a reference electrode (8) and a trans-impedance amplifier (9);
the laser assembly (2) comprises a laser diode (3) and a micro lens (5); the laser diode (3), the half-reflecting and half-transmitting mirror (11) and the micro lens (5) are arranged on the same light path, the laser diode (3) is connected with the integrated active driver (1), a light source of the laser diode (3) reaches one end of the optical fiber (6) through the micro lens (5), the other end of the optical fiber (6) is provided with a LAPS chip (7), and the LAPS chip (7) is used for measuring the concentration of glucose;
the LAPS chip (7) comprises a first gold layer, a silicon dioxide layer, an N-type silicon substrate and a second gold layer which are sequentially connected; diluting isolation epoxy glue with acetone, coating the isolation epoxy glue on other areas except the surface of the first gold layer, and dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer in sequence to serve as a sensing surface;
the semi-reflecting and semi-transmitting lens (11) belongs to a laser assembly (2), the laser assembly (2) further comprises a photodiode (4) and a reflector (12), the photodiode (4) is connected with the integrated active driver (1), part of light sources of the laser diode (3) reach one end of the optical fiber (6) through the semi-reflecting and semi-transmitting lens (11) and the micro lens (5), and the other part of light sources of the laser diode (3) are reflected to the photodiode (4) through the reflector (12);
the LAPS chip (7) and the reference electrode (8) are respectively connected with one end of a transimpedance amplifier (9), and the other end of the transimpedance amplifier (9) is used for connecting external equipment.
2. The photoaddressable electrochemical sensor according to claim 1, characterized in that the LAPS chip (7) comprises a first gold layer, a silicon dioxide layer, an N-type silicon substrate and a second gold layer connected in sequence; and diluting the isolating epoxy glue with acetone, coating the isolating epoxy glue on other areas except the surface of the first gold layer, and dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer in sequence to serve as a sensing surface.
3. An optically addressed electrochemical sensor according to claim 1, characterized in that the laser assembly (2) further comprises a photodiode (4), a semi-reflective and semi-transparent mirror (11) and a reflective mirror (12);
the photodiode (4) is connected with the integrated active driver (1), part of light sources of the laser diode (3) reach one end of the optical fiber (6) through the semi-reflecting and semi-transmitting mirror (11) and the micro lens (5), and the other part of light sources of the laser diode (3) are reflected to the photodiode (4) through the reflecting mirror (12).
4. A method of fabricating a photoaddressable electrochemical sensor according to any one of claims 1 to 3, comprising the steps of:
and (3) constructing a topological structure: the optical addressing electrochemical sensor comprises an integrated active driver (1), a laser component (2), an optical fiber (6), a LAPS chip (7), a reference electrode (8) and a trans-impedance amplifier (9);
the laser assembly (2) comprises a laser diode (3) and a micro lens (5); the laser diode (3), the half-reflecting and half-transmitting mirror (11) and the micro lens (5) are arranged on the same light path, the laser diode (3) is connected with the integrated active driver (1), a light source of the laser diode (3) reaches one end of the optical fiber (6) through the micro lens (5), the other end of the optical fiber (6) is provided with a LAPS chip (7), and the LAPS chip (7) is used for measuring the concentration of glucose;
the LAPS chip (7) and the reference electrode (8) are respectively connected with one end of a transimpedance amplifier (9), and the other end of the transimpedance amplifier (9) is used for connecting external equipment;
manufacturing and assembling the LAP S chip (7): taking an N-type silicon wafer as a substrate, growing a layer of silicon dioxide on each of two sides of the N-type silicon wafer under thermal dry oxidation, completely removing the layer of silicon dioxide by hydrofluoric acid (HF), depositing a first gold layer and a second gold layer on each of two sides by using a thermal evaporation method, photoetching a micro-ring on the second gold layer by photolithography, wherein the second gold layer is directly contacted with the surface of the N-type silicon wafer, and meanwhile, obtaining a solid gold surface, namely a first gold layer, on the opposite side; cutting the chip into a round chip, placing the optical fiber (6) in the center of a gold ring of the round chip, assembling the LAPS chip (7) and the optical fiber (6) by using silver glue, connecting the LAPS chip (7) and the transimpedance amplifier (9) by using the silver glue through a silver paint line, and coating the isolated epoxy glue on other areas except the surface of the first gold layer by using acetone to dilute the isolated epoxy glue;
functional modification of the sensor: sequentially dripping glucose oxidase GOx solution and perfluorosulfonic acid-polytetrafluoroethylene copolymer on the surface of the first gold layer to serve as a sensing surface;
manufacturing a reference electrode: the bare silver wire is placed in hydrochloric acid to generate an Ag-AgCl thin layer, and the Ag-AgCl thin layer is assembled near the LAPS chip (7) by epoxy resin to be used as a reference electrode;
assembling the sensor: the integrated active driver (1), the laser assembly (2) and the transimpedance amplifier (9) are assembled on a printed circuit board.
5. The method for manufacturing the optical addressing electrochemical sensor according to the claim 4, characterized in that, the laser component (2) in the step of constructing the topological structure further comprises a photodiode (4), a semi-reflecting and semi-transparent mirror (11) and a reflective mirror (12);
the photodiode (4) is connected with the integrated active driver (1), part of light sources of the laser diode (3) reach one end of the optical fiber (6) through the semi-reflecting and semi-transparent mirror (11) and the micro lens (5), and the other part of light sources of the laser diode (3) are reflected to the photodiode (4) through the reflecting mirror (12).
6. The method for manufacturing the photoaddressable electrochemical sensor according to claim 4, wherein the concentration of the glucose oxidase GOx solution in the step of modifying the sensor function is 10-50g/L, and the dosage is 0.5-1.0 μmL.
7. The method for manufacturing an optically addressed electrochemical sensor according to claim 4, wherein the perfluorosulfonic acid-polytetrafluoroethylene copolymer in the step of modifying the sensor function is a diluent prepared by diluting the perfluorosulfonic acid-polytetrafluoroethylene copolymer with 95% ethanol to a mass fraction of 1%, and the amount of the diluent is 0.4 μmL.
8. A method for detecting glucose using the photoaddressable electrochemical sensor according to any one of claims 1 to 3, comprising the steps of:
establishing an I/V curve of the photocurrent: the sensing surface of the LAPS chip (7) is directly contacted with a sample to be detected, and under the action of glucose oxidase GOx, the oxidation-reduction potential is changed along with the change of the glucose concentration of the sample, so that an I/V standard curve of photocurrent is established;
and measuring the concentration of the glucose to be measured according to the established I/V curve of the photocurrent: the sensing areas of the LAPS chip (7) and the reference electrode (8) are contacted with a sample to be tested, a depletion layer is generated after a direct current bias voltage is applied to the interface of an N-type silicon crystal substrate and a silicon dioxide layer, when the LAPS chip (7) is irradiated by modulated light, a photocurrent signal is amplified by a transimpedance amplifier (9) and transmitted to a computer for data analysis, the photocurrent is determined by the direct current bias voltage and the redox potential of a metal layer, and when glucose oxidase GOx exists on the sensing surface, enzyme catalysis reaction is carried out and H is induced to be generated2O2Due to H2O2The glucose concentration can be measured by the displacement of the standard I/V curve established on the photocurrent and the bias current.
9. The method as claimed in claim 8, wherein the measurement range is 0.01-100M, the glucose concentration has a good log-linear relationship with the signal of the electrochemical photo-addressable sensor, and the standard curve of the I/V of the photocurrent is: y958-101.11 gX, R20.992, X is glucose in the sampleThe concentration mM and Y are the voltage mV obtained after the concentration sample is detected by the photoaddress electrochemical sensor.
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