CN111781264A - Preparation method of PtNPs-based 3D paper-based electrochemical glucose sensor - Google Patents
Preparation method of PtNPs-based 3D paper-based electrochemical glucose sensor Download PDFInfo
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- 239000008103 glucose Substances 0.000 title claims abstract description 58
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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
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Abstract
The invention discloses a preparation method of a PtNPs-based 3D paper-based electrochemical glucose sensor, which comprises the steps of firstly preparing a 3D paper-based microfluidic screen printing electrode by a photoetching technology and a screen printing technology, wherein the 3D paper-based microfluidic screen printing electrode comprises a working electrode layer and a counter/reference electrode layer; and then modifying a working electrode of the 3D paper-based microfluidic screen printing electrode by adopting an electrodeposition platinum nano particle method, and then covalently fixing glucose oxidase in an aldehyde hydrophilic area of the counter/reference electrode to prepare the portable 3D paper-based microfluidic glucose sensor. Compared with the prior art, the preparation method of the 3D paper-based microfluidic glucose sensor is simple, convenient and quick, glucose oxidase is covalently immobilized through hydroformylation, and the 3D paper-based microfluidic glucose sensor has better repeatability, selectivity and stability for glucose and can be used for detecting glucose in body surface sweat.
Description
Technical Field
The invention belongs to the technical field of biomedical sensing, and particularly relates to a preparation method of a PtNPs-based 3D paper-based microfluidic electrochemical glucose sensor and a method for detecting glucose.
Background
Diabetes is one of three important diseases threatening the health of human beings, and is particularly important for controlling diabetes and complications thereof, and the clinical diagnosis of diabetes is mainly judged by measuring the concentration of glucose in blood. Therefore, accurate and rapid detection of glucose concentration in blood is of great importance for clinical control of diabetes and its complications. Currently, significant success has been achieved in the detection of glucose based on electrochemical methods, and thus commercial electrochemical glucose detectors have been developed. However, these products have the limitations of high cost, large batch-to-batch variation, invasiveness, need of frequent disinfection measures to avoid cross infection, incapability of continuous monitoring and the like. There are several valuable health-related analytes in human sweat, such as lactic acid, ammonium salts, and glucose. There have been a number of studies to date showing that there is some correlation between glucose concentration in blood and glucose concentration in sweat, and that glucose in sweat is in the concentration range of 10 μ M to 0.7 mM. Therefore, it is the focus of researchers to establish a fast, simple, accurate, painless and non-invasive method for detecting glucose from sweat.
The paper-based micro-fluidic chip is a novel micro-fluidic chip, and uses paper materials to replace other materials (such as silicon, glass, high polymer and the like) as a reaction substrate, so that the paper-based micro-fluidic chip forms a structure with hydrophilic/hydrophobic micro-channels and other related analytical devices. The paper-based microfluidic analytical device has the advantages of easy carrying, simple operation, low cost, less sample consumption, capability of performing multi-element detection, no need of external equipment and the like, and is expected to become the cheapest analytical detection device in the future. The 2D paper-based microfluidic chip is formed by preparing a closed hydrophobic boundary on paper by a physical or chemical method to form a microfluidic channel. The 3D paper-based microfluidic chip is prepared by stacking multiple layers of paper with microfluidic channel patterns and ensuring that the microfluidic channels between adjacent paper layers are communicated with each other. Both can be used as a substrate for filtering samples, carrying out chromatographic separation and carrying out biochemical reaction, but the 3D paper-based microfluidic chip has the capability of integrating complex microfluidic network channels, and provides more functions for the preparation of microfluidic channels. The analysis method applied to the paper-based microfluidic analytical device comprises a colorimetric method, an electrochemiluminescence method, a chemiluminescence method and a surface enhanced Raman spectroscopy. The use of paper-based microfluidic devices in combination with electrochemical analysis techniques is a new trend in modern analytical chemistry. The paper-based micro-fluidic electrochemical detection device has been rapidly developed in the fields of medicine, food safety, environmental monitoring and the like due to the advantages of less sample consumption, high analysis speed, low cost, easy miniaturization, integration, portability and the like of on-site POCT.
In recent years, the nano material is widely applied to electrochemical sensors and has a good effect of improving the analysis performance of the sensors. Among them, PtNPs have been widely used in the research of enzyme sensors because of their excellent electrocatalytic reduction performance for hydrogen peroxide. The invention modifies the working electrode of the 3D paper-based microfluidic control electrode by the method of electrodeposition of PtNPs, and then covalently fixes glucose oxidase in the hydroformylation hydrophilic region of the counter/reference electrode layer to prepare the portable 3D paper-based microfluidic glucose sensor.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of a novel disposable and environment-friendly PtNPs-based 3D paper-based microfluidic sensor which can be used for quantitatively detecting glucose in body surface sweat.
The technical scheme for realizing the purpose of the invention is as follows:
the invention relates to a PtNPs-based 3D paper-based microfluidic electrochemical sensor, which is prepared by modifying a working electrode of a 3D paper-based microfluidic electrode by adopting an electrodeposition PtNPs method and then fixing glucose oxidase in an aldehyde hydrophilic area of a counter/reference electrode.
The PtNPs-based 3D paper-based electrochemical glucose sensor comprises two layers of different paper-based microfluidic channels and screen-printed electrodes (SPEs) printed on the paper-based microfluidic channels; wherein, one layer of paper-based microfluidic channel is used for printing a carbon working electrode, and the other layer of paper-based microfluidic channel is used for printing a carbon counter electrode and a silver/silver chloride reference electrode; the PtNPs are electrodeposited on the working electrode, and the glucose oxidase is fixed on a microfluidic channel containing a counter electrode and a reference electrode.
The preparation method of the PtNPs-based 3D paper-based electrochemical glucose sensor comprises the following steps:
1. aldehyde treatment of filter paper
First, filter paper is treated with KIO4Soaking in a thermostat for a period of time until the filter paper and the KIO are mixed4After complete reaction, washing with deionized water for 3 times, and soaking in deionized water for 1 minute each time; finally, the excess water on the filter paper was blotted with a paper towel and placed in a desiccator to completely dry the filter paper.
The filter paper is Whatman No.1 filter paper;
the KIO4The concentration of the (A) is 0.01-0.1M, the temperature in the thermostat is 50-75 ℃, and the soaking time is 1-4 hours.
2. Preparation of paper-based microfluidic channel
A piece of filter paper is placed on a tray of a spin coater, and SU-82007 photoresist is dripped in the center of the filter paper. Under the action of the spin coater, the photoresist is uniformly distributed on the filter paper. The filter paper is first soft baked at 95 deg.c for 10 min, then covered with prepared mask and aligned under ultraviolet lamp for 30 min. And (3) after exposure, hard baking for 2-3 minutes at 95 ℃, firstly putting the filter paper into acetone for soaking for 1 minute, and then washing for three times by using isopropanol so as to thoroughly wash the unpolymerized photoresist. The parts which are not shielded are polymerized by the photoresist under the irradiation of an ultraviolet lamp to form a hydrophobic barrier, the areas which are surrounded by the hydrophobic barrier and are not covered by the photoresist form a hydrophilic area, and finally the patterned paper-based microfluidic channel is formed.
The using amount of the SU-8 photoresist is 1-5 mL;
the dosage of the acetone and the isopropanol is 8-30 mL respectively.
3. Preparation of paper-based screen printing electrode
The paper-based screen printing electrode is a three-electrode system, namely a working electrode, a counter electrode and a reference electrode, wherein the working electrode is a layer, and the counter electrode and the reference electrode are a layer, namely a counter/reference electrode layer for short. Before printing, printing inks are prepared, mainly including carbon ink and silver/silver chloride (Ag/AgCl) ink. The working part of the paper-based screen-printed electrode is printed on the hydrophilic area of the paper-based microfluidic device, and the contact part is printed on the hydrophobic area of the paper-based microfluidic device. The screen printing process is prepared from three layers: the first layer was carbon ink printed for use as a working electrode, the second layer was carbon ink printed for use as a counter electrode, and the third layer was Ag/AgCl ink printed for use as a reference electrode. After each layer was printed on the screen printer, it was dried in a 60 ℃ oven for 30 minutes and cooled at room temperature before proceeding to the next step.
4. Modification of working electrodes
10 μ L of 4.0 mM H was added dropwise to the hydrophilic region of the working electrode layer2PtCl6And 10. mu.L of 0.5M KCl solution, stabilized at room temperature for 5 minutes, and applied with a voltage of-0.6V for 100 seconds to electrodeposit PtNPs on the surface of the working electrode.
5. Immobilization of glucose oxidase
Preparing glucose oxidase by using PBS buffer solution, dripping glucose oxidase solution into an aldehydized hydrophilic region of the paper-based screen printing electrode pair/reference electrode layer, and incubating overnight in a refrigerator at 4 ℃;
the concentration of the glucose oxidase solution is 1 mg/mL-10 mg/mL.
6. Electrochemical detection
(1) Assembling the working electrode modified with PtNPs and a counter/reference electrode fixed with glucose oxidase to form a complete 3D paper-based electrochemical glucose sensor;
(2) preparing glucose standard solutions with different concentrations by using a PBS buffer solution;
(3) recording the current generated by the 3D paper-based electrochemical glucose sensor in the glucose solution with different concentrations through an I-t curve method to obtain a corresponding regression equation and a relevant coefficient;
the initial potential of the I-t curve method is-0.4V;
(4) and (4) detecting the glucose solution to be detected according to the step (3) to obtain an I-t curve current, and substituting the I-t curve current into the standard curve established in the step (3) to obtain the concentration of glucose in the sample to be detected.
Compared with the prior art, the invention has the following beneficial effects:
1. the paper-based micro-fluidic channel is used for covalent fixation of glucose oxidase by performing hydroformylation treatment on the hydrophilic region of the paper-based micro-fluidic channel for the first time, so that the complex process that other materials are used for modifying electrodes to fix biomolecules on the traditional electrochemical sensor is overcome, and the repeatability of the sensor is improved.
2. According to the invention, the working electrode of the 3D paper-based microfluidic substrate electrode is modified by an electrodeposition PtNPs method, the modified electrode with large specific surface area and good conductivity is prepared, and a sensitive electron transfer channel is constructed, so that the detection potential of the sensor is reduced, the anti-interference performance of the sensor is improved, and the detection sensitivity of the sensor is greatly improved.
3. The permeability of the 3D paper-based microfluidic electrochemical sensor prepared by the invention provides a simpler, more convenient and safer noninvasive body surface online sweat and glucose quantitative detection device.
Drawings
FIG. 1 is a schematic diagram of the structure of a 3D paper-based electrochemical glucose sensor;
in the figure, (a) a working electrode layer, (b) a counter/reference electrode layer, a carbon working electrode 1, a carbon counter electrode 2, a silver/silver chloride reference electrode 3, a hydrophobic region 4, and a hydrophilic region 5.
FIG. 2 (A) is a scanning electron microscope image of a bare working electrode, (B) is a scanning electron microscope image of a PtNPs-modified working electrode, (C) is an XPS image of (a) a bare working electrode and (B) a PtNPs-modified working electrode, and (D) is an XPS image of PtNPs.
FIG. 3 shows bare SPEs andPtNPs modify SPEs at H2O2Cyclic voltammograms in solution, in particular bare electrodes in (a) PBS and (b) 1.0 mM H2O2Cyclic voltammograms of (1); PtNPs modified electrodes in (c) PBS, (d) 0.25 mMH2O2And (e) 0.4 mM H2O2Cyclic voltammogram of (1).
FIG. 4 shows the continuous dropwise addition of H onto a PtNPs-modified electrode2O2The latter current response;
FIG. 4 (A) PtNPs modified electrode 1.0 mM H was continuously added dropwise at intervals of 50s2O2Graph of (A), (B) response current and H2O2Linear relationship between concentrations.
FIG. 5 (A) I-t plots of PtNPs modified electrodes in glucose solutions of different concentrations (0, 0.001, 1, 3, 5, 7, 9, 11mM), (B) standard curves for glucose.
Detailed Description
The invention will be further described with reference to the following drawings and examples, which are given for illustration and not for limitation of the invention.
Example 1
A PtNPs-based 3D paper-based electrochemical glucose sensor is shown in figure 1 and comprises two layers of paper-based microfluidic channels, namely a working electrode layer (figure 1 a) and a counter/reference electrode layer (figure 1 b), wherein a working part of a paper-based screen printing electrode is printed in a hydrophilic area 5, a contact part is printed in a hydrophobic area 4, a carbon working electrode 1 is printed on the working electrode layer, and a carbon counter electrode 2 and a silver/silver chloride reference electrode 3 are printed on the counter/reference electrode layer; PtNPs are electrodeposited on the working electrode, and glucose oxidase is fixed on the hydrophilic region of the counter/reference electrode.
Example 2
Preparation of a PtNPs-based 3D paper-based electrochemical glucose sensor, which comprises the following steps:
(1) firstly, a plurality of Whatman No.1 filter papers are soaked in 0.03M KIO4And (3) incubating the solution for 2h at 65 ℃ in a thermostat to realize the hydroformylation of hydroxyl groups on the surface of the filter paper. After the hydroformylation is complete, fresh deionization is usedThe filter paper was rinsed with water 2 times for 1min each time. And finally, absorbing excessive water on the surface of the washed aldehyde filter paper by using dry absorbent paper, and fully drying in a dryer.
(2) Fig. 1a and fig. 1b show the hydrophilic area and the hydrophobic area of the 3D paper-based microfluidic channel, respectively, and the preparation process of the paper-based microfluidic channel is as follows: 2.5 mL of SU-82007 negative photoresist is poured onto the center of the aldehyde-modified Whatman No.1 filter paper, and the photoresist is uniformly distributed and permeated into the filter paper under the action of a spin coater (500 rpm, 15s; 6500 rpm, 60 s). The photoresist-coated filter paper was first baked on a bake station at 95 ℃ for 10 min to remove the solvent on the filter paper. Then, a pre-designed ultraviolet mask plate is covered on the filter paper, tightly attached and exposed for 1min under an ultraviolet exposure lamp. And baking the exposed filter paper on a glue drying table at 95 ℃ for 3 min, soaking the filter paper in 15 mL of acetone and washing the filter paper with isopropanol for three times to completely remove unpolymerized photoresist on the surface of the filter paper, and finally forming the patterned microfluidic channel on the filter paper.
(3) The structure of the working electrode, the counter electrode and the reference electrode of the 3D paper-based screen-printed electrode is shown in figure 1. The working portion of the paper-based screen-printed electrode was printed on the hydrophilic area of the glyoxal functionalized microfluidic device and the contact portion was printed on the hydrophobic area of the microfluidic device. The 3D paper-based screen-printed electrode consists of 2 layers of microfluidic devices (fig. 1), one layer (fig. 1 a) being used to print the working electrode, i.e. the working electrode layer; the other layer (fig. 1 b) is used to print the counter and reference electrodes, i.e. the counter/reference electrode layer. The silk-screen process is prepared by three steps: firstly, printing carbon ink on a working electrode layer (figure 1 a) to prepare a carbon working electrode 1, secondly, printing carbon ink on a counter/reference electrode layer (figure 1 b) to prepare a carbon counter electrode 2, and finally, printing silver/silver chloride ink on the counter/reference electrode layer to prepare a reference electrode 3, wherein after each layer is printed, the carbon working electrode is dried at 60 ℃ for 30 minutes, and then the next step is carried out after the carbon working electrode is cooled at room temperature.
(4) 10 mu L of 4.0 mM H is dripped into the hydrophilic region of the working electrode layer2PtCl6And 10. mu.L of 0.5M KCl solution, stabilized at room temperature for 5 minutes, and applied with-0.6V for 100 seconds to allow Pt to standNPs are electrodeposited on the surface of the working electrode. Then 20 mu L of 6 mg/mL glucose oxidase solution is dripped into the aldehyde hydrophilic region of the counter/reference electrode layer, and the mixture is incubated for 12h in a refrigerator at 4 ℃. After preparation, 2 layers are stacked and used, namely, a complete 3D paper-based microfluidic detection substrate electrode.
The surface morphology of the carbon working electrode before and after modification is characterized by a scanning electron microscope and X-ray photoelectron spectroscopy, and it can be seen from the figure that the surface of the bare working electrode (figure 2A) of the 3D paper-based microfluidic substrate electrode is in a uniform sheet structure, and the surface of the working electrode modified by PtNPs is obviously added with a plurality of nano particles (figure 2B). In addition, Pt element is added to the X-ray photoelectron spectrum of the modified working electrode, which indicates that PtNPs are successfully deposited on the surface of the working electrode, as shown in FIGS. 2C and 2D.
FIG. 3 shows naked SPEs and PtNPs modified SPEs at H2O2Cyclic voltammograms in solution. As can be seen, the bare SPEs are at H2O2None of the solutions (curve b) had a significant redox peak. Whereas PtNPs modify SPEs at H2O2The reduction current in the solution is obviously increased and follows with H2O2The reduction current increases with increasing concentration (curves d, e). This is due to H2O2The catalytic reduction of Pt on the surface of the electrode indicates that the modified electrode pair H2O2Has obvious catalytic response.
FIG. 4 shows the continuous dropwise addition of H onto a PtNPs-modified electrode2O2The latter current response. FIG. 4A shows a graph with H2O2The reduction current tends to increase stepwise as the concentration increases. And the reduction current with increased H can be seen in FIG. 4B2O2The concentration is in good linear relation, which shows that the PtNPs modified electrode can be used for H2O2Continuous detection of (2).
The current response of glucose solutions with different concentrations was measured by the I-t curve method, and the results are shown in FIG. 5A; when the glucose solution is between 1 μ M and 12 mM, a good linear relationship exists between the obtained response current and the glucose concentration, as shown in FIG. 5B.
In order to evaluate the practical application value of the 3D paper-based microfluidic glucose sensor, the 3D paper-based microfluidic glucose sensor is used for measuring glucose in human sweat. Selecting 3 healthy volunteers, carrying out 30 min vigorous exercise, collecting sweat samples from the skin surfaces of the 3 volunteers to a detection area of the sensor after the body sweats slightly, and detecting the response current of the sensors at-0.4V by using a chronoamperometry. In order to verify the reliability of the method, 3 sweat samples are also subjected to spectrophotometry by using a commercial kit, the comparative detection results are shown in table 1, and the data in the table show that the detection results of the 2 methods are small in difference, which indicates that the 3D paper-based microfluidic glucose sensor has high feasibility for detecting glucose in sweat.
TABLE 1 comparison of results of electrochemical and spectrophotometric measurements of glucose in sweat
Claims (7)
1. The PtNPs-based 3D paper-based electrochemical glucose sensor is characterized by comprising two layers of different paper-based microfluidic channels and screen-printed electrodes printed on the paper-based microfluidic channels; wherein, one layer of paper-based microfluidic channel is used for printing a carbon working electrode, and the other layer of paper-based microfluidic channel is used for printing a carbon counter electrode and a silver/silver chloride reference electrode; platinum nano particles PtNPs are electrodeposited on the working electrode, and glucose oxidase is fixed on a microfluidic channel containing a counter electrode and a reference electrode.
2. The 3D paper-based electrochemical glucose sensor according to claim 1, wherein the paper-based microfluidic channel is prepared from Whatman No.1 filter paper, and the hydrophobic region of the paper-based microfluidic channel is prepared by photolithography, and the detailed process is as follows: firstly, 1-5 mL of SU-8 photoresist is uniformly distributed on filter paper, then a designed ultraviolet mask plate is covered on the filter paper, and the filter paper is exposed for 2-30 min under an ultraviolet exposure lamp;
then baking the exposed filter paper on a glue drying table at 95 ℃ for 3-10 min, and then sequentially washing the filter paper for 3-5 times by using 8-30 mL of acetone and isopropanol to remove the unpolymerized photoresist on the filter paper;
finally, a micro-fluidic paper-based analysis device with patterns is formed;
the region of the microfluidic paper-based analysis device on which the photoresist was polymerized forms a hydrophobic barrier, and the region surrounded by the hydrophobic barrier that is not covered by the photoresist forms a hydrophilic region.
3. The 3D paper-based electrochemical glucose sensor according to claim 2, characterized in that the working part of the paper-based screen-printed electrode is printed in a hydrophilic area, the contact part is printed in a hydrophobic area, carbon ink used as a working electrode and a counter electrode is firstly printed, then silver/silver chloride ink used as a reference electrode and a conductive contact is printed, each layer is dried in a drying oven for 20-60 min after being printed, and the next step is carried out after being cooled at room temperature.
4. The 3D paper-based electrochemical glucose sensor as claimed in claim 1, wherein the filter paper for printing the counter electrode and the reference electrode is previously subjected to an aldehyde treatment before use, and the detailed process is as follows: firstly, soaking filter paper in 0.01-0.1M KIO4Soaking in the solution for 1-4 hours in a thermostat with the temperature of 50-75 ℃ until the filter paper and the KIO are mixed4After complete reaction, washing for 3 times by deionized water, soaking in deionized water for 1 minute each time, and then washing; finally, the excess water on the washed filter paper was blotted with a paper towel and placed in a desiccator for 12 hours to completely dry the filter paper.
5. The 3D paper-based electrochemical glucose sensor of claim 1, wherein: the deposition method of the PtNPs is as follows: 15 mu L of 4.0 mM H is dripped into the hydrophilic area of the paper-based screen printing electrode2PtCl6And 15. mu.L of a 0.5 MKCl solution, stabilized at room temperature for 5 min, deposited at a potential of-0.6V for 100s to deposit PtNPs on the surface of the working electrode, followed by washing with PBS,and drying at room temperature for later use.
6. The 3D paper-based electrochemical glucose sensor of claim 1, wherein: the fixing method of the glucose oxidase comprises the following steps: preparing glucose oxidase by using PBS buffer solution, dripping 20 mu L of glucose oxidase solution into a hydrophilic region of a paper-based screen printing electrode pair/reference electrode layer, and reacting for 12 hours in a refrigerator at 4 ℃; the concentration of the glucose oxidase solution is 1 mg/mL-10 mg/mL.
7. The PtNPs-based 3D paper-based electrochemical glucose sensor according to any one of claims 1 to 6, applied to the detection of glucose, comprising the following steps:
(1) assembling the working electrode modified with PtNPs and a counter/reference electrode fixed with glucose oxidase to form a complete 3D paper-based electrochemical glucose sensor;
(2) preparing glucose standard solutions with the concentrations of 0, 0.001, 1, 3, 5, 7, 9 and 11mM by using a PBS buffer solution;
(3) recording an I-t curve graph of the 3D paper-based electrochemical glucose sensor in glucose solutions with different concentrations through an I-t curve method to obtain a corresponding regression equation and a relevant coefficient;
the initial potential of the I-t curve method is-0.4V;
(4) and (4) detecting the glucose solution with the concentration to be detected according to the step (3) to obtain an I-t curve current, and substituting the I-t curve current into the standard curve established in the step (3) to obtain the concentration of the glucose in the sample to be detected.
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