CN111781264A - Preparation of 3D paper-based electrochemical glucose sensor based on PtNPs - Google Patents

Abstract

The invention discloses a preparation method of a 3D paper-based electrochemical glucose sensor based on PtNPs. The method firstly prepares a 3D paper-based microfluidic screen printing electrode by photolithography technology and screen printing technology, including a working electrode layer and counter/ Reference electrode layer; then the working electrode of the 3D paper-based microfluidic screen-printed electrode was modified by electrodeposition of platinum nanoparticles, followed by covalent immobilization of glucose in the aldehyde-based hydrophilic region of the counter/reference electrode oxidase, a portable 3D paper-based microfluidic glucose sensor was fabricated. Compared with the prior art, the preparation method of the 3D paper-based microfluidic glucose sensor of the present invention is simple and fast, and the glucose oxidase is covalently immobilized by acetaldehyde, and has good repeatability, selectivity and stability to glucose. , and can be used for the detection of glucose in surface sweat.

Description

Preparation of 3D paper-based electrochemical glucose sensor based on PtNPs

technical field

The invention belongs to the technical field of biomedical sensing, and in particular relates to a preparation method of a PtNPs-based 3D paper-based microfluidic electrochemical glucose sensor and a method for glucose detection.

Background technique

Diabetes is one of the three major diseases that threaten our human health. It is particularly important to control diabetes and its complications. The clinical diagnosis of diabetes is mainly judged by measuring the concentration of glucose in the blood. Therefore, accurate and rapid detection of glucose concentration in blood is of great significance to the clinical control of diabetes and its complications. At present, the detection of glucose based on electrochemical methods has achieved remarkable results, and thus a commercial electrochemical glucose detector has been developed. However, these products have limitations such as high cost, large batch-to-batch variation, invasiveness, frequent use of disinfection measures to avoid cross-infection, and inability to continuously monitor. Several valuable analytes such as lactic acid, ammonium salts, and glucose, which are related to health conditions, are present in human sweat. A large number of studies have shown that there is a certain correlation between the glucose concentration in blood and the glucose concentration in sweat, and the glucose concentration in sweat is in the range of 10 μM to 0.7 mM. Therefore, establishing a fast, simple, accurate, painless and non-invasive method to detect glucose from sweat is the focus of researchers.

Paper-based microfluidic chip is a new type of microfluidic chip, which uses paper material to replace other materials (such as silicon, glass, polymer, etc.) as the reaction substrate, so that it can form hydrophilic/hydrophobic microchannels and other related Analyze the structure of the device. The paper-based microfluidic analytical device has the advantages of easy portability, simple operation, low cost, low sample consumption, multivariate detection, and no need for external equipment. It is expected to become the cheapest analytical detection device in the future. The 2D paper-based microfluidic chip forms a microfluidic channel by preparing a closed hydrophobic boundary on paper by physical or chemical methods. The 3D paper-based microfluidic chip is fabricated by stacking multiple layers of paper patterned with microfluidic channels and ensuring that the microfluidic channels between adjacent paper layers are connected to each other. Both can be used as substrates for filtration of samples, chromatographic separation and biochemical reactions, but the 3D paper-based microfluidic chip has the ability to integrate complex microfluidic network channels, providing more functions for the preparation of microfluidic channels. The analytical methods applied on paper-based microfluidic analytical devices include colorimetry, electrochemiluminescence, chemiluminescence, and surface-enhanced Raman spectroscopy. Combining paper-based microfluidic devices with electrochemical analysis techniques is a new trend in modern analytical chemistry. Paper-based microfluidic electrochemical detection devices have been rapidly developed in the fields of medicine, food safety and environmental monitoring due to their low sample consumption, fast analysis speed, low cost, easy miniaturization, integration, portability and other field POCT advantages. .

In recent years, nanomaterials have been widely used in electrochemical sensors, which have a good effect on improving the analytical performance of sensors. Among them, PtNPs have been widely used in the research of enzyme sensors due to their good electrocatalytic reduction performance for hydrogen peroxide. In the present invention, the working electrode of the 3D paper-based microfluidic electrode is modified by the method of electrodepositing PtNPs, and then the glucose oxidase is covalently fixed in the aldehyde-based hydrophilic region of the counter/reference electrode layer to prepare a portable The 3D paper-based microfluidic glucose sensor has a simple and fast preparation method, has good repeatability, selectivity and stability for glucose, and can be used for the detection of glucose in body surface sweat.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a new type of disposable and environmentally friendly preparation method of a PtNPs-based 3D paper-based microfluidic sensor that can be used for quantitative detection of glucose in body surface sweat .

The technical scheme that realizes the object of the present invention is:

The present invention is a 3D paper-based microfluidic electrochemical sensor based on PtNPs. The working electrode of the 3D paper-based microfluidic electrode is modified by the method of electrodepositing PtNPs, and then the aldehyde-based hydrophilicity of the control/reference electrode is applied. A portable 3D paper-based glucose sensor was fabricated by immobilizing glucose oxidase in the sexual region.

The PtNPs-based 3D paper-based electrochemical glucose sensor includes 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 channels The microfluidic channel was used to print the carbon working electrode, and another layer of paper-based microfluidic channel was used to print the carbon counter electrode and the silver/silver chloride reference electrode; Glucose oxidase is immobilized on the microfluidic channel of the electrode and the reference electrode.

The preparation method of the PtNPs-based 3D paper-based electrochemical glucose sensor includes the following processes:

1. Formaldehyde treatment of filter paper

First, soak the filter paper with KIO ₄ and soak it in a constant temperature box for a period of time. After the filter paper and KIO ₄ are completely reacted, they are washed 3 times with deionized water, and soaked in deionized water for 1 minute each time; Absorb excess moisture and place in a desiccator to allow the filter paper to dry completely.

Described filter paper is Whatman No.1 filter paper;

The concentration of the KIO ₄ is 0.01 M~0.1 M, the temperature in the incubator is 50~75°C, and the soaking time is 1~4 hours.

2. Fabrication of Paper-Based Microfluidic Channels

Take a piece of filter paper and place it on the tray of the dispenser, and drop SU-8 2007 photoresist in the center of the filter paper. Under the action of the glue dispenser, the photoresist is evenly distributed on the filter paper. First put the filter paper in a soft bake at 95°C for 10 minutes, then cover the filter paper with the prepared mask and expose it under UV light for 30 minutes. After exposure, hard bake at 95°C for 2~3 minutes, soak the filter paper in acetone for 1 minute, and then wash it with isopropanol three times to completely wash off the unpolymerized photoresist. The parts that are not blocked are polymerized by the photoresist under the irradiation of the UV lamp to form a hydrophobic barrier, and the area surrounded by the hydrophobic barrier that is not covered by the photoresist forms a hydrophilic area, and finally a patterned area is formed. Paper-based microfluidic channels.

The amount of the SU-8 photoresist is 1-5 mL;

The consumption of described acetone and isopropanol is respectively 8～30 mL.

3. Preparation of Paper-Based Screen-Printed Electrodes

The paper-based screen printing electrode is a three-electrode system, that is, a working electrode, a counter electrode and a reference electrode, in which the working electrode is one layer, and the counter electrode and the reference electrode are one layer, referred to as the counter/reference electrode layer. Before printing, the ink for printing should be prepared first, mainly carbon ink and silver/silver chloride (Ag/AgCl) ink. The working part of the paper-based screen-printed electrode is printed on the hydrophilic region of the paper-based microfluidic device, and the contact part is printed on the hydrophobic region of the paper-based microfluidic device. The screen printing process is prepared by three layers: the first layer is printed carbon ink used as working electrode, the second layer is printed carbon ink used as counter electrode, and the third layer is printed Ag used as reference electrode /AgCl ink. After each layer was printed on the screen printer, it was dried in a 60°C incubator for 30 minutes, and cooled at room temperature before proceeding to the next step.

4. Modification of the Working Electrode

10 μL of 4.0 mM H ₂ PtCl ₆ and 10 μL of 0.5 M KCl solution were added dropwise to the hydrophilic area of the working electrode layer, stabilized at room temperature for 5 min, and applied a voltage of -0.6 V for 100 s to electrodeposit PtNPs on the surface of the working electrode. .

5. Immobilization of Glucose Oxidase

Glucose oxidase was prepared with PBS buffer, glucose oxidase solution was added dropwise to the aldehyde-based hydrophilic area of the paper-based screen-printed electrode pair/reference electrode layer, and incubated overnight in a 4°C refrigerator;

The concentration of the glucose oxidase solution is 1 mg/mL to 10 mg/mL.

6. Electrochemical detection

(1) The modified PtNPs working electrode and the glucose oxidase-immobilized counter/reference electrode were assembled to form a complete 3D paper-based electrochemical glucose sensor;

(2) Use PBS buffer to configure glucose standard solutions of different concentrations;

(3) The current generated by the reaction of the 3D paper-based electrochemical glucose sensor in different concentrations of glucose solution was recorded by the I-t curve method, and the corresponding regression equation and correlation coefficient were obtained;

The initial potential of the I-t curve method is -0.4 V;

(4) Detect the glucose solution to be tested according to step (3) to obtain the I-t curve current, which is substituted into the standard curve established in step (3) to obtain the concentration of glucose in the sample to be tested.

Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

1. For the first time, the present invention is used to covalently immobilize glucose oxidase by performing aldehyde treatment on the hydrophilic region of the paper-based microfluidic channel, which overcomes the complex need of modifying electrodes with other materials to immobilize biomolecules on traditional electrochemical sensors. process, which improves the reproducibility of the sensor.

2. In the invention, the working electrode of the 3D paper-based microfluidic substrate electrode is modified by the method of electrodepositing PtNPs, the modified electrode with large specific surface area and good conductivity is prepared, and a sensitive electron transfer channel is constructed, which not only reduces the detection of the sensor The potential of the sensor improves the anti-interference performance of the sensor, and also greatly improves the detection sensitivity of the sensor.

3. By utilizing the permeability of the 3D paper-based microfluidic electrochemical sensor prepared by the invention, a simpler and safer non-invasive body surface online sweat glucose quantitative detection device is provided.

Description of drawings

Figure 1 is a schematic structural diagram of a 3D paper-based electrochemical glucose sensor;

In the figure, (a) working electrode layer, (b) counter/reference electrode layer, carbon working electrode 1, carbon counter electrode 2, silver/silver chloride reference electrode 3, hydrophobic region 4, hydrophilic region 5 .

Figure 2 (A) SEM image of bare working electrode, (B) SEM image of PtNPs modified working electrode, (C) XPS image of (a) bare working electrode and (b) PtNPs modified working electrode, (D) XPS plot of PtNPs.

Figure 3 shows the cyclic voltammograms of bare SPEs and PtNPs-modified SPEs in H ₂ O ₂ solution, specifically the cyclic voltammograms of bare electrodes in (a) PBS and (b) 1.0 mM H ₂ O ₂ ; PtNPs _{Cyclic voltammograms} of modified electrodes in (c) PBS, (d) 0.25 mM H2O2 and ₍ e ₎ 0.4 mM H2O2.

Figure 4 shows the current response after continuous dropwise addition of H ₂ O ₂ on the PtNPs modified electrode;

Figure 4 (A) It curve of PtNPs-modified electrode with continuous dropwise addition of 1.0 mM H ₂ O ₂ at intervals of 50 s, (B) the linear relationship between the response current and the concentration of H ₂ O ₂ .

Figure 5 (A) I-t curves of PtNPs modified electrodes in different concentrations of glucose solution (0, 0.001, 1, 3, 5, 7, 9, 11 mM), (B) standard curve of glucose.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and examples, and the examples are only used to illustrate the present invention and not to limit the present invention.

Example 1

A 3D paper-based electrochemical glucose sensor based on PtNPs, as shown in Fig. 1, includes two layers of paper-based microfluidic channels, namely the working electrode layer (Fig. 1a) and the counter/reference electrode layer (Fig. 1b), The working part of the paper-based screen printed electrode is printed on the hydrophilic area 5, the contact part is printed on the hydrophobic area 4, the carbon working electrode 1 is printed on the working electrode layer, and the carbon counter electrode 2 is printed on the counter/reference electrode layer and silver/silver chloride reference electrode 3; PtNPs were electrodeposited on the working electrode, and glucose oxidase was immobilized on the hydrophilic region of the counter/reference electrode.

Example 2

The preparation of a 3D paper-based electrochemical glucose sensor based on PtNPs includes the following steps:

(1) First, soak several sheets of Whatman No. 1 filter paper in 0.03 M KIO ₄ solution and incubate at 65 °C for 2 h in an incubator to achieve the aldehyde grouping of hydroxyl groups on the surface of the filter paper. After the aldehydeylation was completed, the filter paper was rinsed twice with fresh deionized water for 1 min each time. Finally, use dry absorbent paper to absorb the excess water on the surface of the washed aldehyde-based filter paper and fully dry it in a desiccator.

(2) Figure 1a and Figure 1b show the hydrophilic and hydrophobic regions of the 3D paper-based microfluidic channel, respectively. The preparation process of the paper-based microfluidic channel is as follows: take 2.5 mL of SU-8 2007 negative photoresist Pour it on the center of whatman No. 1 filter paper modified with aldehyde groups, and under the action of a glue dispenser (500 rpm, 15s; 6500 rpm, 60s), the photoresist is evenly distributed and penetrated into the filter paper. First, the filter paper covered with photoresist was baked on a glue baking table at 95 °C for 10 min to remove the solvent on the filter paper. Then, cover the pre-designed UV mask on the filter paper, stick it tightly, and expose it under the UV exposure lamp for 1 min. After the exposed filter paper was baked at 95 °C for 3 min on the glue drying table, it was soaked in 15 mL of acetone and washed with isopropanol for three times to completely remove the unpolymerized photoresist on the surface of the filter paper. Finally, a band formed on the filter paper. Patterned microfluidic channels.

(3) The structures of the working electrode, counter electrode and reference electrode of the 3D paper-based screen-printed electrode are shown in Figure 1. The working part of the paper-based screen-printed electrode is printed on the hydrophilic region of the acetaldehyde-based functionalized microfluidic device, while the contact part is printed on the hydrophobic region of the microfluidic device. The 3D paper-based screen-printed electrode consists of a 2-layer microfluidic device (Fig. 1), one layer (Fig. 1a) is used to print the working electrode, the working electrode layer; the other layer (Fig. 1b) is used to print the counter electrode and the The reference electrode, the counter/reference electrode layer. The screen printing process was prepared in three steps: firstly, carbon ink was printed on the working electrode layer (Fig. 1a) to prepare carbon working electrode 1, and secondly, carbon ink was printed on the counter/reference electrode layer (Fig. 1b) to prepare carbon pair. Electrode 2, and finally the reference electrode 3 was prepared by printing silver/silver chloride ink on the counter/reference electrode layer. step.

(4) Add 10 μL of 4.0 mM H ₂ PtCl ₆ and 10 μL of 0.5 M KCl solution dropwise to the hydrophilic area of the working electrode layer, stabilize at room temperature for 5 minutes, and apply a voltage of -0.6 V for 100 s to electrodeposit PtNPs on the working electrode. surface. Then, 20 μL of 6 mg/mL glucose oxidase solution was added dropwise to the aldehyde-based hydrophilic area of the counter/reference electrode layer, and incubated at 4 °C for 12 h. After preparation, the two layers are stacked together to form a complete 3D paper-based microfluidic detection substrate electrode.

Scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the surface morphology of the carbon working electrode before and after modification. It can be seen from the figure that the surface of the bare working electrode (Fig. 2A) of the 3D paper-based microfluidic substrate electrode is a uniform sheet However, the surface of the PtNPs-modified working electrode obviously increased many nanoparticles (Fig. 2B). In addition, Pt element was added to the X-ray photoelectron spectrum of the modified working electrode, indicating that PtNPs were successfully deposited on the surface of the working electrode, as shown in Figures 2C and 2D.

Figure 3 shows the cyclic voltammograms of bare SPEs and PtNPs-modified SPEs in H ₂ O ₂ solution. It can be seen from the figure that the bare SPEs have no obvious redox peaks in the H2O2 solution ₍ curve b ₎ . However, the reduction current of PtNPs-modified SPEs in H ₂ O ₂ solution increased significantly, and the reduction current also increased with the increase of H ₂ O ₂ concentration (curves d, e). This is due to the catalytic reduction of H ₂ O ₂ by Pt on the electrode surface, indicating that the modified electrode has an obvious catalytic response to H ₂ O ₂ .

Figure 4 shows the current response after continuous dropwise addition of H2O2 _on the PtNPs _- modified electrode. Figure 4A shows a stepwise increase in the reduction current with increasing _{H2O2 concentration} . And it can be seen from Fig. 4B that there is a good linear relationship between the reduction current and the increasing _H2O2 concentration, indicating that the _{PtNPs modified} electrode can be used for the continuous detection of _H2O2 .

The I-t curve method was used to detect the current response of glucose solutions with different concentrations, and the results are shown in Figure 5A; when the glucose solution was between 1 μM and 12 mM, there was a good linear relationship between the response current and the glucose concentration, as shown in Figure 5B shown.

In order to evaluate the practical application value of the 3D paper-based microfluidic glucose sensor, the 3D paper-based microfluidic sensor was used to measure glucose in human sweat. Three healthy volunteers were selected, after 30 minutes of vigorous exercise, and after sweating a little, the sweat samples were collected from the skin surface of the three volunteers to the detection area of the sensor, and the chronoamperometry was used to detect the temperature at -0.4 V. response current. In order to verify the reliability of this method, the three sweat samples were also measured spectrophotometrically with a commercial kit. The comparative test results are shown in Table 1. From the data in the table, it can be seen that the difference between the test results of the two methods is small, indicating that The 3D paper-based microfluidic glucose sensor has high feasibility for the determination of glucose in sweat.

Table 1 Comparison of the results of electrochemical and spectrophotometric determination of glucose in sweat

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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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