CN111812170A - Three-dimensional paper-based electrochemical sensing device and preparation method and application thereof - Google Patents
Three-dimensional paper-based electrochemical sensing device and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B25/00—Phosphorus; Compounds thereof
- C01B25/16—Oxyacids of phosphorus; Salts thereof
- C01B25/26—Phosphates
- C01B25/45—Phosphates containing plural metal, or metal and ammonium
Abstract
The invention discloses a preparation method of a three-dimensional paper-based electrochemical sensing device, which comprises the following steps: the method comprises the following steps: providing fiber paper, an upper plastic package film and a lower plastic package film, wherein the upper plastic package film is provided with holes with the shape consistent with that of the fiber paper; step two: clamping fiber paper between the upper plastic package film and the lower plastic package film to make the opening aligned with the center of the fiber paper; packaging the plastic packaging film with the fiber paper, so that the fiber paper is fixed by the plastic packaging film; step three: printing the conductive paste on the packaged plastic packaging film to obtain an electrode; step four: drying and sterilizing the printed electrode, and modifying the working area of the electrode with (Fe, Mn)3(PO4)2And obtaining the three-dimensional paper-based electrochemical sensing device by using the/N-CNRs nano material. The invention also discloses a preparation method of the catalystA three-dimensional paper-based electrochemical sensing device and application thereof are provided. The three-dimensional paper-based electrochemical sensing device can realize in-situ, real-time and high-sensitivity monitoring of small molecules released by three-dimensional cultured cells.
Description
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to a three-dimensional paper-based electrochemical sensing device and a preparation method and application thereof.
Background
Cells are the basic functional unit in life research, and biochemical analysis of them can provide critical information for research in the fields of biology and medicine. However, at present, the cell culture mainly depends on a two-dimensional culture system, and the changes of physiological functions, structures and the like of living cells in the three-dimensional growth process cannot be monitored in real time. Microfabrication techniques have been used to construct complex scaffolds for three-dimensional culture of cells, however, these methods typically involve some unusually large equipment. Therefore, it is necessary to construct a simple and stable portable sensor device for real-time and highly sensitive monitoring of small molecule release from three-dimensional cultured cells.
In the traditional method for detecting the release of small molecules by cells, the cells are firstly cultured in a culture dish, and then the constructed electrochemical sensor is placed in a culture solution in the culture dish for testing. This method greatly limits the sensitivity and accuracy of detection due to the long distance between the cell and the sensor and the very short half-life of the small molecules released by the cell. In addition, it is reported that cells are directly grown on a two-dimensional sensing membrane to detect the release of small molecules from the cells, but since the cells are in a three-dimensional growth state in an organism, the method can cause the mismatch of the growth dimensions of the cells, so that the accuracy of the detection result is not high.
Disclosure of Invention
The invention aims to provide a three-dimensional paper-based electrochemical sensing device which can realize in-situ real-time and high-sensitivity monitoring of small molecules released by three-dimensional cultured cells.
In order to solve the technical problems, the first aspect of the invention provides a preparation method of a three-dimensional paper-based electrochemical sensing device, which comprises the following steps:
the method comprises the following steps: providing fiber paper, an upper plastic package film and a lower plastic package film, wherein the thickness of the fiber paper is 0.15-0.25 mu m, and the size of intercepted particles is 10-30 mu m; the upper layer plastic packaging film is provided with openings with the shape consistent with that of the fiber paper, and the area of the openings is smaller than that of the fiber paper;
step two: clamping the fiber paper between the upper layer plastic packaging film and the lower layer plastic packaging film to enable the opening to be aligned with the fiber paper, wherein the edge of the opening of the fiber paper and the edge of the opening of the upper layer plastic packaging film have a certain overlapping area; packaging the plastic packaging film clamped with the fiber paper by a plastic packaging machine to enable the fiber paper to be fixed by the plastic packaging film;
step three: printing the conductive paste on the packaged plastic packaging film in a printing mode to obtain an electrode, and ensuring that a working area of the electrode is printed on the fiber paper in the open hole;
step four: drying and sterilizing the printed electrode, and modifying the working area of the electrode with (Fe, Mn)3(PO4)2the/N-CNRs nano material is used for obtaining the three-dimensional paper-based cell culture electrochemical sensing device;
wherein, the (Fe, Mn)3(PO4)2the/N-CNRs nano-material is prepared by the following method:
s1, preparing a nitrogen-doped porous carbon material N-CNRs into an aqueous solution with the concentration of 1-3 mg/mL, and performing ultrasonic dispersion for 20-80 min; the N-CNRs are prepared according to the method described in Chinese patent with the application number of 201910510602.4;
s2, adding divalent Fe salt, divalent manganese salt and a reducing agent into the solution obtained in the step S1, fully dissolving to obtain a mixed solution, and controlling Fe in the mixed solution2+With Mn2+The concentration ratio of (A) to (B) is 1: 1-1: 15;
s3, mixing the mixed solution obtained in the step S2 with ethylene glycol, placing the mixture in a water bath at the temperature of 50-70 ℃, and adding phosphate solution under the condition of continuous stirring to enable Mn in the solution to be in the form of Mn2+And Fe2+And PO4 3-In a concentration ratio of3: 2; stirring and reacting for 1-5 h, centrifuging the mixed solution, washing and freeze-drying the obtained precipitate to obtain the (Fe, Mn)3(PO4)2/N-CNRs。
The paper has a three-dimensional cellulose network structure, has the advantages of low cost, good biocompatibility, easy operation, convenient storage and the like, and can be used for manufacturing disposable biosensing devices. The fiber paper with the thickness of 0.15-0.25 μm and the size of the trapped particle of 10-30 μm is very suitable for the attached growth of cells in the surface or internal reticular structure. Furthermore, the porous cellulose network structure of the paper can achieve the flow of liquid by capillary action without the use of a pump. Therefore, the paper is selected to be used as the electrochemical sensing device, and the nano material capable of specifically detecting the small molecules released by the cells is modified on the working electrode of the paper-based electrochemical sensing device, so that the in-situ real-time and high-sensitivity detection of the small molecules released by the cells growing in the three-dimensional paper fiber can be realized.
(Fe,Mn)3(PO4)2In the/N-CNRs nano material, (Fe, Mn)3(PO4)2To H2O2Has strong catalytic decomposition effect, and the N-CNTs have high specific surface area due to the large amount of nano-pore structures, which greatly increases (Fe, Mn) deposited on the surface of the N-CNTs3(PO4)2And H2O2Contact area of equal small molecules, therefore (Fe, Mn)3(PO4)2the/N-CNRs nano material has the characteristics of high electrocatalytic activity, high conductivity, good stability, good biocompatibility and the like. The invention uses (Fe, Mn)3(PO4)2N-CNRs nano material as H2O2The small-molecule electro-catalyst does not need to use the traditional enzyme catalyst, overcomes the defects of the enzyme catalyst, and has strong anti-interference capability on other interferents.
Further, in the first step, the shape of the fiber paper is round or square; furthermore, the open holes are round holes or square holes with the diameter or width being 2-4 mm narrower than that of the fiber paper.
Further, in the second step, the packaging temperature of the plastic packaging machine is 105-115 ℃.
Further, in the third step, the conductive paste is carbon paste, silver paste or conductive gel.
Further, in the fourth step, the drying temperature is 80-110 ℃, and the drying time is 3-10 min.
Further, in the fourth step, the modification is specifically: will (Fe, Mn)3(PO4)2the/N-CNRs nano-material is prepared into a solution with the concentration of 2-5 mg/mL, 1-3 mu L of the solution is dripped onto an electrode, and the solution is dried.
Further, in step S2, Fe2+And Mn2+The total concentration of (B) is 1 to 3 mM.
Further, in step S2, Fe in the mixed liquid is controlled2+With Mn2+The concentration ratio of (A) to (B) is 1: 7.
In step S2 of the present invention, the reducing agent is added to prevent Fe2+With Mn2+Oxidation takes place. The reducing agent may be ascorbic acid and/or sodium borohydride, and the addition amount thereof is preferably 0.5 to 1.5 mM.
Further, in step S3, the phosphate may be ammonium phosphate, sodium phosphate, potassium phosphate, or the like.
In a second aspect, the invention provides a three-dimensional paper-based electrochemical sensing device prepared by the method of the first aspect.
In a third aspect, the invention provides an application of the three-dimensional paper-based electrochemical sensing device in the second aspect in cell release small molecule detection, wherein the small molecule can be H2O2。
The invention has the beneficial effects that:
1. the three-dimensional paper-based electrochemical sensing device is simple in preparation method and low in cost; in addition, in the modification (Fe, Mn)3(PO4)2The N-CNRs nano material can make the sensing device show good catalytic performance and strong anti-interference capability. Compared with the traditional detection method, the three-dimensional paper-based electrochemical sensing device can release micromolecular sources to cells growing in the three-dimensional paper fibersAnd (3) detecting the position in real time and with high sensitivity.
2. The paper-based electrochemical sensing device has wide application prospect in clinical diagnosis and early monitoring of daily diseases.
Drawings
FIG. 1 is a schematic diagram of the construction of a three-dimensional paper-based cell culture electrochemical sensing device, wherein paper fibers can be used for three-dimensional culture of cells;
FIG. 2 is a fluorescent microscopic image (DAPI staining) of the growth state of cells on three-dimensional paper fibers;
FIG. 3 shows the difference in Fe2+,Mn2+Synthesized in proportion (Fe, Mn)3(PO4)2Per N-CNRs vs. 200. mu.M hydrogen peroxide (H)2O2) Current response histogram of (a);
FIG. 4 is a solution of (Fe, Mn)3(PO4)2Carbon nanotubes and (Fe, Mn)3(PO4)2The sensing device with respectively modified N-CNRs is 200 mu M H2O2Comparison of cyclic voltammograms in 0.01M PBS;
FIG. 5 is a solution of (Fe, Mn)3(PO4)2Anti-interference detection diagram of hydrogen peroxide detection of/N-CNRs modified sensing device (hydrogen peroxide: H)2O2And glucose: glucose, dopamine: DA, ascorbic acid: AA, uric acid: UA);
FIG. 6 is (Fe, Mn)3(PO4)2Paper-based sensing device modified by N-CNRs releases H to cells2O2And (4) detecting the result in real time.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1: preparation (Fe, Mn)3(PO4)2Nitrogen-doped porous carbon material
In this example, N-CNRs were used as the substrate material, and the surface thereof was chemically grown in situ (Fe, Mn)3(PO4)2Preparation (Fe, Mn)3(PO4)2/N-CNRs。(Fe,Mn)3(PO4)2The specific preparation process of the/N-CNRs comprises the following steps:
(1) nitrogen-doped porous carbon materials (N-CNRs) were prepared according to the method described in the published patent (application No. 201910510602.4, published 2019, 9, 17).
(2) Preparing N-CNRs into a water solution with the concentration of 2mg/mL, and ultrasonically dispersing for 30 min; then dividing the solution into multiple groups, adding divalent Fe salt and divalent Mn salt into each group of solution, and controlling Fe2+With Mn2+Has a total concentration of 2mM, Fe2+With Mn2+In the ratio of 1:0, 3:1, 1:3, 1:7, 1:15 and 0:1 respectively; and 1mM ascorbic acid was added to each group of solutions.
(3) Adding the prepared solution into ethylene glycol, and fully mixing; putting the mixed solution into a water bath at 60 ℃ and adding ammonium phosphate solution into the mixed solution under the condition of continuous stirring to ensure that Mn is obtained2+And Fe2+And PO4 3-The final concentration ratio in the mixed solution was 3: 2. Stirring for reaction for 2h, centrifuging, washing with water for 3 times, and freeze drying the precipitate to obtain (Fe, Mn)3(PO4)2/N-CNRs。
In a solution containing 200 mu M H2O2Was subjected to electrochemical performance test in 0.01M PBS. As can be seen from the histogram of the current response in FIG. 3, Fe2+And Mn2+The optimal ratio of (A) to (B) is 1: 7. Fe was used in the following experiments2+、Mn2+In a ratio of 1:7 (Fe, Mn)3(PO4)2N-CNRs nano material.
Will (Fe, Mn)3(PO4)2Grown on N-doped carbon nanotubes (N-CNTs), a common N-doped carbon base material, in the same manner and tested for H2O2Performance of (Fe, Mn)3(PO4)2Comparison was made with respect to the/N-CNRs. As can be seen from the test results of FIG. 4, (Fe, Mn)3(PO4)2the/N-CNRs pair H2O2The peak current response of the current is obviously higher than that of (Fe, Mn)3(PO4)2N-CNTs, indicating (Fe, Mn)3(PO4)2N-CNRs nano material for H2O2Has higher electrocatalytic activity.
Example 2: preparation of three-dimensional paper-based cell culture electrochemical sensing device
FIG. 1 is a schematic diagram of the construction of a three-dimensional paper-based cell culture electrochemical sensing device, which specifically comprises the following steps:
(1) first, paper fiber whatman #114, which can be used for culturing cells, is cut into a circular shape having a diameter of 4mm wider than the designed electrode width by a paper cutter;
(2) cutting a circular hole with the diameter being 2mm narrower than that of the paper fiber on the upper layer of the plastic packaging film by a paper cutter according to the same method;
(3) sandwiching the cut paper fiber between two layers of plastic packaging films, and keeping the paper fiber aligned with the porous surface;
(4) packaging the plastic packaging film with the paper fibers at 110 ℃ by using a plastic packaging machine, wherein the paper fibers can be well fixed by the plastic packaging film due to the certain overlapping area between the paper fibers and the edge of the hole of the plastic packaging film;
(5) printing the carbon paste on the packaged plastic packaging film according to the designed electrode shape by a screen printing technology, and ensuring that the working area of the electrode is printed on the exposed paper fiber;
(6) placing the printed electrode in an oven, heating at 100 deg.C for 5min, taking out, and sterilizing; then, 1. mu.L of (Fe, Mn) with a concentration of 5mg/mL was dropped on the working area of the electrode3(PO4)2N-CNRs, air-dried at room temperature.
Example 3: cell culture experiment of three-dimensional paper-based cell culture electrochemical sensing device
(1) Sterilizing the sensor, inoculating cells in a biosafety cabinet at a cell inoculation density of 40 μ L5 × 105seed/mL, placing the inoculated seed in a cell culture box for culturing for 5 hours;
(2) and after the cells finish attached growth in the three-dimensional space, carrying out performance detection on the sensor.
FIG. 2 is a DAPI-stained fluorescence microscope showing the growth of cells on three-dimensional paper fibers, showing that the cells can attach well to the paper fibers for three-dimensional growth.
Example 4: electrochemical performance research of three-dimensional paper-based cell culture electrochemical sensing device
Firstly, the anti-interference capability of the sensing device is tested by adopting a chronoamperometry, and the selected interferents are researched and commonly released H by cells2O2Some substances coexisting: glucose, DA, AA, UA. The results show (FIG. 5) that the three-dimensional paper-based electrochemical sensing device is modified (Fe, Mn)3(PO4)2Post pair of/N-CNRs H2O2The detection shows strong anti-interference capability.
Subsequently, cell release H was recorded by chronoamperometry2O2Real-time current response. As can be seen in FIG. 6, the addition of 5 μ g/mL to the paper-based sensing device containing cells stimulated the release of H from the cells2O2After the drug (PMA) (cell response 2), a clear current response was observed, indicating that PMA stimulates H released by cells2O2Can be successfully modified with (Fe, Mn)3(PO4)2The paper-based sensing device of the/N-CNRs detects. After the cells in the paper-based sensing device are incubated with 0.1mM inhibitor (DPI) for a period of time, PMA (cell response 1) is added, so that the generated current response is very small, and the fact that the DPI can well inhibit the PMA from stimulating the cells to release H is proved2O2. PMA (polymethyl methacrylate) added into paper-based sensing device containing cells for stimulating cells to release H2O2Then, a certain concentration of catalase (500U @) is addedmL) (cellular response 3) H can be observed2O2Can be rapidly eliminated by catalase.
The results of the experiments show that the catalyst is based on (Fe, Mn)3(PO4)2In-situ detection of cell release H constructed by/N-CNRs2O2The paper-based sensing device can be used for capturing H released by cells in real time2O2And can realize the pair H2O2Rapid and highly sensitive detection.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. A preparation method of a three-dimensional paper-based electrochemical sensing device is characterized by comprising the following steps:
the method comprises the following steps: providing fiber paper, an upper plastic package film and a lower plastic package film, wherein the thickness of the fiber paper is 0.15-0.25 mu m, and the size of intercepted particles is 10-30 mu m; the upper layer plastic packaging film is provided with openings with the shape consistent with that of the fiber paper, and the area of the openings is smaller than that of the fiber paper;
step two: clamping the fiber paper between the upper layer plastic packaging film and the lower layer plastic packaging film to ensure that the opening is aligned with the center of the fiber paper and ensure that the opening edge of the fiber paper and the opening edge of the upper layer plastic packaging film have a certain overlapping area; packaging the plastic packaging film clamped with the fiber paper by a plastic packaging machine to enable the fiber paper to be fixed by the plastic packaging film;
step three: printing the conductive paste on the packaged plastic packaging film in a printing mode to obtain an electrode, and ensuring that a working area of the electrode is printed on the fiber paper in the open hole;
step four: drying and sterilizing the printed electrode, and modifying the working area of the electrode with (Fe, Mn)3(PO4)2the/N-CNRs nano material is used for obtaining the three-dimensional paper base fineCell culture electrochemical sensing devices;
wherein, the (Fe, Mn)3(PO4)2the/N-CNRs nano-material is prepared by the following method:
s1, preparing a nitrogen-doped porous carbon material N-CNRs into an aqueous solution with the concentration of 1-3 mg/mL, and performing ultrasonic dispersion for 20-80 min; the N-CNRs are prepared according to the method described in Chinese patent with the application number of 201910510602.4;
s2, adding divalent Fe salt, divalent Mn salt and a reducing agent into the solution obtained in the step S1, fully dissolving to obtain a mixed solution, and controlling Fe in the mixed solution2+With Mn2+The concentration ratio of (A) to (B) is 1: 1-1: 15;
s3, mixing the mixed solution obtained in the step S2 with ethylene glycol, placing the mixture in a water bath at the temperature of 50-70 ℃, and adding phosphate solution under the condition of continuous stirring to enable Mn in the solution to be in the form of Mn2+And Fe2+And PO4 3-The concentration ratio of (A) to (B) is 3: 2; stirring and reacting for 1-5 h, centrifuging the mixed solution, washing and freeze-drying the obtained precipitate to obtain the (Fe, Mn)3(PO4)2/N-CNRs。
2. The preparation method of the three-dimensional paper-based electrochemical sensor device according to claim 1, wherein in the second step, the packaging temperature of the plastic packaging machine is 105-115 ℃.
3. The method for preparing the three-dimensional paper-based electrochemical sensing device according to claim 1, wherein in the third step, the conductive paste is carbon paste, silver paste or conductive gel.
4. The preparation method of the three-dimensional paper-based electrochemical sensing device according to claim 1, wherein in the fourth step, the drying temperature is 80-110 ℃, and the drying time is 3-10 min.
5. The method for preparing the three-dimensional paper-based electrochemical sensing device according to claim 1, wherein the step four isThe modification is specifically: will (Fe, Mn)3(PO4)2the/N-CNRs nano material is prepared into a solution with the concentration of 2-5 mg/mL, 1-3 mu L of the solution is dripped onto a working area of the electrode, and the electrode is dried.
6. The method for preparing the three-dimensional paper-based electrochemical sensing device according to the claim 1, wherein in the step S2, Fe2+And Mn2+The total concentration of (B) is 1 to 3 mM.
7. The method for preparing the three-dimensional paper-based electrochemical sensing device according to claim 1, wherein in step S2, Fe in the mixed solution is controlled2+With Mn2+The concentration ratio of (A) to (B) is 1: 7.
8. The method for preparing the three-dimensional paper-based electrochemical sensing device according to claim 1, wherein in the step S2, the reducing agent is ascorbic acid and/or sodium borohydride, and the addition amount is 0.5-1.5 mM.
9. A three-dimensional paper-based electrochemical sensing device prepared according to the method of any one of claims 1-8.
10. Use of the three-dimensional paper-based electrochemical sensing device of claim 9 in cell-released small molecule detection.
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Application publication date: 20201023 |