CN110907512A - Construction method of visual paper-based biological cathode photoelectrochemical sensor - Google Patents
Construction method of visual paper-based biological cathode photoelectrochemical sensor Download PDFInfo
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
The invention discloses a construction method of a visual paper-based biocathode photoelectrochemical sensor, and relates to the technical field of electrochemical detection. The silver iodide/cerium dioxide nano composite material is prepared by a one-step method to be used as a photoelectric anode, a paper chip is used as a substrate to modify biomolecules to construct a biological cathode, and an external coil is connected with a portable digital multimeter to measure two biomarkers MUC1 and miRNA-21. TMB color developing agent is dripped into the colorimetric area of the paper chip and utilized in H2O2Under the existing condition, the target object MUC1 can oxidize TMB to become a blue product oxTMB, so that the prejudgment of naked eye visual colorimetric display of the biomarker is realized; under the irradiation of light, the transfer speed of silver iodide/cerium dioxide photoelectrons of the anode material is accelerated, and the instantaneous current generated in the process can be changed from ten thousands of numbersThe table is read immediately, and high-sensitivity detection of MUC1 and miRNA-21 is realized by combining a foldable, portable and easily-operated paper-based biological cathode.
Description
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to a construction method of a visual paper-based biocathode photoelectrochemical sensor.
Background
In recent years, with the improvement of economic level of people, cancer patients in China are increasing year by year due to environmental pollution, bad life style, excessive mental stress and the like. It is statistically estimated that nearly one-sixth of our deaths are caused by cancer and it is predicted that twenty cancer morbidity and mortality will continue to rise in the future. In order to effectively realize early cancer screening, especially to carry out ultra-sensitive detection on low-level tumor markers, it is necessary to find a method for rapidly and quantitatively detecting tumor targets. However, common clinical diagnosis means all involve expensive detection instruments and complex professional operation, so the invention utilizes the advantages of low cost, rapidness, portability, small reagent consumption and the like of the paper chip to realize the on-site efficient detection of the tumor marker.
As colorimetric techniques are continuously applied in analytical chemistry, the main principle is that the analyte promotes the change of a substance from colorless to colored in a certain way, and the concentration of the substance is detected by observing the intensity of the color. Therefore, the paper chip is used as a substrate to provide a faster platform for colorimetric display, the method does not need a large expensive instrument for detection, and early prejudgment on the concentration of the target object can be realized through visual observation, so that the detection of the tumor marker is simpler and more efficient.
In order to improve the sensitivity and accuracy of sensor detection, the photosensitive nano material has attracted great interest to researchers due to its large specific surface area and good light absorption. The photosensitive nano composite material is greatly applied to photoelectrochemical analysis due to the advantages of uniform distribution and better electron transfer rate. Among them, a nanocomposite material composed of silver iodide and cerium oxide has received much attention in electrochemical applications.
Disclosure of Invention
Aiming at the existing problems, the invention provides a construction method of a visual paper-based biocathode photoelectrochemical sensor, which is characterized by comprising the following steps:
(1) cutting fluorine-doped tin oxide (FTO) conductive glass into a size of 1 cm multiplied by 5 cm, sequentially adding acetone, absolute ethyl alcohol and ultrapure water, performing ultrasonic treatment for 10-20 min, and then drying in an oven at 40-60 ℃ for 30 min;
(2) taking conductive glass as a substrate, and preparing a silver iodide/cerium dioxide nano composite material on the surface of the conductive glass by using a hydrothermal synthesis method to serve as a photoelectric anode;
(3) designing a paper chip capable of realizing visual detection of two biomarkers as a biological cathode;
(4) modifying aptamer chains corresponding to the two markers on the surface of the prepared paper-based biological cathode for capturing cancer cells, and then sealing active sites on the surface of the working electrode by using bovine serum albumin;
(5) folding the paper base to corresponding position, adding cancer cells with different concentrations to the surface of the modified paper base biological cathode working area, and adding TMB and H into the paper base color development area2O2A color developing agent formed by the solution is used for carrying out visual prejudgment on the target object;
(6) and (3) washing the biological cathode after the color reaction is finished with ultrapure water for three times, and connecting the biological cathode with the photoelectric anode prepared in the step (2) through an external lead and a digital multimeter to construct the paper-based biological cathode photoelectrochemical sensor.
The preparation process of the invention, which takes conductive glass as a substrate and utilizes a hydrothermal synthesis method to prepare the silver iodide/cerium dioxide nano composite material on the surface of the conductive glass as a photoelectric anode, comprises the following steps: firstly, dissolving 0.20 g of cerium dioxide and 0.033 g of potassium iodide in 60 mL of ultrapure water, stirring for 30 min to fully dissolve and mix the cerium dioxide and the potassium iodide, then adding 2 mL of 0.1M silver nitrate solution, transferring the mixed solution into a 100 mL stainless steel reaction kettle with a polytetrafluoroethylene lining, placing the treated FTO conductive glass in the step (1) into the reaction kettle, closing and screwing the FTO conductive glass, placing the reaction kettle in an oven preheated by 180 ℃ for reaction for 12 h, cooling to room temperature after the reaction is finished, taking out the FTO conductive glass, washing with secondary water for three times, and drying at room temperature to obtain the silver iodide/cerium dioxide nanocomposite.
The preparation process of the paper chip as the biological cathode, which is designed to realize visual detection of two biomarkers, comprises the following steps: the method comprises the steps of designing a hydrophobic wax batch printing pattern of a paper chip shown in the attached drawing 1 on a computer by utilizing AI software, cutting the paper chip into paper with the size of A4, printing the designed hydrophobic wax batch printing pattern on A4 paper by utilizing a wax printer, then placing the paper chip in an oven, heating until the wax is melted and the whole paper thickness is soaked to form a hydrophobic area, and finally sequentially printing working electrodes on the paper chip by adopting a screen printing method.
The preparation process of modifying the prepared paper-based biological cathode surface with aptamer chains corresponding to the two markers for capturing cancer cells and then sealing the active sites on the surface of the working electrode with bovine serum albumin comprises the following steps: and (4) functionalizing the paper chip prepared in the step (3), firstly growing gold nanoparticles in situ in a working area to facilitate connection of an aptamer, then dripping 10 mu L of a combined signal probe constructed by the aptamer 1 and the aptamer 2 on the surface of the paper chip, and reacting for 15 hours in a dark room-temperature environment.
The paper base is folded to a corresponding position, cancer cells with different concentrations are added to the surface of a modified paper base biological cathode working area, and then TMB and H are added to a paper base color development area2O2The process of pre-judging the object visually comprises the following steps: washing the paper chip treated in the step (4) with Phosphate Buffer Solution (PBS), drying, folding to make the working area contact with the color development area, dripping mixed solution consisting of 5 mu L of MUC1 and 5 mu L of LmiRNA-21 target substance into the working area 1, reacting for 30 min at room temperature, dripping PBS and 1% of Bovine Serum Albumin (BSA) to prevent nonspecific binding sites, unfolding the paper chip after the reaction is finished, dripping 10 mu L of TMB and H2O2The color developing agent formed by the solution can be used for visually prejudging the concentration of the target object by observing the color depth degree in the corresponding color developing area within 3 minutesSimilarly, the visual colorimetric detection can also be realized by respectively dripping 5 μ L of the No. 1 target substance MUC1 and 5 μ L of the No. 2 target substance miRNA-21 on the surfaces of the working area 2 and the working area 3.
The invention has the beneficial effects that:
(1) the experimentally synthesized silver iodide/cerium dioxide composite material has larger specific surface area, and is favorable for expanding the absorption of light.
(2) The paper-based biological cathode capable of detecting two target objects is constructed, so that the sensor can realize instant visual colorimetric display except for quantitative detection of a digital multimeter.
(3) The preparation method has the advantages that the paper material with low cost is adopted, so that the preparation steps of the photoelectrochemical paper chip are simplified, the preparation cost is reduced, and the repeatability of the preparation and detection of the paper chip is improved;
(4) the photoelectrochemistry analysis device can greatly reduce background signals and improve the detection sensitivity when being used for detecting the target object.
Drawings
The invention will be described in further detail below with reference to the accompanying drawings and specific embodiments, and fig. 1 is a design diagram of a paper-based biocathode of the photoelectrochemical sensor.
Fig. 2 is a mechanism diagram of the visualized paper-based biocathode photoelectrochemical sensor.
Detailed Description
Example 1: construction of visual paper-based biocathode photoelectrochemical sensor
(1) Cutting fluorine-doped tin oxide (FTO) conductive glass into a size of 1 cm multiplied by 5 cm, sequentially adding acetone, absolute ethyl alcohol and ultrapure water, performing ultrasonic treatment for 10 min, and then placing the glass in a 40 ℃ oven for 30 min for drying;
(2) taking conductive glass as a substrate, preparing a silver iodide/cerium dioxide nano composite material on the surface of the conductive glass as a photoelectric anode by using a hydrothermal synthesis method, firstly dissolving 0.20 g of cerium dioxide and 0.033 g of potassium iodide in 60 mL of ultrapure water, stirring for 30 min to fully dissolve and mix, then adding 2 mL of 0.1M silver nitrate solution, then transferring the mixed solution into a 100 mL stainless steel reaction kettle lined with polytetrafluoroethylene, placing the FTO conductive glass treated in the step (1) in the reaction kettle, sealing and screwing, placing in a 180-C preheating oven for reaction for 12 h, cooling to room temperature after the reaction is finished, taking out the FTO conductive glass, washing with secondary water for three times, and drying at room temperature to obtain the silver iodide/cerium dioxide nano composite material;
(3) designing a paper chip capable of visually detecting two biomarkers as a biological cathode, specifically, designing a hydrophobic wax batch printing pattern of the paper chip as shown in figure 1 on a computer by using AI software, cutting the paper chip into paper with the size of A4, printing the designed hydrophobic wax batch printing pattern on A4 paper by using a wax printer, then placing the paper chip in an oven for heating until the wax is melted and permeates the thickness of the whole paper to form a hydrophobic area, and finally, sequentially printing working electrodes on the paper chip by using a screen printing method;
(4) modifying an aptamer chain corresponding to the two markers on the surface of the prepared paper-based biological cathode for capturing cancer cells, and then sealing active sites on the surface of a working electrode by using bovine serum albumin, wherein the specific operation comprises the steps of functionalizing the paper chip prepared in the step (3), firstly growing gold nanoparticles in situ in a working area to facilitate connection of aptamers, then dropwise adding 10 mu L of a combined signal probe constructed by the aptamer 1 and the aptamer 2 on the surface of the paper chip, and reacting for 15 h in a dark room-temperature environment;
(5) folding the paper base to corresponding position, adding cancer cells with different concentrations to the surface of the modified paper base biological cathode working area, and adding TMB and H into the paper base color development area2O2Washing the paper chip treated in the step (4) with Phosphate Buffer Solution (PBS), drying, folding to make the working area contact with the color development area, dripping the mixed solution of 5 muL MUC1 and 5 muL miRNA-21 target substance into the working area 1, reacting at room temperature for 30 min, dripping PBS and 1% Bovine Serum Albumin (BSA) to prevent nonspecific binding sites, unfolding the paper chip after the reaction is finished, and dripping 10 muL TMB and H2O2The concentration of the target substance can be visually judged by observing the color depth of the color developing agent formed by the solution in a corresponding color developing area within 3 minutes, and similarly, 5 muL of No. 1 target substance MUC1 and 5 muL of No. 2 target substance miRNA-21 are respectively dripped on the surfaces of a working area 2 and a working area 3 to realize visual colorimetric detection;
(6) and (3) washing the biological cathode after the color reaction is finished with ultrapure water for three times, and connecting the biological cathode with the photoelectric anode prepared in the step (2) through an external lead and a digital multimeter, so that the two target objects can be detected immediately.
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Claims (7)
1. A construction method of a visual paper-based biocathode photoelectrochemical sensor is characterized by comprising the following steps:
(1) cutting fluorine-doped tin oxide (FTO) conductive glass into a size of 1 cm multiplied by 5 cm, sequentially adding acetone, absolute ethyl alcohol and ultrapure water, performing ultrasonic treatment for 10-20 min, and then drying in an oven at 40-60 ℃ for 30 min;
(2) taking conductive glass as a substrate, and preparing a silver iodide/cerium dioxide nano composite material on the surface of the conductive glass by using a hydrothermal synthesis method to serve as a photoelectric anode;
(3) designing a paper chip capable of realizing visual detection of two biomarkers as a biological cathode;
(4) modifying aptamer chains corresponding to the two markers on the surface of the prepared paper-based biological cathode for capturing cancer cells, and then sealing active sites on the surface of the working electrode by using bovine serum albumin;
(5) folding the paper base to corresponding position, adding cancer cells with different concentrations to the surface of the modified paper base biological cathode working area, and adding TMB and H into the paper base color development area2O2A color developing agent formed by the solution is used for carrying out visual prejudgment on the target object;
(6) and (3) washing the biological cathode after the color reaction is finished with ultrapure water for three times, and connecting the biological cathode with the photoelectric anode prepared in the step (2) through an external lead and a digital multimeter to construct the paper-based biological cathode photoelectrochemical sensor.
2. The construction method of the visual paper-based biocathode photoelectrochemical sensor according to claim 1, which is characterized in that: in the step (1), the FTO conductive glass is cut into the size of 1 cm multiplied by 5 cm, and then is sequentially put into acetone, absolute ethyl alcohol and ultrapure water for ultrasonic treatment for 10 min and then is put into a 60 ℃ oven for drying for 30 min.
3. The construction method of the visualized paper-based biocathode photoelectrochemical sensor according to claim 1, which comprises the steps of preparing a silver iodide/cerium dioxide nano composite material; the method is characterized in that: firstly, dissolving 0.20 g of cerium dioxide and 0.033 g of potassium iodide in 60 mL of ultrapure water, stirring for 30 min to fully dissolve and mix the cerium dioxide and the potassium iodide, then adding 2 mL of 0.1M silver nitrate solution, transferring the mixed solution into a 100 mL stainless steel reaction kettle with a polytetrafluoroethylene lining, placing the treated FTO conductive glass in the step (1) into the reaction kettle, closing and screwing the FTO conductive glass, placing the reaction kettle in an oven preheated by 180 ℃ for reaction for 12 h, cooling to room temperature after the reaction is finished, taking out the FTO conductive glass, washing with secondary water for three times, and drying at room temperature to obtain the silver iodide/cerium dioxide nanocomposite.
4. The construction method of the visual paper-based biocathode photoelectrochemical sensor according to claim 1, wherein a paper chip capable of visually detecting two biomarkers is designed as the biocathode, and the construction method is characterized in that: the method comprises the steps of designing a hydrophobic wax batch printing pattern of a paper chip shown in the attached drawing 1 on a computer by utilizing AI software, cutting the paper chip into paper with the size of A4, printing the designed hydrophobic wax batch printing pattern on A4 paper by utilizing a wax printer, then placing the paper chip in an oven, heating until the wax is melted and the whole paper thickness is soaked to form a hydrophobic area, and finally sequentially printing working electrodes on the paper chip by adopting a screen printing method.
5. The construction method of the visual paper-based biocathode photoelectrochemical sensor according to claim 1, which comprises the steps of modifying the prepared paper-based biocathode surface with aptamer chains corresponding to two markers for capturing cancer cells, and then sealing the active sites on the surface of the working electrode with bovine serum albumin, wherein the method comprises the following steps: and (4) functionalizing the paper chip prepared in the step (3), firstly growing gold nanoparticles in situ in a working area to facilitate connection of an aptamer, then dripping 10 mu L of a combined signal probe constructed by the aptamer 1 and the aptamer 2 on the surface of the paper chip, and reacting for 15 hours in a dark room-temperature environment.
6. The method of claim 1A visual paper-based biological cathode photoelectrochemical sensor construction method comprises folding a paper base to a corresponding position, adding cancer cells with different concentrations to the surface of a modified paper-based biological cathode working area, and adding TMB and H into a paper-based color development area2O2The color developing agent formed by the solution is used for carrying out visual prejudgment on the target object, and is characterized in that: washing the paper chip processed in the step (4) with Phosphate Buffer Solution (PBS), drying, folding to make the working area contact with the color development area, dripping mixed solution consisting of 5 muL MUC1 and 5 muL miRNA-21 target substance into the working area 1, reacting for 30 min at room temperature, dripping PBS and 1% Bovine Serum Albumin (BSA) to prevent nonspecific binding sites, unfolding the paper chip after the reaction is finished, and dripping 10 muL TMB and H2O2And (3) observing the color depth of a color developing agent formed by the solution in a corresponding color developing area for 3 minutes to pre-judge the concentration of the target object in a visual mode, and respectively dripping 5 mu L of No. 1 target object MUC1 and 5 mu L of No. 2 target object miRNA-21 on the surfaces of the working area 2 and the working area 3 to realize visual colorimetric detection similarly.
7. The construction method of the visual paper-based biocathode photoelectrochemical sensor according to claim 1, which is characterized in that: and (3) washing the biological cathode working area after the colorimetric reaction in the step (5) with ultrapure water for three times, and then connecting the biological cathode working area with the photoelectric anode constructed in the step (2) through a lead and a digital multimeter, so that the two targets can be detected immediately.
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Cited By (1)
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| CN113295741A (en) * | 2021-06-16 | 2021-08-24 | 江苏大学 | Method for constructing photoelectric photochromic visual sensor for simultaneously detecting multiple organic pollutants |
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