CN107064118B - Construction method of paper-based high-flux photo-electrochemical biosensor - Google Patents
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Abstract
The invention discloses a photo-electrochemical biosensor which is simple to operate and high in flux and is successfully used for simultaneously detecting three kinds of cancer cells. Firstly, a layer of gold nanoparticles is modified on the surface of a paper fiber through gold nano autocatalytic reduction, then a cadmium sulfide-graphene-zinc oxide rod ternary composite material is prepared on the surface of a long-gold-modified paper working electrode to serve as a photoelectrode, a traditional xenon lamp is replaced by chemiluminescence, the photoelectrode is sequentially excited by controlling the sequence of chemiluminescence reaction, and three kinds of cancer cells are simultaneously detected by sequentially detecting three photocurrent peaks.
Description
Technical Field
The invention relates to the field of paper-based high-throughput photoelectrochemical analysis and detection, in particular to a construction method of a paper microfluidic chip analysis and detection platform based on a chemiluminescence photoelectrochemical analysis method.
Background
Cancer is one of the most major diseases threatening human health at present, and an effective method for improving the survival rate of patients can be realized by making a timely diagnosis at the early stage of cancer. The cancer cell is a variant cell and is a source of cancer, and different from a normal cell, the cancer cell has three characteristics of unlimited proliferation, transformation and easy metastasis, and can be unlimited proliferation and damage normal cell tissues. Therefore, the construction of a rapid and accurate cancer cell analysis and detection method has very important significance. So far, various analytical methods are endless, and based on the existing detection principle, people continuously seek methods with faster reaction time, shorter detection time and higher sensitivity.
At present, methods for detecting cancer cells mainly comprise methods such as electrochemistry, electrochemiluminescence, fluorescence and the like. However, the method is expensive and complex in operation, even needs professional operation, is not favorable for realizing portable high-flux rapid field detection, and limits the wide application and development of the method. Therefore, it is necessary to develop a portable, rapid, high-throughput assay.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a method for simultaneously detecting three types of cancer cells by using a photoelectric biosensor, which is simple in operation, fast in detection speed, and low in cost.
In order to solve the technical problems, the invention realizes the simultaneous detection of three kinds of cancer cells by constructing a paper-based high-flux photo-electrochemical biosensor, and the preparation method of the photo-electrochemical biosensor comprises the following steps:
(1) designing a wax batch printing pattern of a three-dimensional hollow channel microfluidic paper chip on a computer, wherein the wax batch printing pattern consists of four parts, a working chip (A), a hollow channel chip (B), a semi-hydrophilic channel chip (C) and an auxiliary chip (D), the pattern is shown in figure 1, punching and cutting a wax batch printing pattern grid area by using a puncher and a laser cutting machine to prepare a solution inlet and a hollow channel, and printing carbon electrode patterns on the working area and the auxiliary electrode area which are designed on the back of the wax batch printing pattern respectively as shown in figure 2;
(2) modifying a layer of gold nanoparticles in a working area by a gold nano autocatalytic reduction method to serve as a paper working electrode substrate;
(3) preparing a zinc oxide nanorod on the surface of a paper working electrode for modifying gold nanoparticles by a hydrothermal method;
(4) modifying a layer of graphene on the surface of a zinc oxide nanorod;
(5) preparing cadmium sulfide on the surface of graphene by a continuous ion adsorption method;
(6) preparing a hemiconcanavalin-gold nano-luminol composite material;
(7) modifying an aptamer chain corresponding to the cancer cell on the surface of the paper working electrode to capture the cancer cell, and then sealing an active site on the surface of the working electrode by using bovine serum albumin;
(8) adding cancer cells with different concentrations to the surface of the modified paper working electrode, fixing MCF-7 cancer cells by the working electrode 1, fixing K562 cancer cells by the working electrode 2, and fixing HL-60 cancer cells by the working electrode 3;
(9) fixing the prepared hemiconcanavalin-gold nano-luminol composite material on a working electrode;
(10) the modified paper chip is folded as shown in figure 3, then is fixed by an automatic conducting clamp as shown in figure 4, is connected to an electrochemical workstation, hydrogen peroxide is added through an inlet, chemical reactions on the three working electrodes are sequentially initiated due to different time when the hydrogen peroxide reaches the working electrodes, and the three cancer cells are simultaneously detected by measuring the photocurrent intensity of the three working electrodes.
The paper material of the invention is common filter paper, absorbent paper or chromatographic paper, the cancer cells are MCF-7, K562 and HL-60 cancer cells, and the MCF-7 cancer cell aptamer: 5' -GCA GTT GAT CCT TTG GAT ACC CTG GTT TTT TTT TTT-H2N-3', K562 cancer cell aptamers: 5' -H2N-TTT TTT TTT TAC AGC AGA TCA GTC TAT CTT CTC CTG ATG GGT TCC TAT TTA TAG GTG AAG CTG T-3', HL-60 cancer cell aptamers: 5' -H2N-TTT TTT TTT ATC CAG AGT GAC GCA GCA TGC CCT AGT TAC TAC TAC TCT TTT TAG CAA AC。
The preparation process of the three-dimensional hollow channel microfluidic paper chip comprises the following steps: designing a hydrophobic wax batch printing pattern of a microfluidic paper chip on a computer, wherein the pattern is shown in figure 1, the wax batch printing pattern is composed of four parts, namely a working chip (A), a hollow channel chip (B), a semi-hydrophilic channel chip (C) and an auxiliary chip (D), 1 is a solution inlet, 2-4 are working electrodes 1-3, 5 are hollow channels, 6-8 are semi-hydrophilic channels, 6 is printing 60% of wax, 7 is printing 40% of wax, 8 is printing 80% of wax, and 9 is an auxiliary electrode printing area; then, punching and cutting the grid area in the attached figure 1 by using a puncher and a laser cutting machine to prepare a hollow channel; placing the chromatographic paper printed with the hydrophobic wax batch printing patterns into an oven, and heating for 30 s at 130 ℃ to ensure that the wax permeates the interior of the paper fiber to construct a completely hydrophobic part and a semi-hydrophobic part; the carbon electrode patterns are printed on the working area and the auxiliary electrode area designed on the back of the wax batch printing pattern respectively as shown in figure 2.
The gold nano autocatalytic reduction method of the invention modifies a layer of gold nano particles in a paper electrode working area and comprises the following steps: (1) preparing 5 nm gold nano-seeds: firstly, 5 mL of chloroauric acid with the mass concentration of 1% and 10 mL of 0.03M sodium citrate are stirred for 10 min, then 250 mL of secondary water are added to continue stirring for 10 mL, finally 5 mL of 0.1M sodium borohydride is added to stir for 24 h at room temperature, centrifugation is carried out for 30 min at 12000 rpm, and the gold nanoparticles are re-dispersed into 10 mL of secondary water, so that the gold nanoparticles are obtained.
(2) Growing gold nano-particles on the surface of the working electrode: and dropwise adding the prepared gold nano seeds (10 mu L) to the surface of the working electrode, airing at room temperature, repeating for 5 times, dropwise adding a mixed solution (20 mu L) of sodium citrate with the mass concentration of 1% and 0.01M hydroxylamine hydrochloride to the surface of the electrode, reacting for 30 min at room temperature, and washing with secondary water to obtain the long-gold-modified paper working electrode.
The preparation process of growing zinc oxide nano-rods on the surface of a gold nano-modified paper working electrode comprises the following steps: firstly, 40 nM zinc acetate solution is hung on the surface of a paper working electrode modified by gold nanoparticles (1000 rpm, 60 s), then the paper working electrode is dried at 95 ℃, the drying is repeated for 6 times, 50 mL of mixed solution of 25 mM zinc nitrate and 25 mM hexamethylenetetramine is prepared and placed in an autoclave, the paper working electrode is placed in the autoclave, the conductive surface faces downwards, the autoclave is placed in an oven at 95 ℃ for reaction for 6 hours, the reaction is washed by secondary water after the reaction is finished, and then the zinc oxide nanorod is obtained after the drying at 60 ℃.
The preparation process of the graphene modified on the surface of the zinc oxide comprises the following steps: and (3) coating the 0.5 mg/mL graphene solution on the surface of a paper electrode for growing the zinc oxide nano rod in a suspending way (3000 rpm, 30 s), and then drying in a 75 ℃ oven for 3 h to modify a layer of graphene on the surface of the zinc oxide nano rod.
The process for preparing the cadmium sulfide on the surface of the graphene by the continuous ion adsorption method comprises the following steps: firstly, 100 mM of cadmium sulfate aqueous solution (10 mu L) is dripped on the surface of a paper electrode, the mixture is kept stand for 30 s, the solution is sucked out from the back surface of the paper electrode by using absorbent paper, then 100 mM of sodium sulfide aqueous solution (10 mu L) is dripped on the surface of the electrode to react for 30 s, the solution is sucked out from the back surface of the paper electrode by using the absorbent paper, and the step is repeated for 30 times, so that a layer of cadmium sulfide can be obtained on the surface of graphene.
The preparation method of the semi-concanavalin-gold nano-luminol composite material comprises the following steps: mixing the gold nanoparticles (1 mL) prepared above, 5 mM hemiconcanavalin (500. mu.L), and 5 mM luminol (500. mu.L), stirring at 4 ℃ for 5 h, centrifuging, washing with water, and then re-dissolving in 5 mL secondary water to obtain a hemiconcanavalin-gold nano-luminol composite material.
The paper-based high-flux photo-electrochemical biosensor determination process comprises the following steps: firstly, respectively adding 10 mu L of EDC-NHS mixed solution to the surface of a working electrode for reaction for 3 h, then dropwise adding 10 mu L of aptamer chain to the surface of the working electrode for reaction for 30 min, fixing the MCF-7 cancer cell aptamer chain by using a working electrode 1, fixing the K562 cancer cell aptamer chain by using a working electrode 2, fixing the HL-60 cancer cell aptamer chain by using a working electrode 3, then flushing by using a pH 7.4 buffer solution, then adding 5 mg/mL bovine serum albumin (10 mu L) to seal an electrode surface active site, reacting for 1 h, and flushing by using a pH 7.4 buffer solution; adding corresponding cancer cells with different concentrations into the working electrodes 1, 2 and 3 respectively to react for 30 min, washing with buffer solution with pH 7.4 to remove the cancer cells which are not captured; dripping 10 μ L of the semi-concanavalin-gold nano-luminol composite material prepared above onto the surfaces of working electrodes 1, 2 and 3 respectively for reaction for 30 min, and washing with a buffer solution with pH 7.4 to remove the non-fixed semi-concanavalin-gold nano-luminol composite material; the modified paper chip is folded as shown in the attached drawing 3, then is fixed by an automatic conducting clamp as shown in the attached drawing 4, is connected to an electrochemical workstation, hydrogen peroxide is added through an inlet, chemical reactions on the three working electrodes are sequentially initiated due to different time when the hydrogen peroxide reaches the working electrodes, the three cancer cells are simultaneously detected by measuring the photocurrent intensities of the three working electrodes, and a working curve is drawn according to different cancer cell concentrations and the photocurrent intensities.
The invention has the beneficial effects that:
(1) a layer of gold nano is prepared on the surface of the paper electrode through autocatalysis in-situ growth, so that the conductivity of the electrode is improved, and the detection sensitivity is improved;
(2) preparing a cadmium sulfide-graphene-zinc oxide ternary composite material, and improving electron-hole separation;
(3) the chemiluminescence is used as an internal light source to replace the traditional xenon lamp, so that the detection cost is saved;
(4) the flow rate of the liquid on the paper chip is regulated and controlled to control the chemiluminescence reaction sequence, so that multi-component measurement is realized.
Drawings
Fig. 1 shows batch printing patterns of hydrophobic wax of a three-dimensional hollow channel microfluidic paper chip, wherein 1 is a solution inlet, 2-4 are working electrodes 1-3, 5 are hollow channels, 6-8 are semi-hydrophilic channels, 6 is printing 60% wax, and 7 is printing 40% wax. 8 is printing 80% wax, 9 is the auxiliary electrode printing area.
Fig. 2 is a three-dimensional hollow channel microfluidic paper chip after printing electrodes.
Fig. 3 shows a folding method of a three-dimensional hollow channel microfluidic paper chip.
Fig. 4 shows a folded three-dimensional hollow channel microfluidic paper chip clamped by self-conducting clamps.
The invention is described in further detail below with reference to the figures and specific embodiments.
Detailed Description
Example 1: simultaneously detecting three cancer cells of MCF-7, K562 and HL-60 in human blood.
(1) Designing a hydrophobic wax batch printing pattern of a microfluidic paper chip on a computer, wherein the pattern is shown in figure 1, the wax batch printing pattern is composed of four parts, namely a working chip (A), a hollow channel chip (B), a semi-hydrophilic channel chip (C) and an auxiliary chip (D), 1 is a solution inlet, 2-4 are working electrodes 1-3, 5 are hollow channels, 6-8 are semi-hydrophilic channels, 6 is printing 60% of wax, 7 is printing 40% of wax, 8 is printing 80% of wax, and 9 is an auxiliary electrode printing area; then, punching and cutting the grid area in the attached figure 1 by using a puncher and a laser cutting machine to prepare a hollow channel; placing the chromatographic paper printed with the hydrophobic wax batch printing patterns into an oven, and heating for 30 s at 130 ℃ to ensure that the wax permeates the interior of the paper fiber to construct a completely hydrophobic part and a semi-hydrophobic part; printing carbon electrode patterns on a working area and an auxiliary electrode area designed on the back of the wax batch printing pattern respectively as shown in the attached figure 2;
(2) preparing 5 nm gold nano-seeds: firstly, 5 mL of chloroauric acid with the mass concentration of 1% and 10 mL of 0.03M sodium citrate are stirred for 10 min, then 250 mL of secondary water are added to continue stirring for 10 mL, finally 5 mL of 0.1M sodium borohydride is added to stir for 24 h at room temperature, centrifugation is carried out for 30 min at 12000 rpm, and the gold nanoparticles are re-dispersed into 10 mL of secondary water, so that the gold nanoparticles are obtained.
(3) Growing gold nano-particles on the surface of the working electrode: and dropwise adding the prepared gold nano seeds (10 mu L) to the surface of the working electrode, airing at room temperature, repeating for 5 times, dropwise adding a mixed solution (20 mu L) of sodium citrate with the mass concentration of 1% and 0.01M hydroxylamine hydrochloride to the surface of the electrode, reacting for 30 min at room temperature, and washing with secondary water to obtain the long-gold-modified paper working electrode.
(4) Growing a zinc oxide nanorod on the surface of a gold nano-modified paper working electrode, firstly, suspending a 40 nM zinc acetate solution on the surface of the gold nano-modified paper working electrode (1000 rpm, 60 s), then drying at 95 ℃, repeating for 6 times, preparing 50 mL of a mixed solution of 25 mM zinc nitrate and 25 mM hexamethylenetetramine, placing the paper working electrode in an autoclave, placing the paper working electrode in the autoclave with a conductive surface facing downwards, placing the autoclave in an oven at 95 ℃ for reacting for 6 h, washing with secondary water after the reaction is finished, and then drying at 60 ℃ to obtain the zinc oxide nanorod.
(5) The surface of zinc oxide is modified with a layer of graphene, 0.5 mg/mL graphene solution is coated on the surface of a paper electrode (3000 rpm, 30 s) for growing a zinc oxide nanorod in a suspending way, and then the surface of the zinc oxide nanorod is modified with a layer of graphene after being dried in a 75 ℃ oven for 3 h.
(6) Preparing cadmium sulfide on the surface of graphene by a continuous ion adsorption method, firstly, dripping 100 mM aqueous solution (10 mu L) of cadmium sulfate on the surface of a paper electrode, standing for 30 s, sucking the solution from the back of the paper electrode by using absorbent paper, then dripping 100 mM aqueous solution (10 mu L) of sodium sulfide on the surface of the electrode for reaction for 30 s, sucking the solution from the back of the paper electrode by using the absorbent paper, and repeating the steps for 30 times to obtain a layer of cadmium sulfide on the surface of the graphene.
(7) Preparing hemiconcanavalin-gold nano-luminol composite material, mixing the gold nano (1 mL), 5 mM hemiconcanavalin (500 muL) and 5 mM luminol (500 muL), stirring for 5 h at 4 ℃, centrifuging, washing with water, and then re-dissolving in 5 mL secondary water to obtain the hemiconcanavalin-gold nano-luminol composite material.
(8) Fixing a cancer cell aptamer chain on the surface of a paper working electrode, blocking an active site by using bovine serum albumin, firstly adding 10 mu L of EDC-NHS mixed solution to the surface of the working electrode respectively for reaction for 3 h, then dropwise adding 10 mu L of the aptamer chain to the surface of the working electrode for reaction for 30 min, fixing an MCF-7 cancer cell aptamer chain by using a working electrode 1, fixing a K562 cancer cell aptamer chain by using a working electrode 2, fixing an HL-60 cancer cell aptamer chain by using a working electrode 3, then washing by using a pH 7.4 buffer solution, then adding 5 mg/mL of bovine serum albumin (10 mu L) to block the active site on the surface of the electrode, reacting for 1 h, and washing by using a pH 7.4 buffer solution.
(9) Capturing cancer cells on the surface of the working electrode, adding MCF-7, K562 and HL-60 cancer cells with different concentrations into the working electrodes 1, 2 and 3 respectively to react for 30 min, and washing with a buffer solution with pH 7.4 to remove the cancer cells which are not captured.
(10) Fixing the hemiconcanavalin-gold nano-luminol composite material on a working electrode for capturing cancer cells, respectively dripping 10 mu L of the prepared hemiconcanavalin-gold nano-luminol composite material on the surfaces of the working electrodes 1, 2 and 3 for reaction for 30 min, and then flushing and washing by using a buffer solution with pH 7.4 to remove the non-fixed hemiconcanavalin-gold nano-luminol composite material.
(11) The paper-based high-flux photoelectrochemical biosensor simultaneously detects three cancer cells, a modified paper chip is folded as shown in an attached drawing 3, then is fixed by an automatic conducting clamp as shown in an attached drawing 4 and is connected to an electrochemical workstation, hydrogen peroxide is added through an inlet, chemical reactions on three working electrodes are sequentially triggered due to different time when the hydrogen peroxide reaches the working electrodes, the three cancer cells are simultaneously detected by detecting the photocurrent intensity of the three working electrodes, and a working curve is drawn according to different cancer cell concentrations and photocurrent intensities.
Claims (9)
1. A construction method of a paper-based high-flux photo-electrochemical biosensor is characterized by comprising the following steps:
(1) designing a wax batch printing pattern of a three-dimensional hollow channel microfluidic paper chip on a computer, wherein the wax batch printing pattern consists of four parts, a working chip (A), a hollow channel chip (B), a semi-hydrophilic channel chip (C) and an auxiliary chip (D), the pattern is shown in figure 1, punching and cutting a wax batch printing pattern grid area by using a puncher and a laser cutting machine to prepare a solution inlet and a hollow channel, and printing carbon electrode patterns on the working area and the auxiliary electrode area which are designed on the back of the wax batch printing pattern respectively as shown in figure 2;
(2) modifying a layer of gold nanoparticles in a working area by a gold nano autocatalytic reduction method to serve as a paper working electrode substrate;
(3) preparing a zinc oxide nanorod on the surface of a paper working electrode for modifying gold nanoparticles by a hydrothermal method;
(4) modifying a layer of graphene on the surface of a zinc oxide nanorod;
(5) preparing cadmium sulfide on the surface of graphene by a continuous ion adsorption method;
(6) preparing a hemiconcanavalin-gold nano-luminol composite material;
(7) modifying an aptamer chain corresponding to the cancer cell on the surface of the paper working electrode to capture the cancer cell, and then sealing an active site on the surface of the working electrode by using bovine serum albumin;
(8) adding cancer cells with different concentrations to the surface of the modified paper working electrode, fixing MCF-7 cancer cells by the working electrode 1, fixing K562 cancer cells by the working electrode 2, and fixing HL-60 cancer cells by the working electrode 3;
(9) fixing the prepared hemiconcanavalin-gold nano-luminol composite material on a working electrode;
(10) the modified paper chip is folded as shown in figure 3, then is fixed by an automatic conducting clamp as shown in figure 4, is connected to an electrochemical workstation, hydrogen peroxide is added through an inlet, chemical reactions on the three working electrodes are sequentially initiated due to different time when the hydrogen peroxide reaches the working electrodes, and the three cancer cells are simultaneously detected by measuring the photocurrent intensity of the three working electrodes.
2. The construction method of the paper-based high-throughput photoelectrochemical biosensor according to claim 1, wherein the paper material is common filter paper, absorbent paper or chromatographic paper, the cancer cells are MCF-7, K562 and HL-60 cancer cells, and the MCF-7 cancer cell aptamers: 5' -GCA GTT GAT CCT TTG GAT ACC CTG GTT TTT TTT TTT-H2N-3', K562 cancer cell aptamers: 5' -H2N-TTT TTT TTT TAC AGC AGA TCA GTC TAT CTT CTC CTG ATG GGT TCC TAT TTA TAG GTG AAG CTG T-3', HL-60 cancer cell aptamers: 5' -H2N-TTT TTT TTT ATC CAG AGT GAC GCA GCA TGC CCT AGT TAC TAC TAC TCT TTT TAG CAA AC。
3. The construction method of the paper-based high-throughput photoelectrochemical biosensor, according to claim 1, is used for preparing a three-dimensional hollow channel microfluidic paper chip, and is characterized in that: the hydrophobic wax of designing micro-fluidic paper chip prints the pattern in batches on the computer, the style is as shown in figure 1, this wax prints the pattern in batches and comprises four bibliographic categories, is work chip (A), hollow channel chip (B), semi-hydrophilic channel chip (C) and supplementary chip (D) respectively, includes: a solution inlet (1), working electrodes 1-3 (2-4), a hollow channel (5), semi-hydrophilic channels (6-8), wherein the first semi-hydrophilic channel (6) is printed with 60% wax, the second semi-hydrophilic channel (7) is printed with 40% wax, the third semi-hydrophilic channel (8) is printed with 80% wax, and an auxiliary electrode printing area (9); then, punching and cutting the grid area in the attached figure 1 by using a puncher and a laser cutting machine to prepare a hollow channel; placing the chromatographic paper printed with the hydrophobic wax batch printing patterns into an oven, and heating for 30 s at 130 ℃ to ensure that the wax permeates the interior of the paper fiber to construct a completely hydrophobic part and a semi-hydrophobic part; the carbon electrode patterns are printed on the working area and the auxiliary electrode area designed on the back of the wax batch printing pattern respectively as shown in figure 2.
4. The construction method of the paper-based high-throughput photoelectrochemical biosensor, according to claim 1, a layer of gold nanoparticles is modified in a working area of a paper electrode by a gold nano autocatalytic reduction method, and the construction method is characterized in that: firstly, stirring 5 mL of chloroauric acid with the mass concentration of 1% and 10 mL of sodium citrate with the concentration of 0.03M for 10 min, then adding 250 mL of secondary water, continuing to stir 10 mL, finally adding 5 mL of sodium borohydride with the concentration of 0.1M, stirring at room temperature for 24 h, centrifuging at 12000 rpm for 30 min, re-dispersing in 10 mL of secondary water, preparing to obtain gold nano seeds, dropwise adding 10 mu L of prepared gold nano seeds to the surface of a working electrode, drying at room temperature, repeating for 5 times, dropwise adding 20 mu L of a mixed solution of sodium citrate with the mass concentration of 1% and 0.01M hydroxylamine hydrochloride to the surface of the electrode, reacting at room temperature for 30 min, and washing with secondary water to obtain the long-gold modified paper working electrode.
5. The construction method of the paper-based high-throughput photoelectrochemical biosensor according to claim 1, wherein the zinc oxide nanorod grows on the surface of the paper working electrode modified by gold nanoparticles, and the construction method is characterized in that: firstly, spin-coating a zinc acetate solution with the concentration of 40 nM on the surface of a paper working electrode modified by gold nanoparticles at the rotation speed of 1000 rpm for 60s, then drying at 95 ℃, repeating for 6 times, preparing 50 mL of a mixed solution of 25 mM zinc nitrate and 25 mM hexamethylenetetramine, placing the mixed solution in an autoclave, placing the paper working electrode in the autoclave with the conductive surface facing downwards, placing the autoclave in an oven with the temperature of 95 ℃ for reaction for 6 h, washing with secondary water after the reaction is finished, and then drying at 60 ℃ to obtain the zinc oxide nanorod.
6. The construction method of the paper-based high-throughput photoelectrochemical biosensor according to claim 1, wherein a layer of graphene is modified on the surface of zinc oxide, and the construction method is characterized in that: and (3) coating the graphene solution with the mass concentration of 0.5 mg/mL on the surface of a paper electrode for growing the zinc oxide nano rod in a spinning way, wherein the spinning speed is 3000 rpm, the spinning time is 30 s, and then drying in a 75 ℃ oven for 3 h to modify a layer of graphene on the surface of the zinc oxide nano rod.
7. The construction method of the paper-based high-throughput photoelectrochemical biosensor according to claim 1, wherein cadmium sulfide is prepared on the surface of graphene by a continuous ion adsorption method, and the construction method is characterized in that: firstly, 10 mu L of aqueous solution of cadmium sulfate with the concentration of 100 mM is dripped on the surface of a paper electrode, the mixture is kept stand for 30 s, the solution is sucked out from the back surface of the paper electrode by using absorbent paper, then 10 mu L of aqueous solution of sodium sulfide with the concentration of 100 mM is dripped on the surface of the electrode to react for 30 s, the solution is sucked out from the back surface of the paper electrode by using the absorbent paper, and the step is repeated for 30 times, so that a layer of cadmium sulfide can be obtained on the surface of graphene.
8. The construction method of the paper-based high-throughput photoelectrochemical biosensor according to claim 1, wherein the semi-concanavalin-gold nano-luminol composite material is prepared by the following steps: mixing 1 mL of gold nanoparticles prepared above, 500. mu.L of hemiconcanavalin with a concentration of 5 mM, and 500. mu.L of luminol with a concentration of 5 mM, stirring at 4 ℃ for 5 h, centrifuging, washing with water, and then dissolving in 5 mL of secondary water again to obtain the hemiconcanavalin-gold nanoparticles-luminol composite material.
9. The method for constructing the paper-based high-throughput photoelectrochemical biosensor as claimed in claim 1, wherein the paper-based high-throughput photoelectrochemical biosensor comprises the following steps: firstly, respectively adding 10 mu L of EDC-NHS mixed solution to the surface of a working electrode for reaction for 3 h, then dropwise adding 10 mu L of aptamer chain to the surface of the working electrode for reaction for 30 min, fixing the MCF-7 cancer cell aptamer chain by using a working electrode 1, fixing the K562 cancer cell aptamer chain by using a working electrode 2, fixing the HL-60 cancer cell aptamer chain by using a working electrode 3, then flushing by using a pH 7.4 buffer solution, then adding 10 mu L of bovine serum albumin with the mass concentration of 5 mg/mL to seal an electrode surface active site, reacting for 1 h, and flushing by using a pH 7.4 buffer solution; adding corresponding cancer cells with different concentrations into the working electrodes 1, 2 and 3 respectively to react for 30 min, washing with buffer solution with pH 7.4 to remove the cancer cells which are not captured; dripping 10 μ L of the semi-concanavalin-gold nano-luminol composite material prepared above onto the surfaces of working electrodes 1, 2 and 3 respectively for reaction for 30 min, and then washing with a pH 7.4 buffer solution to remove the non-fixed semi-concanavalin-gold nano-luminol composite material; the modified paper chip is folded as shown in the attached drawing 3, then is fixed by an automatic conducting clamp as shown in the attached drawing 4, is connected to an electrochemical workstation, hydrogen peroxide is added through a solution inlet, chemical reactions on the three working electrodes are sequentially initiated due to different time when the hydrogen peroxide reaches the working electrodes, the three cancer cells are simultaneously detected by measuring the photocurrent intensities of the three working electrodes, and a working curve is drawn according to different cancer cell concentrations and the photocurrent intensities.
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