CN111830104B - Photoelectrochemistry biosensor and preparation method and application thereof - Google Patents
Photoelectrochemistry biosensor and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a photoelectrochemistry biosensor and a preparation method and application thereof, belonging to the technical field of detection and analysis. The photoelectrochemical biosensor includes: an electrode comprising an electrode substrate and a covalent organic polymer film coated on the electrode substrate; the covalent organic polymer membrane is modified with palladium nanoparticles and an aptamer; the covalent organic polymer film is prepared by polymerizing 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde and tri (4-aminophenyl) amine. The cathode photoelectrochemical cell sensor is successfully constructed by in-situ synthesis of the covalent organic polymer film, can be used for detecting various cells such as cancer cells and the like by changing the types of the aptamers, and has high sensitivity and strong specificity, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of detection and analysis, and particularly relates to a photoelectrochemical biosensor and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The photoelectrochemical biosensor has the advantages of simple instrument, high sensitivity, easy miniaturization and the like, and is widely applied in recent years. Among them, semiconductor nanomaterials are the most widely used photoactive materials. The quantum size effect of the nano material can split the energy level of the material into a negative Conduction Band (CB) and a positive Valence Band (VB), and the potential change on the conduction band and the valence band can improve the redox capability and the photocatalytic performance of the nano material. The reported heterostructure-based Photoelectrochemical (PEC) biosensors have so far focused mainly on the coupling of various n-type semiconductors such as TiO2, znO and CdS quantum dots. Unlike n-type semiconductors, which use electrons as charge carriers, p-type semiconductors interact with electron acceptors, so that they use holes as the predominant carrier in a cathodic PEC sensor, effectively eliminating interference from reducing species.
Recently, covalent organic polymers have received increasing attention due to their unique properties, including well-defined chemical composition, interconnectivity of building blocks of different sizes, controllable structure, tunable function and high stability. Covalent Organic Polymers (COPs) have been widely used in the fields of gas storage and separation, catalysis, sensing, cancer therapy, opto-electronics and energy conversion.
Currently, methods for cancer detection include fluorescence, fluorescence imaging, raman spectroscopy, and colorimetry. However, the inventors have found that these methods still have the disadvantages of using expensive instruments and consuming a long time. Compared with the method, the photoelectrochemistry biosensor has the advantages of low cost, simple equipment, high sensitivity, quick operation and the like, and gradually draws attention of people.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a photoelectrochemical biosensor and a preparation method and application thereof, the invention successfully constructs a cathode photoelectrochemical cell sensor by in-situ synthesis of a covalent organic polymer film, can be used for detecting various cells such as cancer cells and the like by changing the types of aptamers, and has high sensitivity and strong specificity, thereby having good value of practical application.
In order to achieve the technical purpose, the technical scheme of the invention is as follows:
in a first aspect of the present invention, there is provided a photoelectrochemical biosensor comprising:
an electrode comprising an electrode substrate and covalent organic polymer films (COPs) coated on the electrode substrate;
the covalent organic polymer membrane is modified with palladium nanoparticles (PdNPs) and an aptamer;
wherein, the covalent organic polymer film is prepared by the polymerization reaction of 2, 6-dihydroxy naphthalene-1, 5-diformaldehyde (DHNDA) and tri (4-aminophenyl) amine (TAPA).
In a second aspect of the present invention, there is provided a method for preparing the above photoelectrochemical biosensor, the method comprising:
s1, preparing a covalent organic polymer film on an electrode substrate in situ;
s2, modifying the palladium nanoparticles and the aptamer on an electrode substrate coated by a covalent organic polymer film.
In a third aspect of the present invention, there is provided a use of the above-mentioned photoelectrochemical biosensor in cell detection.
In a fourth aspect of the invention, there is provided a method for detecting cells in vitro, the method comprising:
and incubating the sample to be detected and the photoelectrochemistry biosensor together, and measuring a photoelectric signal.
In a fifth aspect of the present invention, there is provided a use of the above-described photoelectrochemical biosensor and/or detection method in any one of:
1) Screening anti-cancer drugs;
2) Biological sample cancer cell analysis.
The beneficial technical effects of one or more technical schemes are as follows:
1. synthesis of covalent organic Polymer films (COP): under the condition of room temperature, a COP film is synthesized by in-situ growth on a transparent indium tin oxide coated glass (ITO) slice through a one-pot method, the obtained COP film has good cathode PEC performance, and PdNPs can catalyze H 2 O 2 Oxidation of dopamine to produce amino pigments and O 2 And can be used as electron acceptors of COPs to generate enhanced photocurrent when irradiated with light. Further, the technical scheme develops a cathode photoelectrochemical cell sensor, and a specially designed aptamer of the cathode photoelectrochemical cell sensor is used as a recognition element to detect the human lung cancer cell line.
2. High sensitivity: the technical scheme uses the aptamer with high selectivity and specificity, greatly improves the sensitivity of the sensor, can sensitively detect cancer cells, and has detection lines of 8 cells per milliliter.
3. High specificity: in the technical scheme, the aptamer with high specificity and selectivity is used as the capture probe of the sensor, so that non-specific combination of other non-target cells and the aptamer is avoided to a great extent, and the specificity of the invention is greatly improved.
4. A wide range of potential applications: the PEC cell sensor designed according to the above technical solution can be further applied to various cancers by simply changing the kinds of aptamers, and thus, the PEC cell sensor can provide a simple, low-cost and rapid strategy to sensitively detect various cells without involving any extraction of nucleic acids and proteins, and provide a basis and possibility for early detection and diagnosis of cancers, thereby having good practical application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.
FIG. 1 is a diagram of the preparation and detection of a photoelectrochemical biosensor of the present invention; wherein, A is a schematic diagram of in-situ synthesis of COP thin films on transparent indium tin oxide coated glass (ITO), B is a schematic diagram of photoelectrochemistry PEC cell sensor structure, and C is a schematic diagram of detection of A549 cells by using the photoelectrochemistry PEC cell sensor;
FIG. 2 is a diagram showing the characteristics of materials used in examples of the present invention, wherein A is an infrared spectrum of tris (4-aminophenyl) amine (TAPA), 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde DHNDA) and COP, B is an SEM image of a COP film, C is a TEM image of PdNPs, D is XRD data of the COP film, and E and F are Bu at 0.1 mol/L, respectively 4 NPF 6 Diffuse Reflectance Spectra (DRS) and CV curves of ITO glass modified by COP in acetonitrile at a potential ranging from-1.5V to 2V (Ag/Ag was used as potential) + Electrodes for measurement); the scan rate was 0.1 volts per second; inset in F shows that the ITO glass contains 1 mM Fc and 0.1M Bu 4 NPF 6 CV curve in acetonitrile solution;
FIG. 3 shows the photocurrent of different modified electrodes in the present invention, where a is COP/ITO, b is PdNPs/COP/ITO, and a and b are both 10 mmol/L or moreThe scan was performed in 0.1 mol/l phosphate buffered saline (pH = 7.4) of dopamine, c is COP/ITO, d is PdNPs/COP/ITO, e is Aptamer/PdNPs/COP/ITO, f is A549 cells/MCH/Aptamer/PdNPs/COP/ITO, c, d, e, f are all 10 mmol/l dopamine and 10 mmol/l H 2 O 2 0.1 moles per liter phosphate buffered solution (pH = 7.4);
FIG. 4 is the photocurrent response of COP/ITO and PdNPs/COP/ITO electrodes in 0.1 mol/L phosphoric acid buffer solution (pH = 7.4) in the example of the invention, where a is by purifying N 2 Oxygen removal, b is a compound containing dissolved O 2 C is 10 millimoles of H per liter 2 O 2 D is 10 mmoles of dopamine per liter and 10 mmoles of H per liter 2 O 2 E is saturated O 2 Error bars show the standard deviation of the three experiments;
FIG. 5 is a graph showing the results of sensitivity experiments in examples of the present invention, wherein A is the change in photocurrent (from a to g:0, 10) of the sensor for various concentrations of the lung adenocarcinoma cell line (A549 cells) 2 ，10 3 ，10 4 ，10 5 ，10 6 Per ml of individual cells); b is a linear relationship between photocurrent and log concentration of lung adenocarcinoma cells (a 549 cells); error bars represent standard deviations of three independent experiments;
FIG. 6 is a graph showing the results of selective experiments and the characterization of stability results in examples of the present invention, wherein A is a graph measuring photocurrents of the cathodic PEC cell sensor against human breast cancer cell lines (MCF-7 cells), human cervical cancer cell lines (Hela cells), human liver cancer cell lines (HepG 2 cells) and lung adenocarcinoma cell lines (A549 cells); the concentration of each type of cells was 10 6 Per ml of individual cells; error bars show the standard deviation of three experiments; b is a cathode PEC biosensor containing 10 millimoles of dopamine per liter and 10 millimoles of H per liter 2 O 2 Of phosphoric acid buffer solution (pH = 7.4) for 11 cycles in succession.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. 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.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As described above, methods currently used for cancer detection include fluorescence, fluorescence imaging, raman spectroscopy, colorimetry, and the like. However, these methods still have the disadvantages of using expensive instruments and consuming a long time.
In view of the above, in one embodiment of the present invention, there is provided a photoelectrochemical biosensor including:
an electrode comprising an electrode substrate coated with covalent organic polymer films (COPs);
the covalent organic polymer membrane is modified with palladium nanoparticles (PdNPs) and aptamers.
The covalent organic polymer film is prepared by polymerizing 2, 6-dihydroxy naphthalene-1, 5-diformaldehyde (DHNDA) and tri (4-aminophenyl) amine (TAPA).
In still another embodiment of the present invention, the electrode substrate may be made of a material having transparency and excellent conductivity, but is not limited thereto. Specific examples of the electrode substrate include metal oxides such as zinc oxide, indium Tin Oxide (ITO), and Indium Zinc Oxide (IZO), preferably Indium Tin Oxide (ITO) coated glass.
In yet another embodiment of the present invention, the aptamer is chemically immobilized on the surface of the electrode by palladium sulfide.
The aptamer can be specifically combined with an object to be detected, so that the aptamer is used for capturing the object to be detected to further realize detection of the object to be detected; preferably, the aptamer is a nucleic acid aptamer.
In another embodiment of the present invention, the test substance is preferably a cell, such as a cancer cell, and further a lung cancer cell.
In yet another embodiment of the present invention, the palladium nanoparticle has a diameter of 4 to 10nm.
In another embodiment of the present invention, there is provided a method for preparing the above-mentioned photoelectrochemical biosensor, the method comprising:
s1, preparing a covalent organic polymer film on an electrode substrate in situ;
s2, modifying the palladium nanoparticles and the aptamer onto an electrode substrate coated with a covalent organic polymer film.
In another embodiment of the present invention, the step S1 specifically comprises the following steps:
dispersing 2, 6-dihydroxynaphthalene-1, 5-diformaldehyde (DHNDA) and tris (4-aminophenyl) amine (TAPA) in a solution containing xylene, anhydrous ethanol and glacial acetic acid, uniformly dispersing to obtain a mixed solution, and immersing the electrode substrate in the mixed solution.
The mass ratio of DHNDA to TAPA is 1;
the volume ratio of the xylene, the anhydrous ethanol and the glacial acetic acid is 1-10, and is as follows, 1.
The mass volume ratio of the DHNDA to the dimethylbenzene is 1-10 mu L; such as 1. Mu.L of 1mg;
immersing the mixed solution for 1-30 min, such as 1min, 5min, 10min, 20min and 30min; preferably 10min, and the covalent organic polymer film is uniformly covered on the electrode substrate by controlling the immersion time, thereby being beneficial to later detection.
In another embodiment of the present invention, the step S2 includes:
and dripping the palladium nanoparticle solution on the covalent organic polymer film-coated electrode substrate, and dripping the aptamer on the palladium nanoparticle modified covalent organic polymer film-coated electrode substrate after drying.
Wherein the aptamer is denatured and thiolated (activated).
Wherein the specific denaturation method comprises the following steps: modifying the aptamer at 90-95 ℃ for 1-10 minutes, then cooling at 0-5 ℃ for 1-20 minutes, and finally cooling at 20-30 ℃ for 1-10 minutes;
the specific method for thiolation treatment comprises the following steps: the disulfide-bonded aptamer is reduced with tris (2-carboxyethyl) phosphine hydrochloride for 0.5 to 2 hours (preferably 1 hour).
After aptamer immobilization modification to the electrode, the unmodified site was blocked with 6-Mercaptohexanol (MCH).
The palladium nanoparticles can be obtained by commercial or self-preparation; the preparation method is not particularly limited, and the sodium citrate is preferably prepared by a sodium citrate reduction method.
In another embodiment of the present invention, there is provided a use of the above-described photoelectrochemical biosensor in cell detection.
Wherein the cell is a cancer cell, further a lung cancer cell.
When the cell is a lung cancer cell (a 549), the aptamer nucleotide sequence is:
5’-HS-GGT TGC ATG CCG TGG GGA GGG GGG TGG GTT TTA TAG CGT ACT CAG-3’(SEQ ID NO.1)。
in another embodiment of the present invention, there is provided a method for detecting cells in vitro, the method comprising:
and incubating the sample to be detected and the photoelectrochemistry biosensor together, and measuring a photoelectric signal.
The incubation specific conditions are as follows: incubating at 30-40 deg.C (preferably 37 deg.C) for 15-75 min (preferably 30 min); thereby leading the aptamer to be fully combined with the cells to be detected and improving the detection accuracy and sensitivity.
In another embodiment of the present invention, there is provided a use of the above-mentioned photoelectrochemical biosensor and/or detection method in any one of the following:
1) Screening drugs;
2) Analysis of cells in a biological sample;
wherein the drug comprises an anti-cancer drug.
The cells include cancer cells, such as lung cancer cells.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The nucleotide sequence of the A549 aptamer is as follows: 5'-HS-GGT TGC ATG CCG TGG GGA GGG GGG TGG GTT TTA TAG CGT ACT CAG-3' (SEQ ID No. 1).
Examples
The principle of the embodiment is as shown in fig. 1:
The specific process comprises the following steps:
synthesis of COP film on ITO glass: cutting ITO glass into 4.7 cm × 1cm pieces, 10% at 1.0 mol/l NaOH 2 O 2 And acetoneMedium sonication, followed by thorough rinsing with ultra pure water and blow drying with nitrogen before use. 1mg of 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) and 1mg of tris (4-aminophenyl) amine (TAPA) were dispersed in 250. Mu.l of xylene, 250. Mu.l of absolute ethanol and 50. Mu.l of glacial acetic acid. After the ultrasonic treatment to obtain a uniformly dispersed solution, the ITO electrode was immersed in the above mixed solution for 10 minutes, and when the color changed from dark to red, a COP film was obtained. The obtained ITO electrode (COP/ITO) modified with a COP thin film was washed with methylene chloride.
Synthesis of Palladium nanoparticles (PdNPs) (diameter 4-10 nm): magnetic stirring was maintained throughout the synthesis. First, pdCl is added 2 Dissolving in hydrochloric acid to prepare Pd precursor stock solution. Then 16.6 ml of 0.2% precursor solution was added to 483.4 ml of boiling deionized water, after 1 minute, 11.6 ml of 1% sodium citrate solution was added, and 5.5 ml of freshly prepared NaBH in 50 ml of water was rapidly injected 4 (0.038 g) and sodium citrate (0.5 g). The dark brown solution was held at boiling point for 10 minutes and then cooled to room temperature.
Preparation of photoelectrochemical cell sensor: 20 ml of PdNPs solution was dropped onto COP/ITO and dried at room temperature. Then 6 micromoles per liter of A549 aptamer were denatured at 95 ℃ for 5 minutes, then cooled at 4 ℃ for 15 minutes, and finally at 25 ℃ for 5 minutes. After 25 micromole per liter of TCEP is added to open the palladium-sulfur bond of the aptamer, 20 microliters of aptamers with different concentrations are dripped on PdNPs/COP/ITO electrodes and incubated for 12 hours at room temperature, so that the aptamer is chemically fixed on the surface of the electrode through palladium-sulfur. After washing with 10 mM phosphate buffer (pH 7.4), 20. Mu.L MCH (1 mM) was dropped on the electrode for 60 minutes to block unmodified sites. After washing with 10 mmol/l phosphate buffer solution (pH = 7.4), the electrodes were incubated with lung adenocarcinoma cell line (a 549 cells) for 30 minutes. The prepared electrode was washed with 10 mmol/l phosphoric acid buffer solution (pH = 7.4), stored at 4 ℃, and dried under a nitrogen stream.
Culturing and electrochemical detection of lung adenocarcinoma cell line (A549 cell): an electrochemical biosensor is constructed on the ITO electrode. Lung adenocarcinoma cell line (A549 cell) in 10% fetal calf bloodDulbecco's Modified Eagle's Medium (DMEM) containing 1% penicillin-streptomycin and serum (FBS) were incubated at 37 ℃ in an incubator containing 5% carbon dioxide. Cell density was calculated using a cell counter. The cell concentration reaches 10 6 After each ml of cells, the cells were harvested by 800 rpm/min centrifugation for 5 minutes, and then added to a549 aptamer/PdNPs/COP/ITO electrodes and incubated for 30 minutes. Subsequently, the electrodes were rinsed with 10 mmol per ml of phosphate buffer solution (pH = 7.4) and a cathodic PEC photocurrent signal measurement was performed.
Results of the experiment
1. Characterization of materials
This example investigated the synthesis of COP films using infrared spectroscopy (FTIR) (fig. 2A). The infrared spectrum of tris (4-aminophenyl) amine (TAPA) was 3338cm -1 And 162cm -1 There is one stretching frequency corresponding to N-H and C = C for the amine and phenyl rings, respectively. The infrared spectrum of 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) has a characteristic tensile vibration at 2918cm -1 Characteristic tensile vibration at 639cm -1 Characteristic stretching vibration of carbonyl group. Tris (4-aminophenyl) amine (TAPA) after polymerization was at 3338cm -1 The N-H stretched tape and 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) were measured at 1639cm -1 C = O tensile band disappeared at 1603cm -1 A new C-N tensile belt appears at 2911cm -1 The C-H stretching frequency corresponding to the phenyl ring appeared, confirming that the monomers of tris (4-aminophenyl) amine (TAPA) and 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde (DHNDA) have been converted to COP.
The present example also uses a Scanning Electron Microscope (SEM) to study the morphology and structure of COP films and PdNPs. SEM of COP films showed that they were packed nanospheres (FIG. 2B) with diameters of 300-400 nm, pdNPs were spherical with diameters of 4-10nm (FIG. 2C). This example investigated the crystal structure of a COP film using an X-ray diffraction pattern (XRD) (fig. 2D). The two broad peaks at 11.5 ℃ and 21.8 ℃ represent the (100) and (001) crystal planes, respectively. The broad peak at the higher 2 theta angle (21.8 ℃) corresponds to the (001) plane caused by pi stacking between COP layers.
In nearly the entire visible range, the uv diffuse reflection of COP films has a strong absorption capacity in the range of 200-800 nm and a broad peak in the range of 250-450 nm (fig. 2E). The band gap of the COP film can be derived from the absorption edge using the following equation:
E g (eV)＝hc/λ (1)
where h is the Planck constant, c is the speed of light, and λ is the wavelength. The band gap (Eg) for the calculated COP is 1.84 electron volts. 0.1 mol/L Bu of ITO glass modified by COP film 4 NPF 6 Energy level (E) of Cyclic Voltammetry (CV) on COP film in acetonitrile solution onset Ox 、E onset Red And E Fc/Fc+ ) Evaluation was made wherein the solution used N 2 Bubbling for 30 minutes (fig. 2F). The Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO) levels were calculated using equations 2, 3.
E HOMO ＝–(E onset Ox –E Fc/Fc+ +4.8)eV (2)
E LUMO ＝–(E onset Red -E Fc/Fc+ +4.8)eV (3)
Wherein E HOMO Is the highest energy occupying the molecular orbital, E LUMO Is the energy of the lowest unoccupied molecular orbital, E onset Ox Is the initial potential of oxidation, E onset Red Is the initial potential for reduction. According to Eg = E LUMO -E HOMO ，(E FC/FC+ ) = 0.32V, use Ag/Ag + Electrode), the band gap (Eg) of the COP film was calculated to be 1.50 ev. The obtained value (1.50 electron volts) is consistent with the value (1.84 electron volts) obtained by the ultraviolet diffuse reflection theoretical algorithm.
2. Experimental verification of principle
To demonstrate the feasibility of this approach, this example used a method of preparing a cathodic PEC cell sensor using a different modified electrode (fig. 3). After in situ growth of COP films on ITO electrodes, scanning was performed in 10 mmol/l phosphoric acid buffer solution (pH = 7.4) and a photocurrent of-442 microamperes was observed, indicating that COP had good photo-electrical properties (figure 3, curve a). When the COP/ITO electrode contained 10 millimoles of dopamine per liter and 10 millimoles of H per liter 2 O 2 When scanned in 10 millimoles per liter of phosphate buffer (pH = 7.4) (fig. 3, curve c), the photocurrent increased to-624 microamps. PdNPs were scanned in 10 mmol per liter phosphate buffer (pH = 7.4) after fixation on COP/ITO, with a further increase in photocurrent to-1153 microamperes (fig. 3, curve b). As the PdNPs have good conductivity, when the PdNPs/COP/ITO electrode contains 10 millimole per liter of dopamine and 10 millimole per liter of H 2 O 2 When scanned in 10 mmoles per liter phosphate buffer solution (pH = 7.4) (fig. 3, curve d), the photocurrent increased to-3116 microamperes, indicating successful deposition of PdNPs on COP/ITO surface. The increase in photocurrent may be interpreted as being at H 2 O 2 In the presence of PdNPs, the PdNPs can catalyze dopamine oxidation to generate amino pigments and O 2 They can act as electron acceptors for COPs, producing enhanced photocurrent when irradiated with light. It is noted that when 10 is present 6 The photocurrent was reduced to-1185 microamperes per ml of lung adenocarcinoma cell line (a 549 cells) (fig. 3, curve f), which was much less than the photocurrent (-2541 microamperes) in the absence of lung adenocarcinoma cell line (a 549 cells) (fig. 3, curve e). This may be explained by that the captured lung adenocarcinoma cell line (a 549 cell) may cover the PdNPs modified COP/ITO electrode surface, blocking dopamine and H 2 O 2 Into the electrode surface, resulting in a reduction in photocurrent.
3. PEC Performance mechanistic experiments
Control experiments were performed by measuring the photocurrent response of bare PdNPs/COP/ITO electrodes in 0.1 millimole per liter solutions of phosphate buffer (pH = 7.4) containing (1) purified N 2 Oxygen removal, (2) dissolution of O 2 (3) 10 mmoles of H per liter 2 O 2 (4) 10 mmoles of dopamine per liter and 10 mmoles of H per liter 2 O 2 And (5) saturated O 2 . As shown in FIG. 4, the photocurrent of COP/ITO electrode was-442 microampere (FIG. 4 b), and PdNPs/COP/ITO electrode was exposed to dissolved O 2 Was scanned in 0.1 millimoles per liter of phosphate buffer solution and the photocurrent was-1153 microamps (fig. 4 b). Adding H into electrolyte 2 O 2 Then, the photocurrent of COP/ITO and PdNPs/COP/ITO electrodes is respectively increased to-555 microamperes (figure)4c) And-1899 microamperes (FIG. 4 c). In the presence of H 2 O 2 And dopamine, the photoelectric current of the COP/ITO electrode and the PdNPs/COP/ITO electrode is further increased to-624 microamperes and-3116 microamperes. This may be explained by PdNPs catalyzing H 2 O 2 Oxidation of dopamine to produce amino pigments and O 2 They can act as electron acceptors for COPs to produce enhanced photocurrent. In contrast, when the solution of the air-saturated electrolyte was deoxygenated by nitrogen purging, a suppressing effect of cathode photocurrent was generated on the COP/ITO and PdNPs/COP/ITO electrodes (fig. 4 a). The influence of oxygen on the photocurrent of COP/ITO and PdNPs/COP/ITO electrodes was further investigated. Introducing O into the solution 2 The photocurrent of the COP/ITO electrode and the PdNPs/COP/ITO electrode can be increased to-963 microampere (fig. 4 e) and-4588 microampere (fig. 4 e), respectively. These results indicate that oxygen can act as an electron acceptor to produce the cathode photocurrent. Under illumination, photo-generated electrons are transferred from the Conduction Band (CB) of the COP to electron acceptors in the electrolyte, and photo-generated Valence Band (VB) holes are captured by the ITO electrode, thereby generating a cathode photocurrent. Since the solution contains a large amount of dissolved oxygen, the dissolved oxygen can act as an electron acceptor to accept photo-generated electrons from COP, which in turn enhances photocurrent. In addition, pdNPs can also catalyze the reduction of oxygen, which is constantly supplied to the solution as an electron acceptor, resulting in a higher photocurrent (fig. 4 e).
3. Sensitivity test
In order to evaluate the sensitivity of the present technical scheme for detecting lung adenocarcinoma cell lines (a 549 cells), photocurrents of lung adenocarcinoma cell lines (a 549 cells) at different concentrations were measured under the optimal experimental conditions. As shown in fig. 5A, the photocurrent decreased with the increase in the concentration of the lung adenocarcinoma cell line (a 549 cells). In the range of 10 to 10 6 The photocurrent intensity had a good linear relationship to the logarithm of the cell concentration per milliliter range of cells (fig. 5B). The corresponding linear equation is I =227.6log 10 C-2617 (correlation coefficient 0.996), where I is the photocurrent of the cathodic PEC cell sensor and C is the concentration of the lung adenocarcinoma cell line (A549 cells) per ml. The limit of detection was calculated as 8 cells per ml, calculated as three times the standard deviation of the blank signal.
4. Specificity, reproducibility and stability experiments
To investigate the selectivity of the cathode PEC cell transporter, a human breast cancer cell line (MCF-7 cells), a human liver cancer cell line (HepG 2 cells), and a human cervical cancer cell line (Hela cells) were used as control groups. As shown in fig. 6A, the photocurrent difference of a549 cells (Δ I = -1273 microampere) was much larger than those of MCF-7 cells (Δ I = -163 microampere), hepG2 cells (Δ I = -156 microampere) and Hela cells (Δ I = -286 microampere) at the same concentration for different types of cells. Photocurrent difference was defined as Δ I = I 0 -I, wherein I is the photocurrent value of the cathode PEC cell sensor in response to the cell, I 0 Is a blank signal without cells (Δ I = -1273 microamps). These results may be explained by the high affinity of the designed aptamers for the a549 cell target membrane protein, indicating that the proposed cathodic PEC cell sensor has good specificity for a549 cells.
To evaluate the reproducibility of the proposed cathodic PEC cell sensor, 10 mmoles per liter dopamine and 10 mmoles per liter H were included 2 O 2 Five replicates were performed per liter of 0.1 moles phosphate buffer (pH = 7.4). The Relative Standard Deviation (RSD) was 4.87%, indicating that the proposed cell sensor has good reproducibility. The stability of the proposed cell sensor was further investigated. FIG. 6B shows a PEC biosensor containing 10 mmoles of dopamine per liter and 10 mmoles of H per liter 2 O 2 0.1 mol per liter of phosphate buffer (pH = 7.4) was scanned continuously for photocurrent response curves at 11 cycles. The photocurrent remains the same value during each switching lamp period. The Relative Standard Deviation (RSD) was 4.80%, indicating that the proposed cathodic PEC cell sensor has good stability for the detection of a549 cells.
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> university of Shandong Master
<120> photoelectrochemistry biosensor and preparation method and application thereof
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<170> PatentIn version 3.3
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<213> Artificial sequence
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Claims (26)
1. An photoelectrochemical biosensor, comprising:
an electrode comprising an electrode substrate and a covalent organic polymer film coated on the electrode substrate;
the covalent organic polymer membrane is modified with palladium nanoparticles and an aptamer;
the covalent organic polymer film is prepared by polymerizing 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde and tri (4-aminophenyl) amine.
2. The photoelectrochemical biosensor of claim 1, wherein said electrode substrate comprises a metal oxide.
3. The photoelectrochemical biosensor of claim 2, wherein said metal oxide comprises zinc oxide, indium tin oxide, and indium zinc oxide.
4. The photoelectrochemical biosensor of claim 2, wherein said metal oxide is indium tin oxide.
5. The photoelectrochemical biosensor of claim 1, wherein said aptamer is chemically immobilized on the surface of the electrode by palladium sulfide;
or, the aptamer can be specifically combined with an analyte;
or, the test substance is a cell;
or the diameter of the palladium nano-particles is 4-10nm.
6. The photoelectrochemical biosensor of claim 5, wherein said aptamer is a nucleic acid aptamer.
7. The photoelectrochemical biosensor of claim 5, wherein said analyte cell is a cancer cell.
8. The photoelectrochemical biosensor of claim 7, wherein said cancer cell is a lung cancer cell.
9. The method for preparing the photoelectrochemical biosensor according to any one of claims 1 to 8, wherein the method comprises the following steps:
s1, preparing a covalent organic polymer film on an electrode substrate in situ;
s2, modifying the palladium nanoparticles and the aptamer on an electrode substrate coated by a covalent organic polymer film.
10. The method according to claim 9, wherein the step S1 comprises:
dispersing 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde and tris (4-aminophenyl) amine into a solution containing xylene, anhydrous ethanol and glacial acetic acid, uniformly dispersing to obtain a mixed solution, and immersing the electrode substrate into the mixed solution.
11. The method according to claim 10, wherein the mass ratio of the 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde to the tris (4-aminophenyl) amine is 1.
12. The production method according to claim 10, wherein the volume ratio of the xylene to the absolute ethyl alcohol to the glacial acetic acid is 1 to 10.
13. The method according to claim 10, wherein the mass volume ratio of the 2, 6-dihydroxynaphthalene-1, 5-dicarboxaldehyde to the xylene is 1mg to 10 μ L.
14. The method according to claim 10, wherein the mixed solution is immersed for 1 to 30 minutes.
15. The method according to claim 9, wherein the step S2 comprises:
and dripping the palladium nanoparticle solution on the covalent organic polymer film-coated electrode substrate, drying, and dripping the aptamer on the palladium nanoparticle-modified covalent organic polymer film-coated electrode substrate.
16. The method of claim 15, wherein the palladium nanoparticles are prepared by a sodium citrate reduction method.
17. The method of claim 15, wherein the aptamer is denatured and thiolated;
wherein the specific denaturation method comprises the following steps: modifying the aptamer at 90-95 ℃ for 1-10 minutes, then cooling at 0-5 ℃ for 1-20 minutes, and finally cooling at 20-30 ℃ for 1-10 minutes;
the specific method for thiolation treatment comprises the following steps: reducing the adapter bonded by the disulfide bond for 0.5 to 2 hours by adopting tris (2-carboxyethyl) phosphine hydrochloride.
18. The method of claim 15, wherein the reduction time is 1 hour.
19. Use of the photoelectrochemical biosensor of any one of claims 1 to 8 in cell detection.
20. The use of claim 19, wherein the cell is a cancer cell.
21. The use of claim 20, wherein the cells are lung cancer cells;
the aptamer nucleotide sequence of the lung cancer cell is as follows:
5’-HS-GGT TGC ATG CCG TGG GGA GGG GGG TGG GTT TTA TAG CGT ACT CAG-3’（SEQ ID NO.1）。
22. a method for detecting cells in vitro, the method comprising:
incubating a sample to be tested with the photoelectrochemical biosensor of any one of claims 1 to 8, and performing a photoelectric signal measurement.
23. The method for detecting cells in vitro according to claim 22, wherein said incubation is carried out under specific conditions: incubating for 15 to 75min at the temperature of 30 to 40 ℃.
24. The method for detecting cells in vitro according to claim 22, wherein said incubation is carried out under specific conditions: incubate at 37 ℃ for 30 min.
25. Use of the photoelectrochemical biosensor of any one of claims 1 to 8 and/or the method for in vitro cell detection of claim 22 in any one of the following:
1) Screening drugs;
2) Analysis of cells in a biological sample.
26. The use of claim 25, wherein the medicament comprises an anti-cancer drug; the cells include cancer cells.
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