CN110702750A - PEC aptamer sensor with high specificity and ultrahigh detection sensitivity and preparation method thereof - Google Patents
PEC aptamer sensor with high specificity and ultrahigh detection sensitivity and preparation method thereof Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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Abstract
The invention discloses a PEC aptamer sensor with high specificity and ultrahigh detection sensitivity and a preparation method thereof, wherein the PEC aptamer sensor comprises the following components: BiVO4Film (as photoanode), DNA aptamer and g-C3N4A thin film, the PEC aptamer sensor being in g-C3N4The film is used as an interface mediator between the photoanode and the DNA aptamer, g-C3N4The thickness of the film is 5 nm-15 nm. The invention has the advantages that: BiVO4Film and g-C3N4The difference of band structures exists between the films, and the difference of the band structures can promote BiVO4Photogenerated hole direction g-C on thin film3N4The film is directionally transferred, so that the capability of directionally transporting photogenerated holes to the substance to be detected can be obviously improved, and the PEC aptamer sensor has ultrahigh detection sensitivityDegree, in addition, g-C3N4The film has good pi-pi adsorption property on the DNA aptamer, and can effectively fix the DNA aptamer, thereby realizing high-specificity capture of a substance to be detected, and the PEC aptamer sensor has high specificity.
Description
Technical Field
The invention relates to a PEC aptamer sensor and a preparation method thereof, in particular to a PEC aptamer sensor with high specificity and ultrahigh detection sensitivity and a preparation method thereof, and belongs to the technical field of photoelectrochemistry.
Background
The Photoelectrochemical (PEC) sensor takes a light source as an excitation signal, utilizes the electron transfer between a semiconductor photoelectric material in an excited state and an object to be detected to achieve qualitative or quantitative analysis of the object to be detected, and has the advantages of lower background signal, high sensitivity, simple operation, low detection cost and the like compared with the traditional detection methods (such as chromatography, enzyme labeling, atomic absorption spectroscopy and the like) because the excitation signal (the light source) and the detection signal (the electrochemical signal) are different and do not interfere with each other, so the PEC sensor has important application prospects in the fields of environmental risk monitoring and biomedical detection.
Early PEC sensors generally had the disadvantages of poor specificity, low sensitivity, weak signal against interference, etc., and in order to overcome these disadvantages, researchers combined biotechnology with PEC sensors to form biosensing technology, and this combination proved to be effective in overcoming the above disadvantages. Currently, the major biosensing technologies include: enzyme-linked immunosorbent assay technology, molecular imprinting technology and aptamer technology, wherein the aptamer technology is characterized in that a random oligonucleotide DNA sequence (DNA aptamer) capable of specifically recognizing a target object is fixed on a PEC sensor, and the DNA aptamer is used as a front-end detection probe to capture a substance to be detected, so that the photoelectric signal on the surface of the PEC sensor is changed. Compared with molecular imprinting technology and enzyme-linked immunosorbent assay technology, the aptamer technology has higher specificity and environmental stability, so that the aptamer technology is the best biological sensing technology.
The DNA aptamer is combined with a PEC sensor to form a novel sensor, which is called a PEC aptamer sensor.
At present, DNA aptamers are fixed on a photoanode material of a PEC sensor, and other organic matters (such as-NH) with specific groups are often required to be introduced2HS, -CHO, etc.) as a fixing agent, the presence of which increases the electron transport energy barrier between the semiconducting optoelectronic material and the DNA aptamer, therebyWhile reducing the detection sensitivity and specificity of the PEC aptamer sensor.
Disclosure of Invention
To solve the disadvantages of the prior art, it is an object of the present invention to provide a PEC aptamer sensor having high specificity and ultra-high detection sensitivity and a method for preparing the same.
In order to achieve the above object, the present invention adopts the following technical solutions:
a PEC aptamer sensor with high specificity and ultra-high detection sensitivity, comprising: BiVO4Film and DNA aptamer, wherein, BiVO4The film is used as a photo-anode and is characterized by also comprising: g-C3N4A thin film, the PEC aptamer sensor being in g-C3N4The film acts as an interface mediator between the photoanode and the DNA aptamer.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that the BiVO is4The thickness of the film is 300 nm-400 nm.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that the g-C is3N4The thickness of the film is 5 nm-15 nm.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that the PEC aptamer sensor is used for detecting microcystin MC-LR, and the base sequence of the DNA aptamer is as follows: 5'-GGCGCC AAA CAG GAC CAC CAT GAC AATTAC CCA TAC CACCTC ATT ATG CCC CAT CTC CGC-3' are provided.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized by being used for detecting heavy metal ions Hg2+The base sequence of the DNA aptamer is as follows: 5'-TTC TTT CTT CCC TTG TTG GTT-3' are provided.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that the PEC aptamer sensor is used for detecting antibiotic tetracycline, and the base sequence of the DNA aptamer is as follows: 5'-CGT ACG GAA TTC GCT AGC CCC CCG GCAGGC CAC GGC TTG GGT TGG TCC GAC TGC GCG TGG ATC CGA GCT CCA CGT G-3' are provided.
The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that the PEC aptamer sensor is used for detecting a tumor marker dopamine, and the base sequence of the DNA aptamer is as follows: 5'-GTC TCT TGC GCC AGA GAA CAC TGGGGC AGA TAT GGG CCA GCA CAG AAT GAG GCC C-3' are provided.
The method for preparing the PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized in that a photoelectrochemical anodic oxidation method is adopted to prepare BiVO on a conductive substrate4Thin film, then in BiVO4On the film is decorated with g-C3N4Film, finally using g-C3N4The surface pi-pi adsorption of the film fixes the DNA aptamer for detecting the object to be detected on g-C3N4On the film.
The method for preparing the PEC aptamer sensor with high specificity and ultrahigh detection sensitivity is characterized by comprising the following steps:
(1) cleaning the conductive substrate, and drying for later use;
(2) adding Bi (NO)3)3·5H2Dissolving O and NaI in ultrapure water, adjusting the pH value of the solution to 4, fully dissolving p-benzoquinone in ethanol, mixing the two solutions, uniformly stirring, and storing in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking a conductive substrate as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode;
(4) dripping acetylacetonatovanadyl solution on the BiOI film prepared in the step (3), drying, annealing at 450 ℃ for 1h, and then cleaning with NaOH solution to obtain BiVO4A film;
(5) g to C3N4Dissolving the powder in ultrapure water to form a dispersion liquid, and suspending and coating the dispersion liquid on the BiVO prepared in the step (4)4Keeping the temperature of the film at 350-650 ℃ for 1-5 h to obtain BiVO4/g-C3N4A film;
(6) BiVO prepared in step (5)4/g-C3N4Dripping DNA aptamer solution on the surface of the film, keeping the temperature for 2h at 36 ℃, and then cleaning the DNA aptamer which is not firmly adsorbed on the surface of the film.
The aforementioned method for preparing a PEC aptamer sensor with high specificity and ultra-high detection sensitivity is characterized in that, in step (3), the deposition parameters are: depositing at-0.1 to-0.5V for 20s, and then depositing at-0.1V for 40 s.
The invention has the advantages that:
(1) in BiVO4By modifying g-C on the film3N4Film, preparing BiVO4/g-C3N4Electrode material of heterojunction structure, BiVO4Film and g-C3N4The difference of band structures exists between the films, and the difference of the band structures can promote BiVO4Photogenerated hole direction g-C on thin film3N4The direction of the film is directionally transferred, so that the capability of directionally transporting a photogenerated hole to a substance to be detected can be obviously improved, and the PEC aptamer sensor provided by the invention has ultrahigh detection sensitivity and has the potential of challenging detection of a low-concentration detector;
(2)g-C3N4the film has good pi-pi adsorption property on the DNA aptamer, and can effectively fix the DNA aptamer, thereby realizing high-specificity capture of a substance to be detected, so the PEC aptamer sensor provided by the invention has high specificity;
(3) through changing the DNA aptamer, the PEC aptamer sensor provided by the invention can be used for detecting tumor markers dopamine and heavy metal ions Hg+And the antibiotic tetracycline and the like have extremely high detection sensitivity and excellent application width, and are universal PEC aptamer sensors.
Drawings
FIG. 1 is BiVO4Scanning electron microscope SEM image of the film surface;
FIG. 2 is BiVO4/g-C3N4Scanning electron microscope SEM image of the film surface;
FIG. 3 is BiVO4/g-C3N4A thin film Transmission Electron Microscope (TEM) image;
FIG. 4 shows BiVO obtained in example 14/g-C3N4the/MC-LR aptamer sensor is used for linearly detecting the microcystin MC-LR to be detected;
FIG. 5 shows BiVO prepared in example 14/g-C3N4A specific analysis diagram of the/MC-LR aptamer sensor for microcystin MC-LR to be detected;
FIG. 6 shows BiVO prepared in example 24/g-C3N4/Hg2+Aptamer sensor for heavy metal ions Hg of object to be detected2+A linear detection map of;
FIG. 7 shows BiVO obtained in example 34/g-C3N4The tetracycline aptamer sensor is used for carrying out linear detection on the antibiotic tetracycline to be detected;
FIG. 8 shows BiVO obtained in example 44/g-C3N4The dopamine aptamer sensor is used for carrying out linear detection on dopamine serving as a tumor marker of a to-be-detected object.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
Firstly preparing BiVO on a conductive substrate by adopting a photoelectrochemistry anodic oxidation method4Thin film, then in BiVO4On the film is decorated with g-C3N4Film, finally using g-C3N4The surface pi-pi adsorption of the film fixes the DNA aptamer for detecting microcystin MC-LR at g-C3N4Preparing a PEC aptamer sensor BiVO (BiVO) on the film for detecting microcystin MC-LR4/g-C3N4The specific preparation process of the/MC-LR aptamer sensor is as follows:
(1) sequentially placing a conductive substrate, namely FTO conductive glass (10mm multiplied by 20mm multiplied by 1.6mm, square resistance is less than or equal to 15 omega) in acetone, ultrapure water and ethanol for ultrasonic cleaning, performing ultrasonic cleaning for 10min each time and performing ultrasonic cleaning for 30min in total, and drying for later use after cleaning;
(2) 1.4552g Bi (NO) were weighed out3)3·5H2O and 5.9956g of NaI is dissolved in 100mL of ultrapure water, the pH value of the solution is adjusted to 4, 1.4592g of p-benzoquinone is weighed and dissolved in 45mL of ethanol, ultrasonic treatment is carried out for 10min to fully dissolve the p-benzoquinone, then the two solutions are mixed and stirred for 30min until the solution is uniform, and finally the solution is stored in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking cleaned FTO conductive glass as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode, wherein specifically, the BiOI film is firstly deposited at-0.3V for 20s and then deposited at-0.1V for 40 s;
(4) weighing 0.159g of vanadyl acetylacetonate, dissolving in 3mL of dimethyl sulfoxide to obtain vanadyl acetylacetonate solution, dripping 40 mu L of vanadyl acetylacetonate solution on the BiOI film prepared in the step (3), drying at 50 ℃, annealing at 450 ℃ for 1h, and then cleaning with 1mol/L NaOH solution to obtain BiVO4A film;
(5) weighing 4.28g of urea, dissolving the urea in 50mL of ultrapure water, adding 3g of melamine into the solution, carrying out hydrothermal treatment at 180 ℃ for 24h, filtering and drying the solution obtained in the hydrothermal treatment, and then carrying out heat preservation at 550 ℃ for 2h to obtain g-C3N4Powder of g-C obtained3N4Dissolving the powder in 100mL of ultrapure water to form a dispersion liquid, sucking 60 mu L of the dispersion liquid, and suspending and coating the BiVO prepared in the step (4)4Keeping the temperature of the film at 450 ℃ for 1h to obtain BiVO4/g-C3N4The film is cleaned by ultrapure water for later use;
(6) BiVO prepared in step (5)4/g-C3N4Dripping 20 mu L of DNA aptamer solution with the concentration of 20nmol/L for detecting microcystin MC-LR on the surface of the film, preserving the heat for 2h at 36 ℃, and then washing the DNA aptamer which is not firmly adsorbed on the surface of the film by using phosphate buffer with the pH of 7.4, wherein the base sequence of the DNA aptamer for detecting microcystin MC-LR is as follows: 5'-GGCGCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CACCTC ATT ATGCCC CAT CTC CGC-3' are provided.
In the BiVO4/g-C3N4During the preparation of the/MC-LR aptamer sensor, BiVO prepared in the step (4) is subjected to4The film is subjected to electron microscope scanningDrawing, aiming at the BiVO prepared in the step (5)4/g-C3N4The film is subjected to electron microscope scanning and electron microscope transmission, wherein BiVO4SEM image of the film is shown in FIG. 1, BiVO4/g-C3N4The SEM image of the film is shown in FIG. 2, and the TEM image of the film is shown in FIG. 3.
As can be seen from FIG. 1, BiVO4The surface of the film is a twisted strip-like structure which contributes to g-C3N4Adsorption of (3).
As can be seen from FIGS. 2 and 3, BiVO4The surface of the film is adsorbed with a large amount of lamellar g-C3N4，g-C3N4Tightly wrapped in BiVO4The surface of the film is in good contact with the DNA aptamer, and the structure is favorable for the next step of DNA aptamer adsorption.
For BiVO4The film is sliced, the film longitudinal section is characterized and measured through SEM, and BiVO is measured4The thickness of the film was 300 nm.
Using TEM for g-C3N4The morphology of the film is characterized, and the g-C3N4 is a lamellar structure, the thickness of the film is 5nm after measurement, and the formed BiVO4/g-C3N4The thickness of the film was 305 nm.
To prepare BiVO4/g-C3N4After the/MC-LR aptamer sensor, we analyzed the detection limit and specificity.
(1)BiVO4/g-C3N4Detection limit of/MC-LR aptamer sensor
Preparing solutions to be detected with different concentrations: firstly, 50 mu g of microcystin MC-LR is added into 1mL of methanol to completely dissolve the microcystin MC-LR to prepare 50mg/L MC-LR mother liquor, and then Tris-HCl buffer solution with pH 7.4 and concentration of 0.1mol/L is used for dilution to obtain concentration of 5 multiplied by 10 respectively-8μg/L、5× 10-7μg/L、1×10-6μg/L、1×10-5μg/L、1×10-4Mu g/L, 0.0025 mu g/L, 0.05 mu g/L, 1 mu g/L and 10 mu g/L of MC-LR standard solution to be tested. Storing the obtained standard solution to be tested at 4-6 deg.CIn the environment.
To prepare BiVO4/g-C3N4the/MC-LR aptamer sensor is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and a phosphate buffer solution with pH of 7.4 and concentration of 0.1mol/L is used as an electrolyte. And (3) sucking 20 mu L of standard solution to be detected with different concentrations, dripping the solution on the surface of the working electrode, and drying the solution in a constant-temperature drying box at 36 ℃ for 30 min. And then placing the dried working electrode in an electrolytic bath, applying a bias voltage of 0.05V under the irradiation of visible light, recording the photocurrent of the sensor after the MC-LR with different concentrations is switched on through an electrochemical workstation, and then drawing a linear detection line of the sensor by taking the logarithm of the concentration of the MC-LR as a horizontal coordinate and the recorded photocurrent as a vertical coordinate.
Because the DNA aptamer can specifically adsorb a substance to be detected, the BiVO designed by the invention4/g-C3N4The photo-generated holes or electrons generated by the/MC-LR aptamer sensor under the irradiation of visible light can react with the microcystin MC-LR to be detected, so that the change of photocurrent is triggered. BiVO (BiVO) after different concentrations of to-be-detected objects are adsorbed is determined by an i-t curve method4/g-C3N4The photocurrent value of the/MC-LR aptamer sensor establishes a working curve-linear detection line by utilizing the linear relation between the magnitude of the photocurrent and the logarithm of the concentration of the object to be detected, thereby realizing the quantitative analysis of the object to be detected.
The BiVO4/g-C3N4The linear detection diagram of the/MC-LR aptamer sensor on the microcystin MC-LR to be detected is shown in figure 4.
As can be seen from FIG. 4, this BiVO4/g-C3N4The linear detection area of the/MC-LR aptamer sensor for the microcystin MC-LR to be detected is 5 multiplied by 10-7Mu g/L to 10 mu g/L, the detection limit is 4.486 multiplied by 10-8μg/L。
Thus, the BiVO4/g-C3N4the/MC-LR aptamer sensor has ultrahigh detection sensitivity to microcystin MC-LR to be detected.
(2)BiVO4/g-C3N4Mc-LR aptamer sensingSpecificity of the device
Preparing 1mol/L microcystin MC-LR solution and heavy metal ion Hg2+Solution, microcystin MC-RR solution, ofloxacin solution, heavy metal ion Pb2+Solution, bovine serum albumin BSA solution.
To prepare BiVO4/g-C3N4the/MC-LR aptamer sensor is used as a working electrode, an Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, and a phosphate buffer solution with pH of 7.4 and concentration of 0.1mol/L is used as an electrolyte. Absorbing 20 mu L of different liquid drops to be detected, coating the liquid drops on the surface of the working electrode, and drying the liquid drops in a constant-temperature drying box at 36 ℃ for 30 min. A bias voltage of 0.05V was applied under irradiation of visible light, and then the photocurrent of the working electrode under irradiation of visible light was measured.
The measurement results of the photocurrent of the working electrode under visible light irradiation are shown in fig. 5.
As can be seen from FIG. 5, this BiVO4/g-C3N4the/MC-LR aptamer sensor produces an electro-optical response to microcystin MC-LR, and the electro-optical response is sensitive.
This indicates that the BiVO4/g-C3N4the/MC-LR aptamer sensor has high specificity to microcystin MC-LR.
The outbreak of the cyanobacteria bloom can not be effectively controlled for a long time, and the main reason is that once the cyanobacteria propagation exceeds the slow growth stage, the propagation speed is exponentially increased, the cyanobacteria bloom outbreak is finally caused, and the primary stage of the cyanobacteria propagation needs to be effectively monitored for effectively treating the cyanobacteria bloom. The microcystin MC-LR is an important marker product for blue-green algae propagation and is also a highly toxic carcinogen, and the detection of the microcystin MC-LR can not only ensure the safety of water, but also effectively monitor the slow growth stage of the blue-green algae, thereby effectively controlling the outbreak of the blue-green algae bloom. However, in the slow growth stage of cyanobacteria, the microcystins MC-LR secreted by cyanobacteria have extremely low concentration, and thus development of a highly sensitive detection method is required. BiVO prepared by the invention4/g-C3N4Mc-LR aptamer sensors for detection thereofThe limit is 4.486X 10-8Mu g/L, compared with a similar sensor which is newly reported at present, the detection limit is improved by two orders of magnitude, and the sensor also shows extremely high specificity, and is an excellent sensor for detecting the concentration of microcystin MC-LR in the slow growth stage of blue algae.
Example 2
Prepared for detecting heavy metal ions Hg by the same method as the example 12+BiVO (polyethylene-Electron cyclotron resonance) aptamer sensor4/g-C3N4/Hg2+The aptamer sensor is prepared by the following specific steps:
(1) sequentially placing FTO conductive glass (10mm multiplied by 20mm multiplied by 1.6mm, square resistance is less than or equal to 15 omega) in acetone, ultrapure water and ethanol for ultrasonic cleaning, performing ultrasonic cleaning for 10min each time for 30min in total, cleaning and drying for later use;
(2) 1.4552g Bi (NO) were weighed out3)3·5H2Dissolving O and 5.9956g NaI in 100mL of ultrapure water, adjusting the pH value of the solution to 4, weighing 1.4592g of p-benzoquinone, dissolving in 45mL of ethanol, performing ultrasonic treatment for 10min to fully dissolve the p-benzoquinone, mixing the two solutions, stirring for 30min until the solution is uniform, and finally storing the solution in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking cleaned FTO conductive glass as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode, wherein specifically, the BiOI film is firstly deposited at-0.2V for 20s and then deposited at-0.1V for 40 s;
(4) weighing 0.159g of vanadyl acetylacetonate, dissolving in 3mL of dimethyl sulfoxide to obtain vanadyl acetylacetonate solution, dripping 40 mu L of vanadyl acetylacetonate solution on the BiOI film prepared in the step (3), drying at 50 ℃, annealing at 450 ℃ for 1h, and then cleaning with 1mol/L NaOH solution to obtain BiVO4A film;
(5) weighing 4.28g of urea, dissolving the urea in 50mL of ultrapure water, adding 3g of melamine into the solution, carrying out hydrothermal treatment at 180 ℃ for 24h, filtering and drying the solution obtained in the hydrothermal treatment, and then carrying out heat preservation at 550 ℃ for 2h to obtain g-C3N4Powder of g-C obtained3N4The powder is dissolved in 100mL of ultrapure water to form a dispersion liquidAbsorbing 60 mu L of the dispersion liquid to be suspended and coated on the BiVO prepared in the step (4)4Keeping the temperature of the film at 500 ℃ for 1h to obtain BiVO4/g-C3N4The film is cleaned by ultrapure water for later use;
(6) BiVO prepared in step (5)4/g-C3N430 mu L of the solution with the concentration of 20nmol/L is dripped on the surface of the film and is used for detecting heavy metal ions Hg2+The DNA aptamer solution is kept at 36 ℃ for 2h, and then the DNA aptamer which is not firmly adsorbed on the surface of the film is washed away by phosphate buffer solution with pH of 7.4, wherein the DNA aptamer solution is used for detecting heavy metal ions Hg2+The base sequence of the DNA aptamer of (1) is: 5'-TTC TTT CTT CCC TTG TTG GTT-3' are provided.
We used the same procedure as in example 1 to prepare BiVO4/g-C3N4/Hg2+The linear detection region and detection limit of the aptamer sensor were measured.
Through detection, the BiVO4/g-C3N4/Hg+Aptamer sensor for heavy metal ions Hg of object to be detected2+See fig. 6.
As can be seen from FIG. 6, this BiVO4/g-C3N4/Hg+Aptamer sensor for heavy metal ions Hg of object to be detected2+The linear detection region of (2) was 0.05fM to 1. mu.M, and the detection limit was 0.0179 fM.
Thus, the BiVO4/g-C3N4/Hg+Aptamer sensor for heavy metal ions Hg of object to be detected2+Has ultrahigh detection sensitivity.
For BiVO4The film is sliced, the film longitudinal section is characterized and measured through SEM, and BiVO is measured4The thickness of the film was 350 nm.
Using TEM for g-C3N4The morphology of (A) is characterized, and g-C can be seen3N4Is of a lamellar structure, the thickness of the BiVO is 10nm after measurement, and the formed BiVO4/g-C3N4The thickness of the film was 360 nm.
Example 3
Using the same method as in example 1, a PEC aptamer sensor-BiVO for the detection of antibiotic tetracycline was prepared4/g-C3N4The tetracycline aptamer sensor is prepared by the following specific steps:
(1) sequentially placing FTO conductive glass (10mm multiplied by 20mm multiplied by 1.6mm, square resistance is less than or equal to 15 omega) in acetone, ultrapure water and ethanol for ultrasonic cleaning, performing ultrasonic cleaning for 10min each time for 30min in total, cleaning and drying for later use;
(2) 1.4552g Bi (NO) were weighed out3)3·5H2Dissolving O and 5.9956g NaI in 100mL of ultrapure water, adjusting the pH value of the solution to 4, weighing 1.4592g of p-benzoquinone, dissolving in 45mL of ethanol, performing ultrasonic treatment for 10min to fully dissolve the p-benzoquinone, mixing the two solutions, stirring for 30min until the solution is uniform, and finally storing the solution in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking cleaned FTO conductive glass as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode, wherein specifically, the BiOI film is firstly deposited at-0.3V for 20s and then deposited at-0.1V for 40 s;
(4) weighing 0.159g of vanadyl acetylacetonate, dissolving in 3mL of dimethyl sulfoxide to obtain vanadyl acetylacetonate solution, dripping 40 mu L of vanadyl acetylacetonate solution on the BiOI film prepared in the step (3), drying at 50 ℃, annealing at 450 ℃ for 1h, and then cleaning with 1mol/L NaOH solution to obtain BiVO4A film;
(5) weighing 4.28g of urea, dissolving the urea in 50mL of ultrapure water, adding 3g of melamine into the solution, carrying out hydrothermal treatment at 180 ℃ for 24h, filtering and drying the solution obtained in the hydrothermal treatment, and then carrying out heat preservation at 550 ℃ for 2h to obtain g-C3N4Powder of g-C obtained3N4Dissolving the powder in 100mL of ultrapure water to form a dispersion liquid, sucking 60 mu L of the dispersion liquid, and suspending and coating the BiVO prepared in the step (4)4Keeping the temperature of the film at 400 ℃ for 1h to obtain BiVO4/g-C3N4The film is cleaned by ultrapure water for later use;
(6) BiVO prepared in step (5)4/g-C3N410 mu L of 10nmol/L concentration is dripped on the surface of the filmThe DNA aptamer solution for detecting the antibiotic tetracycline is subjected to heat preservation at 36 ℃ for 2 hours, and then the DNA aptamer which is not firmly adsorbed on the surface of the membrane is washed away by using a phosphate buffer solution with the pH of 7.4, wherein the base sequence of the DNA aptamer for detecting the antibiotic tetracycline is as follows: 5'-CGT ACG GAA TTC GCT AGC CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC GACTGC GCG TGG ATC CGA GCT CCA CGT G-3' are provided.
We used the same procedure as in example 1 to prepare BiVO4/g-C3N4The linear detection region and detection limit of the/tetracycline aptamer sensor were determined.
Through detection, the BiVO4/g-C3N4The linear detection diagram of the tetracycline aptamer sensor on the antibiotic tetracycline to be detected is shown in FIG. 7.
As can be seen from FIG. 7, this BiVO4/g-C3N4The linear detection area of the tetracycline aptamer sensor for the antibiotic tetracycline to be detected is 0.01 fM-1 mu M, and the detection limit is 2.94 fM.
Thus, the BiVO4/g-C3N4The tetracycline aptamer sensor has ultrahigh detection sensitivity to the antibiotic tetracycline to be detected.
For BiVO4The film is sliced, the film longitudinal section is characterized and measured through SEM, and BiVO is measured4The thickness of the film was 380 nm.
Using TEM for g-C3N4The morphology of (A) is characterized, and g-C can be seen3N4Is of a lamellar structure, the thickness of the BiVO is 10nm after measurement, and the formed BiVO4/g-C3N4The thickness of the film was 390 nm.
Example 4
The PEC aptamer sensor-BiVO for detecting tumor marker dopamine was prepared by the same method as in example 14/g-C3N4The dopamine aptamer sensor is prepared by the following specific steps:
(1) sequentially placing FTO conductive glass (10mm multiplied by 20mm multiplied by 1.6mm, square resistance is less than or equal to 15 omega) in acetone, ultrapure water and ethanol for ultrasonic cleaning, performing ultrasonic cleaning for 10min each time for 30min in total, cleaning and drying for later use;
(2) 1.4552g Bi (NO) were weighed out3)3·5H2Dissolving O and 5.9956g NaI in 100mL of ultrapure water, adjusting the pH value of the solution to 4, weighing 1.4592g of p-benzoquinone, dissolving in 45mL of ethanol, performing ultrasonic treatment for 10min to fully dissolve the p-benzoquinone, mixing the two solutions, stirring for 30min until the solution is uniform, and finally storing the solution in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking cleaned FTO conductive glass as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode, wherein specifically, the BiOI film is firstly deposited at-0.15V for 20s and then deposited at-0.1V for 40 s;
(4) weighing 0.159g of vanadyl acetylacetonate, dissolving in 3mL of dimethyl sulfoxide to obtain vanadyl acetylacetonate solution, dripping 40 mu L of vanadyl acetylacetonate solution on the BiOI film prepared in the step (3), drying at 50 ℃, annealing at 450 ℃ for 1h, and then cleaning with 1mol/L NaOH solution to obtain BiVO4A film;
(5) weighing 4.28g of urea, dissolving the urea in 50mL of ultrapure water, adding 3g of melamine into the solution, carrying out hydrothermal treatment at 180 ℃ for 24h, filtering and drying the solution obtained in the hydrothermal treatment, and then carrying out heat preservation at 550 ℃ for 2h to obtain g-C3N4Powder of g-C obtained3N4Dissolving the powder in 100mL of ultrapure water to form a dispersion liquid, sucking 60 mu L of the dispersion liquid, and suspending and coating the BiVO prepared in the step (4)4Keeping the temperature of the film at 550 ℃ for 1h to obtain BiVO4/g-C3N4The film is cleaned by ultrapure water for later use;
(6) BiVO prepared in step (5)4/g-C3N4Dripping 15 mu L of DNA aptamer solution with the concentration of 5nmol/L for detecting tumor marker dopamine on the surface of the film, preserving the heat for 2h at 36 ℃, then washing the DNA aptamer which is not firmly adsorbed on the surface of the film by using phosphate buffer with the pH of 7.4, wherein the base sequence of the DNA aptamer for detecting the tumor marker dopamine is as follows: 5'-GTC TCT TGC GCC AGA GAA CAC TGG GGC AGA TAT GGG CCA GCA CAGAATGAG GCC C-3' are provided.
We used the same procedure as in example 1 to prepare BiVO4/g-C3N4The linear detection area and detection limit of the/dopamine aptamer sensor were determined.
Through detection, the BiVO4/g-C3N4The linear detection diagram of the dopamine/dopamine aptamer sensor on the tumor marker dopamine of the analyte is shown in figure 8.
As can be seen from FIG. 8, this BiVO4/g-C3N4The linear detection area of the dopamine aptamer sensor for the tumor marker dopamine of the to-be-detected object is 0.1 pM-0.1 mM, and the detection limit is 0.04 pM.
Thus, the BiVO4/g-C3N4The dopamine aptamer sensor has ultrahigh detection sensitivity to the tumor marker dopamine of the object to be detected.
For BiVO4The film is sliced, the film longitudinal section is characterized and measured through SEM, and BiVO is measured4The thickness of the film was 400 nm.
Using TEM for g-C3N4The morphology of (A) is characterized, and g-C can be seen3N4Is of a lamellar structure, the thickness of the BiVO is 15nm after measurement, and the formed BiVO4/g-C3N4The thickness of the film was 415 nm.
In summary, the invention firstly combines the organic semiconductor molecules g-C3N4Is introduced into BiVO4The effective blending of inorganic substance-biological molecule interface is completed between the film and the DNA aptamer interface. By analyzing microcystin MC-LR and heavy metal ions Hg+The detection results of the antibiotic tetracycline and the tumor marker dopamine show that the sensitivity of the PEC aptamer sensor designed by the invention is greatly improved compared with the sensitivity of the same type of sensors reported at present, so that the PEC aptamer sensor designed by the invention has good application prospects in the fields of environmental monitoring and biomedical detection.
In addition, the PEC aptamer sensor designed by the invention can analyze the specificity of the microcystin MC-LR and heavy metal ions Hg to be detected+AntiThe biotin tetracycline, the tumor marker dopamine and the like have ultrahigh specificity, and the method has the advantages of quick response, simple operation and low detected background signal.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.
Sequence listing
<110> university of west' an electronic technology
<120> PEC aptamer sensor with high specificity and ultra-high detection sensitivity and preparation method thereof
<141>2019-09-10
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<170>SIPOSequenceListing 1.0
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<213>Artificial Sequence
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ttctttcttc ccttgttggt t 21
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<213>Artificial Sequence
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cgtacggaat tcgctagccc cccggcaggc cacggcttgg gttggtccga ctgcgcgtgg 60
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<213>Artificial Sequence
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gtctcttgcg ccagagaaca ctggggcaga tatgggccag cacagaatga ggccc 55
Claims (10)
1. A PEC aptamer sensor with high specificity and ultra-high detection sensitivity, comprising: BiVO4Film and DNA aptamer, wherein, BiVO4The film is used as a photo-anode and is characterized by also comprising: g-C3N4A thin film, the PEC aptamer sensor being in g-C3N4The film acts as an interface mediator between the photoanode and the DNA aptamer.
2. The PEC aptamer sensor with high specificity and ultra-high detection sensitivity according to claim 1, wherein the BiVO is4The thickness of the film is 300 nm-400 nm.
3. The PEC aptamer sensor with high specificity and ultra-high detection sensitivity according to claim 1, wherein the g-C is3N4The thickness of the film is 5 nm-15 nm.
4. The PEC aptamer sensor with high specificity and ultrahigh detection sensitivity according to claim 1, wherein the base sequence of the DNA aptamer for detecting microcystin MC-LR is:
5′-GGCGCC AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CACCTC ATT ATG CCC CATCTC CGC-3′。
5. the PEC aptamer sensor with high specificity and ultrahigh detection sensitivity according to claim 1, which is used for detecting heavy metal ions Hg2+The base sequence of the DNA aptamer is as follows:
5′-TTC TTT CTT CCC TTG TTG GTT-3′。
6. the PEC aptamer sensor with high specificity and ultrahigh detection sensitivity according to claim 1, wherein for the detection of the antibiotic tetracycline, the base sequence of the DNA aptamer is:
5′-CGT ACG GAA TTC GCT AGC CCC CCG GCA GGC CAC GGC TTG GGT TGG TCC GACTGC GCG TGG ATC CGA GCT CCA CGT G-3′。
7. the PEC aptamer sensor with high specificity and ultrahigh detection sensitivity according to claim 1, wherein the base sequence of the DNA aptamer for detecting tumor marker dopamine is:
5′-GTC TCT TGC GCC AGA GAA CAC TGG GGC AGA TAT GGG CCA GCA CAG AAT GAGGCC C-3′。
8. method for preparing a PEC aptamer sensor with high specificity and ultra-high detection sensitivity according to any one of claims 1 to 7, characterized in that BiVO is prepared on a conductive substrate by a photo-electrochemical anodic oxidation method4Thin film, then in BiVO4On the film is decorated with g-C3N4Film, finally using g-C3N4The surface pi-pi adsorption of the film fixes the DNA aptamer for detecting the object to be detected on g-C3N4On the film.
9. The method for preparing a PEC aptamer sensor with high specificity and ultra-high detection sensitivity according to claim 8, comprising the following steps:
(1) cleaning the conductive substrate, and drying for later use;
(2) adding Bi (NO)3)3·5H2Dissolving O and NaI in ultrapure water, adjusting the pH value of the solution to 4, fully dissolving p-benzoquinone in ethanol, mixing the two solutions, uniformly stirring, and storing in a constant temperature and humidity box at 22 ℃ for later use;
(3) preparing a BiOI film in the solution prepared in the step (2) by electrodeposition by taking a conductive substrate as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode;
(4) dripping acetylacetonatovanadyl solution on the BiOI film prepared in the step (3), drying, annealing at 450 ℃ for 1h, and then cleaning with NaOH solution to obtain BiVO4A film;
(5) g to C3N4Dissolving the powder in ultrapure water to form a dispersion liquid, and suspending and coating the dispersion liquid on the BiVO prepared in the step (4)4Keeping the temperature of the film at 350-650 ℃ for 1-5 h to obtain BiVO4/g-C3N4A film;
(6) BiVO prepared in step (5)4/g-C3N4Dripping DNA aptamer solution on the surface of the film, keeping the temperature for 2h at 36 ℃, and then cleaning the DNA aptamer which is not firmly adsorbed on the surface of the film.
10. The method for preparing a PEC aptamer sensor with high specificity and ultra-high detection sensitivity according to claim 9, wherein in step (3), the deposition parameters are: depositing at-0.1 to-0.5V for 20s, and then depositing at-0.1V for 40 s.
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