CN109030592B - Photoelectrochemical sensor based on carbon nitride signal amplification and preparation and application thereof - Google Patents

Photoelectrochemical sensor based on carbon nitride signal amplification and preparation and application thereof Download PDF

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CN109030592B
CN109030592B CN201810840501.9A CN201810840501A CN109030592B CN 109030592 B CN109030592 B CN 109030592B CN 201810840501 A CN201810840501 A CN 201810840501A CN 109030592 B CN109030592 B CN 109030592B
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tetracycline
signal amplification
carbon nitride
sensor based
electrode
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CN109030592A (en
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王建
江舟
张悦
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Fuzhou University
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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
    • G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
    • 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
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Abstract

The invention belongs to the field of electrochemical sensors, and particularly relates to a carbon nitride signal amplification-based photoelectrochemical sensor and preparation and application thereof. In the invention, g-C3N4Is a photoactive material, takes an aptamer of a substance to be detected as a biological recognition element and takes Na2SO4The solution is electrolyte, and GaN is used as a working electrode; g to C3N4And the aptamer of the substance to be detected is added into the electrolyte, and forms a photoelectrochemical sensor based on carbon nitride signal amplification together with the three-electrode system. The sensor is simple to operate and high in sensitivity.

Description

Photoelectrochemical sensor based on carbon nitride signal amplification and preparation and application thereof
Technical Field
The invention belongs to the field of electrochemical sensors, and particularly relates to a carbon nitride signal amplification-based photoelectrochemical sensor and a preparation method thereof.
Background
Tetracyclines are a broad spectrum antibiotic discovered in the 40's of the 20 th century. The antibiotics are widely applied to the treatment of infections caused by bacteria, intracellular mycoplasma, chlamydia and the like in human bodies or in farm animals. However, abuse of tetracycline has resulted in its accumulation in humans either by direct administration of drugs or by indirect consumption of animal foods, which poses serious risks such as increased tolerance of microbial species to drugs, allergic or toxic reactions to some susceptible populations, and inhibition of bone growth. Moreover, as early as 1956, tetracycline has been found to potentially affect tooth development and formation, not only yellowing the teeth, but also causing enamel dysplasia or tooth malformation. Therefore, the detection of tetracycline and environmental monitoring have important social significance.
The prior detection methods of tetracycline mainly comprise the following methods:
(1) plasma resonance detection method: the method provides a real-time and target mass-dependent signal by closely monitoring the refractive index (Ri). The method has high cost and complicated pretreatment.
(2) And (3) detection by an electrochemical luminescence method: the method has high measurement precision and wide detection range, but needs to be improved in the aspects of stability and specificity.
(3) And (3) detecting by a liquid chromatography-mass spectrometry method: although the method can realize high-sensitivity and high-selectivity detection, the method is complex to operate, relatively expensive in instrument cost and long in detection time.
(4) Photoelectric chemical sensor method: the photoelectrochemistry analysis has high sensitivity, low background signal, convenient operation, simple equipment and low detection cost, but has the problem of insufficient selectivity.
Disclosure of Invention
The invention aims to provide a preparation method of a tetracycline photoelectric chemical sensor based on carbon nitride signal amplification aiming at the defects of the prior art. The sensor has simple structure, low cost and high selectivity.
In order to realize the purpose of the invention, the following technical scheme is adopted:
a method for preparing photoelectrochemical sensor based on carbon nitride signal amplification uses g-C3N4The material is an optical active material, the aptamer of the substance to be detected is taken as a biological recognition element, and GaN is taken as a working electrode; g to C3N4And the aptamer of the substance to be detected is added into the electrolyte, and forms a photoelectrochemical sensor based on carbon nitride signal amplification together with the three-electrode system.
g-C3N4The concentration in the electrolyte was 0.66 ~ 19.8.8 mg/ml.
The aptamer concentration of the test substance in the electrolyte was 40 nM ~ 120 nM.
The three-electrode system takes a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode.
A photoelectrochemical sensor based on carbon nitride signal amplification prepared by the preparation method.
Use of a photoelectrochemical sensor based on carbon nitride signal amplification as described above: for tetracycline detection in solution, the solution referred to herein may be a solution formulated into a food, a pharmaceutical, or the like.
When the sensor is used for detecting tetracycline in a solution, more specifically: adding a system to be detected into the electrolyte of the photoelectrochemical sensor based on carbon nitride signal amplification, irradiating the electrolyte by using an ultraviolet lamp (the wavelength is generally 350-400 nm), enhancing the photocurrent of the photoelectrochemical sensor based on the carbon nitride signal amplification in a system with tetracycline, and determining the concentration of the tetracycline in the system to be detected by the photocurrent detected by the photoelectrochemical sensor based on the carbon nitride signal amplification by drawing a standard curve of the concentration and the photocurrent of the known tetracycline.
For the detection of tetracycline in solution, the sequence of the aptamer used was 5' -CGTAC GGAAT TCGCT AGCCC CCCGG CAGGC CACGG CTTGG GTTGG TCCCA CTGCG CGTGG ATCCG AGCTC CACGT.
When the tetracycline fluorescent probe is used for detecting tetracycline in a solution, the detection range is 0.1-10 nmol/L, and the detection limit of the tetracycline is 0.03 nM.
When the GaN-based detection electrode is used for detecting tetracycline in a solution, the bias voltage of the GaN working electrode is + 0.2-0.6V.
Furthermore, when the sensor of the invention is used for detecting tetracycline, the preparation method comprises the following steps: in g-C3N4Is a photoactive material, a tetracycline aptamer is a biological recognition element, and Na is used2SO4The solution is taken as electrolyte, GaN is taken as a working electrode, a platinum electrode is taken as a counter electrode, and an Ag/AgCl electrode is taken as a reference electrode, so that the tetracycline photoelectrochemical sensor is assembled.
Furthermore, the preparation method specifically comprises the following steps:
the tetracycline DNA aptamer is prepared by PBS buffer solution and then is placed in Na2SO4Adding tetracycline DNA aptamer andg-C3N4obtaining mixed liquid; measuring photocurrent under 365 nm irradiation in a three-electrode system by taking GaN as a working electrode, a platinum electrode as a counter electrode, an Ag/AgCl electrode as a reference electrode and mixed liquid as electrolyte, wherein the photocurrent is background photocurrent;
mixing the mixed solution obtained in the step one with a series of tetracycline standard solutions with different volumes and concentrations, and incubating at room temperature for 5-20min to obtain a series of solutions to be tested;
measuring photocurrent under 365 nm irradiation in a three-electrode system by taking GaN as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode and respectively taking a series of liquids to be tested prepared in the step two as electrolyte; taking the difference value delta I between the photocurrent measured in the step (III) and the background photocurrent of the step (I) as a y value, and the concentration of the tetracycline standard solution as an x value to obtain a linear regression equation;
adding the tetracycline sample to be detected into the mixed solution obtained in the step (I), incubating at room temperature for 5-20min, measuring the photocurrent again, and substituting the difference value delta I between the photocurrent and the background current obtained in the step (I) into the linear regression equation to obtain the tetracycline content in the sample to be detected.
Preferably, in the test solution, the concentration of the tetracycline DNA aptamer is 60 nM; g-C3N4The concentration of (A) is 13.2 mg/ml; the concentration range of the tetracycline standard solution is 0.1-10 nmol/L, and the volume is 5 mu L; in the three-electrode system, the bias voltage on the GaN electrode was 0.4V.
The action mechanism of the sensor for detecting tetracycline is as follows:
the invention adopts g-C with good dispersibility in water3N4Is an optically active material, combines tetracycline DNA aptamer as a biological recognition element, and provides a preparation method of a novel photoelectrochemical aptamer sensor for tetracycline specificity detection. Due to g-C3N4The energy band structures of the GaN and the GaN have good matching performance, and photogenerated holes and electrons are in GaN and g-C3N4Can be easily transferred. Under irradiation of light, electrons are driven from g-C3N4The valence band of (a) is excited to its conduction band,and then transferred to a GaN tape. In addition, in g-C3N4Can generate holes and participate in the oxidation of TET and significantly inhibit the recombination of photogenerated electron-hole carriers. Therefore, the photocurrent is significantly enhanced.
Similarly, when other substances are detected, the aptamer capable of specifically binding the substance is selected.
Compared with the prior art, the invention has the following advantages:
1) the invention is based on g-C3N4The energy band structures of the GaN and the GaN have good matching performance, and photogenerated holes and electrons are in GaN and g-C3N4Can be easily transferred; under irradiation of light, electrons are driven from g-C3N4The valence band of (a) is excited to its conduction band and then transferred to the conduction band of GaN; in addition, in g-C3N4The valence band can generate holes to oxidize the substance to be detected, thereby obviously inhibiting the recombination of photon-generated electron hole carriers; therefore, when the substance to be detected exists in the system to be detected, the photocurrent is obviously enhanced, and the method can be used for measuring the content of the substance to be detected in the system; the sensor has simple structure, low cost and high selectivity;
2) when the sensor is used for detecting tetracycline, the high-selectivity tetracycline is realized, and aureomycin, neomycin sulfate, chloramphenicol, kanamycin sulfate, doxycycline and the like basically have no interference on detection; the response of the sensor of the invention to tetracycline is linear in the range of 0.1-10 nmol/L, and the detection limit (3S/N) is 0.03 nM; the sensor solves the problems of expensive tetracycline detection method or device, complex process or insufficient selectivity and the like in the prior art, provides the tetracycline sensor with simple structure, low cost and high selectivity, and can be applied to medical detection and environmental monitoring of tetracycline.
Drawings
FIG. 1 is a schematic diagram of the operation of the sensor of the present invention; TET-tetracycline; Aptamer-Aptamer;
FIG. 2 is a photocurrent response diagram; (a) a photocurrent response diagram of the GaN electrode; (b) adding 13.2 mg/ml g-C3N4OfA flow response graph; (c) continuously adding 5 nmol/L TET photocurrent response graph; medium: 0.01M Na2SO4(ii) a Bias potential (vs SCE): + 0.4V; wavelength of excitation light: 365 nm; an aptamer: 40 nM;
FIG. 3 shows g-C at different concentrations3N4The effect on the photocurrent response of the TET in a three electrode system; (a) 0.66, (b) 6.6, (c) 13.2, (d) 16.5, (e) 19.8 mg/ml;
FIG. 4 is a graph of the effect of different concentrations of aptamer on the photocurrent response on TET: (a) 40 nM (b) 60 nM (c) 80 nM (d) 100 nM (e) 120 nM; at 0.01M Na2SO 4And 13.2 mg/ml g-C3N4PEC measurements were recorded at a bias potential of + 0.4V, with a differential bar = SD (n = 5);
FIG. 5 is a graph of the effect of bias voltage on photocurrent response for a 40 nM aptamer on a GaN electrode: (a) 0.2, (b) 0.3, (c) 0.4, (d) 0.5, (e) 0.6V; at 0.01M Na2SO4And 13.2 mg/ml g-C3N4PEC measurements were recorded at a bias potential of + 0.4V, error bar = SD (n = 5);
FIG. 6 shows a sensor of the present invention using 60 nM aptamer and 13.2 mg/ml g-C3N4PEC response to various antibiotics; at 0.01M Na2SO4And 13.2 mg/ml g-C3N4PEC measurements were recorded at a bias potential of + 0.4V (n = 5);
FIG. 7 is 13.2 mg/ml g-C towards TET using 60 nM aptamer and on different concentrations of GaN electrode3N4A calibration curve of photocurrent response of; PEC measurements were recorded at 0.01M Na2SO 4Upper, bias potential + 0.4V, error bar = SD (n = 3).
Detailed Description
For further disclosure, but not limitation, the present invention is described in further detail below with reference to examples.
The present invention will be described in detail with reference to the detection of tetracycline as an example. The other substances to be tested can be detected by referring to the detection of tetracycline.
With Na2SO4The solution (0.01M) was used as an electrolyte, and a suitable amount of tetracycline (TET) aptamer (DNA aptamer was prepared from the following sequence: 5' -CGTAC GGAAT TCGCT AGCCC CCCGG CAGGC CACGG CTTGG GTTGG TCCCA CTGCGCGTGG ATCCG AGCTC CACGT) and g-C3N4The photocurrent signal under 365 nm irradiation was measured with a three-electrode system as shown in fig. 1, in which GaN was the working electrode, a platinum electrode was the counter electrode, and an Ag/AgCl electrode was the reference electrode. Each photocurrent signal test was averaged in triplicate. As shown in fig. 2, the photocurrent increases sharply and decreases rapidly with the illumination on and off, respectively, indicating that the photocathode has a rapid photo-response. And after measuring the background photocurrent under 365 nm irradiation, adding the tetracycline sample to be measured into the solution, incubating at room temperature for 5-20min, and measuring the photocurrent again, wherein the difference between the photocurrent and the background current is the response of the corresponding TCT concentration.
Increase in g-C3N4Concentration, photocurrent increased significantly (fig. 3). Based on photoinduced charge carrier separation, allowing electrons to pass from g-C3N4As electrons to the GaN electrode. When g-C3N4When the concentration of (B) exceeds 13.2 mg/ml, the photocurrent decreases. This result may be due to an excess of g-C3N4The transfer of photogenerated electrons was prevented and thus 13.2m g/ml was used to fabricate a PEC sensor.
The DNA molecule has a negatively charged phosphate group on the sugar chain (phosphate backbone) and has a negative charge per base in an aqueous solution. g-C3N4The nanosheet is positively charged, and provides an excellent platform for immobilizing tetracycline aptamers.
Figure 4 shows the effect of aptamer concentration on photocurrent response. A significant increase in PEC response to TET was observed as the aptamer concentration increased from 40 nM to 120 nM. This result is associated with the immobilization in g-C3N4The fact that higher concentrations of aptamers above can capture more TET molecules is consistent. However, when aptamer concentrations exceeded 60 nM, PEC response decreased. Steric hindrance caused by excessive aptamer may cause blocking of electron transfer. Thus 60 nM aptamer was selectedFor the manufacture of sensors.
The photo-generated electrons will be driven to the counter electrode by the bias potential of the external circuit, thus contributing to the generation of a high photocurrent. The bias potential also showed a significant effect on the PEC response of the sensor. As shown in fig. 5, the photocurrent response of TET increases as the bias potential increases from 0.2V to 0.6V, indicating that a larger anode bias potential can drive more photogenerated electrons onto the counter electrode to more effectively suppress hole-electron pair recombination. However, when the applied potential is greater than 0.4V, the response to TET does not show further enhancement, possibly due to saturation of the photo-generated holes consumed by TET. Thus, 0.4V is the optimal potential for PEC sensing.
To investigate the selectivity of this PEC aptamer sensor, we recorded the aptamer/g-C after incubation of 5 nM of various antibiotics (including tetracycline, aureomycin, neomycin sulfate, chloramphenicol, kanamycin sulfate) in 0.01M PBS3N4At 0.01M Na2SO4Response to GaN electrodes in solution. Unlike TET, all these antibiotics were directed to aptamers/g-C on GaN electrodes3N4None showed a clear response (fig. 6), indicating that the proposed sensor has a high selectivity due to the specific recognition between the aptamer and the target TET molecule. In addition, the reproducibility of the PEC aptamer sensors was also evaluated.
The photocurrent intensity gradually increased with increasing tetracycline concentration, and the photocurrent intensity versus target concentration curve had a good linear relationship for the target in the range of 0.1 to 10 nM. Linear regression equation expressed as Δ I/a =1.53e-4 + 2.45 e-4lg [C/（nmol/L）]The correlation coefficient is 0.997, and the range is 0.1-10 nmol/L. The limit of detection (LOD) of tetracycline calculated from 3 δ was 0.03 nM. This is comparable or more sensitive to most methods reported previously for tetracycline analysis.
These results show that the results are based on g-C3N4The photocurrent nanoprobes of (a) can be used to quantify analytes in homogeneous solutions with high sensitivity. Relative Standard Deviation (RSD) of tetracycline detection response of 5 nmol/L (n = 5)) The content was found to be 5.3%.
Experimental results show that the PEC sensing platform adopts 13.2 mg/ml g-C3N4, the aptamer concentration is 60 nM, the bias voltage is 0.4V, and the optimal photocurrent response is shown under the reaction condition of PBS buffer (0.01M) with the pH value of 7.4. Linear regression equation expressed as Δ I/a =1.53e-4+ 2.45e-4lg [C /(nmol/L)]The correlation coefficient is 0.997, and the range is 0.1-10 nmol/L. The limit of detection (LOD) of tetracycline calculated from 3 δ was 0.03 nM. The response signals of the sensing platform to aureomycin, neomycin sulfate, chloramphenicol, kanamycin sulfate and doxycycline are far smaller than those to tetracycline.
Example 1
A preparation method of a tetracycline photoelectrochemical sensor based on carbon nitride signal amplification comprises the following steps:
(1) establishment of a standard curve:
[ solution ] Tetracycline DNA aptamers were prepared in PBS buffer with pH =7.4, and then incubated in 0.01M Na2SO4Adding tetracycline DNA aptamer and g-C into the solution3N4Obtaining mixed liquid; measuring photocurrent under 365 nm irradiation in a three-electrode system by taking GaN as a working electrode, a platinum electrode as a counter electrode, an Ag/AgCl electrode as a reference electrode and mixed liquid as electrolyte, wherein the photocurrent is background photocurrent;
mixing the mixed solution obtained in the step one with a series of tetracycline standard solutions with different volumes and concentrations, and incubating at room temperature for 5-20min to obtain a series of solutions to be tested;
measuring photocurrent under 365 nm irradiation in a three-electrode system by taking GaN as a working electrode, a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode and respectively taking a series of liquids to be tested prepared in the step two as electrolyte; and (3) taking the difference value delta I between the photocurrent measured in the step (III) and the background photocurrent of the step (I) as a y value, and the concentration of the tetracycline standard solution as an x value to obtain a linear regression equation as follows: y (a) =1.53e-4+2.45e-4lg[x/(nmol/L)]The correlation coefficient is 0.997;
(2) detection of the actual sample:
and (3) adding the milk sample to be detected into the mixed solution in the step (I), incubating at room temperature for 5-20min, measuring the photocurrent again, and substituting the difference value delta I between the photocurrent and the background current in the step (I) into the linear regression equation in the step (iii) to obtain the tetracycline content in the sample to be detected.
In the to-be-tested solution, the concentration of the tetracycline DNA aptamer is 60 nM; g-C3N4The concentration of (A) is 13.2 mg/ml; the concentration range of the tetracycline standard solution is 0.1-10 nmol/L, and the volume is 5 muL.
Table 1: experimental result for determining recovery rate of tetracycline in milk sample
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
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cgtacggaat tcgctagccc cccggcaggc cacggcttgg gttggtccca ctgcgcgtgg 60
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Claims (7)

1. A preparation method of a photoelectrochemical sensor based on carbon nitride signal amplification is characterized by comprising the following steps: in g-C3N4Is a photoactive material, takes an aptamer of a substance to be detected as a biological recognition element, and takes GaN asA working electrode; g to C3N4Adding the aptamer of the substance to be detected into the electrolyte, and forming a photoelectrochemical sensor based on carbon nitride signal amplification together with the three-electrode system;
g-C3N4the concentration in the electrolyte is 0.66 ~ 19.8.8 mg/ml;
the concentration of the aptamer of the substance to be detected in the electrolyte is 40 nM ~ 120 nM;
the three-electrode system takes a platinum electrode as a counter electrode and an Ag/AgCl electrode as a reference electrode.
2. A photoelectrochemical sensor based on carbon nitride signal amplification manufactured according to the method of claim 1.
3. Use of a photoelectrochemical sensor based on carbon nitride signal amplification according to claim 2, characterized in that: for tetracycline detection in solution.
4. Use of the photoelectrochemical sensor based on carbon nitride signal amplification according to claim 3, characterized in that: when the sensor is used for detecting tetracycline in a solution, a system to be detected is added into electrolyte of the photoelectrochemical sensor based on carbon nitride signal amplification, an ultraviolet lamp is used for irradiating the electrolyte, in a system with tetracycline, the photoelectrochemical sensor based on carbon nitride signal amplification can generate photocurrent enhancement, and the concentration of the tetracycline in the system to be detected can be determined by drawing a standard curve of the concentration and the photocurrent of the known tetracycline, and the photocurrent detected by the photoelectrochemical sensor based on carbon nitride signal amplification.
5. Use of the photoelectrochemical sensor based on carbon nitride signal amplification according to claim 3, characterized in that: for the detection of tetracycline in solution, the sequence of the aptamer used was 5' -CGTAC GGAAT TCGCT AGCCC CCCGGCAGGC CACGG CTTGG GTTGG TCCCA CTGCG CGTGG ATCCG AGCTC CACGT.
6. Use of the photoelectrochemical sensor based on carbon nitride signal amplification according to claim 3, characterized in that: when the tetracycline fluorescent probe is used for detecting tetracycline in a solution, the detection range is 0.1-10 nmol/L, and the detection limit of the tetracycline is 0.03 nM.
7. Use of the photoelectrochemical sensor based on carbon nitride signal amplification according to claim 3, characterized in that: when the GaN-based detection electrode is used for detecting tetracycline in a solution, the bias voltage of the GaN working electrode is + 0.2-0.6V.
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