CN114113265B - Aptamer sensor and preparation method thereof - Google Patents
Aptamer sensor and preparation method thereof Download PDFInfo
- Publication number
- CN114113265B CN114113265B CN202111531807.4A CN202111531807A CN114113265B CN 114113265 B CN114113265 B CN 114113265B CN 202111531807 A CN202111531807 A CN 202111531807A CN 114113265 B CN114113265 B CN 114113265B
- Authority
- CN
- China
- Prior art keywords
- des
- aptamer
- tfpy
- tapp
- cof
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
-
- 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
- G01N27/416—Systems
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensing. The aptamer sensor comprises an electrode matrix and a covalent organic framework material modified on the surface of the electrode matrix, wherein a nucleic acid aptamer for targeted detection of diethylstilbestrol is adsorbed on the covalent organic framework material; the covalent organic framework material is prepared from 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene by adopting a solvothermal method through Schiff base reaction. The aptamer sensor has higher sensitivity, good selectivity, reproducibility, stability, reproducibility and acceptable applicability in complex environments when being used for detecting DES.
Description
Technical Field
The invention relates to an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensing.
Background
Diethylstilbestrol (DES) is a synthetic estrogen used as a growth promoter in meat animals and poultry. As a clinical medical application, DES has the same pharmacological and therapeutic effects as natural estrogens. DES is more stable and stays in the body longer than natural estrogens. Since DES is dark and difficult to degrade, it severely impairs human health. Thus, the use of DES by food animals is prohibited in the united states, japan, the european union and china. Nevertheless, abuse of DES is still widespread and found in many countries in the environment and aquatic organisms. Therefore, the development of a highly sensitive and reliable method for determining DES in polluted foods has important significance for guaranteeing human health. Currently, there are various methods to detect DES to control its abuse, such as high performance liquid chromatography, enzyme-linked immunosorbent assay, fluorescence, gas chromatography-mass spectrometry (GC-MS), direct electrochemistry, molecular imprinting techniques, a combination of colorimetric and fluorescent chemiluminescence, surface Enhanced Raman Spectroscopy (SERS), quartz crystal microbalances, paper sensors, immunochromatography, electrochemical immunosensors, electrochemiluminescence (ECL), photoelectrochemical techniques, and the like. However, these methods require special equipment and a large number of separate analysis procedures, resulting in a more complex, time-consuming and laborious screening procedure. Among different technologies, the electrochemical method has the advantages of quick response, low cost, low detection limit, easy miniaturization, small sample use amount and the like. However, some direct electrochemical methods tend to have poor selectivity due to the high electronic stability and structural similarity of DES. To improve electrochemical sensors for detecting DES, electrochemical immunosensors can be constructed by adsorbing DES antibodies that can specifically bind to DES by forming antibody-antigen complexes. Alternatively, DNA aptamers that specifically bind to DES may be used to analyze DES. Compared with immunosensor, aptamer sensor constructed by using DNA aptamer has the advantages of high sensitivity, good stability, low cost and the like.
Heretofore, various nanomaterials, such as conductive polymers, semiconductor nanomaterials, quantum dots, graphene, carbon nanotubes, porous silicon nanospheres, and porous organic framework materials, have been used as good platforms for constructing biosensors. Among them, porous scaffold materials, such as Metal Organic Frameworks (MOFs) and Covalent Organic Frameworks (COFs), are of great interest because of their high specific surface area, customizable structure and adjustable pore size.
Most MOFs basedDES sensors all rely on the electrocatalytic properties of the electrode-supported MOFs material to DES, however, most electrode materials typically exhibit effective electrocatalytic properties to other small molecules, such as H 2 O 2 、H 2 S, uric acid or dopamine, thereby reducing the selectivity of DES detection, adding the step of optimizing electrode materials, prolonging the detection period, increasing the detection time and the detection cost, and having lower sensitivity when detecting DES.
Disclosure of Invention
The invention aims to provide an aptamer sensor which is used for solving the problem of low sensitivity of the existing sensor for detecting DES.
Another object of the invention is to provide a method for preparing an aptamer sensor.
In order to achieve the above purpose, the aptamer sensor of the invention adopts the following technical scheme:
an aptamer sensor comprises an electrode matrix and a covalent organic framework material modified on the surface of the electrode matrix, wherein a nucleic acid aptamer for targeted detection of diethylstilbestrol is adsorbed on the covalent organic framework material; the covalent organic framework material is prepared from 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene by adopting a solvothermal method through Schiff base reaction.
The invention uses covalent organic framework material as an electrode material for constructing an aptamer sensor for targeted detection of diethylstilbestrol, and the electrode material is prepared from 5,10,15, 20-tetra (4-aminophenyl) porphyrin (TAPP) and 1,3,6, 8-tetra (4-formylphenyl) pyrene (TFPy) by adopting a solvothermal method through Schiff base reaction, and has a porous nano structure, a conjugated structure and rich amino functional groups. Thus, a large number of aptamer chains for targeted detection of diethylstilbestrol can be anchored to the surface and inside of covalent organic framework materials through complex interactions. The aptamer sensor has higher sensitivity, good selectivity, reproducibility, stability, reproducibility and acceptable applicability in complex environments when being used for detecting DES.
Preferably, the molar ratio of 5,10,15, 20-tetra (4-aminophenyl) porphyrin to 1,3,6, 8-tetra (4-formylphenyl) pyrene is (0.8-1.2): 0.8-1.2. Further, the molar ratio of 5,10,15, 20-tetra (4-aminophenyl) porphyrin to 1,3,6, 8-tetra (4-formylphenyl) pyrene is 1:1.
Preferably, the catalyst used in the schiff base reaction is acetic acid.
Preferably, the solvothermal method comprises the steps of: firstly, 5,10,15, 20-tetra (4-aminophenyl) porphyrin, 1,3,6, 8-tetra (4-formylphenyl) pyrene, a catalyst and a solvent are filled into a Schlenk tube, then, the air in the Schlenk tube is replaced by inert gas, the Schlenk tube is sealed, and then, the Schlenk tube is heated and insulated.
Preferably, the inert gas is N 2 。
Preferably, the solvothermal method has a soak temperature of 120-125 ℃. Further, the solvothermal method has a soak temperature of 120 ℃. Preferably, the solvothermal method has a holding time of 144-168 hours. Further, the heat preservation time of the solvothermal method is 168h.
Preferably, the solvent used in the schiff base reaction consists of N, N-dimethylacetamide and o-dichlorobenzene. Preferably, the volume ratio of the N, N-dimethylacetamide to the o-dichlorobenzene is (2.8-3.2): 1. Further, the volume ratio of the N, N-dimethylacetamide to the o-dichlorobenzene is 3:1.
Preferably, after the Schiff base reaction is finished, the solid-liquid separation is carried out on the system with the Schiff base reaction finished, the solid obtained by the solid-liquid separation is washed, and finally the washed solid is dried.
Preferably, the solid-liquid separation is achieved by filtration.
Preferably, the washing is to wash the solid obtained by solid-liquid separation with tetrahydrofuran for 3 times and then acetone for 3 times.
Preferably, the drying is drying the washed solid in vacuo at 100 ℃ for 18h.
The preparation method of the aptamer sensor adopts the following technical scheme:
the preparation method of the aptamer sensor comprises the following steps: firstly, loading a covalent organic framework material onto an electrode matrix to obtain a modified electrode; the modified electrode is then incubated in diethylstilbestrol aptamer solution.
The preparation method of the aptamer sensor is simple and efficient, and has good reproducibility and stability.
Preferably, the loading is by coating a suspension of the covalent organic framework material onto the electrode substrate, followed by a drying process.
Preferably, the concentration of the suspension of covalent organic framework material is 0.8-1.5mg/mL. Further preferred, the concentration of the suspension of covalent organic framework material is 1mg/mL.
Preferably, the suspension of covalent organic framework material consists essentially of water and covalent organic framework material. Water is used as a dispersing agent, so that the covalent organic framework material can be better dispersed.
Preferably, the covalent organic framework material is coated on the electrode matrix in an amount of 21-39.4 mug/cm 2 。
Preferably, the electrode is a gold electrode.
The incubation is to contact the gold electrode fixed with the covalent organic framework material with the aptamer solution, so that the covalent organic framework material adsorbs and fixes the aptamer and reaches an equilibrium state. Preferably, the incubation process of the modified electrode in the nucleic acid aptamer solution is as follows: immersing the modified electrode into a nucleic acid aptamer solution to obtain the aptamer sensor.
Preferably, the temperature of the incubation is 0-4 ℃; the incubation time is 60-80min. Further, the temperature of the incubation is 4 ℃; the incubation time was 1h.
Preferably, the concentration of the nucleic acid aptamer solution for targeted detection of diethylstilbestrol is 100-200nmol/L. Further preferably, the concentration of the nucleic acid aptamer solution for targeted detection of diethylstilbestrol is 100nmol/L.
Preferably, the aptamer solution for targeted detection of diethylstilbestrol consists essentially of water, phosphate and a solution for targeted detection of diethylstilbestrolDetecting the nucleic acid aptamer composition of diethylstilbestrol. Preferably, the nucleic acid aptamer solution for targeted detection of diethylstilbestrol further comprises an alkali metal chloride. Preferably, the phosphate is KH 2 PO 4 And/or Na 2 HPO 4 ·12H 2 O. Preferably, the alkali chloride is KCl and/or NaCl.
Drawings
Fig. 1: (a) is the Fourier transform infrared spectrum (FTIR) spectrum of different materials (TAPP, TFPy and TAPP-TFPy-COF), (b) is the XRD spectrum of TAPP-TFPy-COF, and (c) is the TAPP-TFPy-COF 13 cCP-MAS NMR spectra;
fig. 2: (a) is a high-resolution XPS spectrum of C1s of TAPP-TFPy-COF, (b) is a high-resolution XPS spectrum of N1s of TAPP-TFPy-COF, (C) is a high-resolution XPS spectrum of C1s of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after the immobilization of DES aptamer, and (d) is a high-resolution XPS spectrum of N1s of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after the immobilization of DES aptamer; (e) High resolution XPS spectrum of P2P after DES aptamer immobilization of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE);
fig. 3: (a) is a low power Scanning Electron Microscope (SEM) image of TAPP-TFPy-COF, (b) is a high power Scanning Electron Microscope (SEM) image of TAPP-TFPy-COF, (c) is a low power Transmission Electron Microscope (TEM) image of TAPP-TFPy-COF, and (d) is a high power Transmission Electron Microscope (TEM) image of TAPP-TFPy-COF;
fig. 4: (a) An EIS Nyquist curve diagram obtained in the process of constructing and detecting DES for the aptamer sensor when the blocking effect of BSA is tested, (b) a C-V curve diagram obtained in the process of constructing and detecting DES for the aptamer sensor when the blocking effect of BSA is tested;
fig. 5: (a) An EIS Nyquist graph obtained by preparing an aptamer sensor and detecting DES in example 2, and (b) a C-V graph obtained by preparing an aptamer sensor and detecting DES in example 2;
fig. 6: (a) ΔR from aptamer sensor preparation Using TAPP-TFPy-COF suspensions at different concentrations ct Value (ΔR before and after bare gold electrode AE immobilization TAPP-TFPy-COF) ct Value and TAPP-TFPy-COF fixed DES adaptationΔR in front of and behind the body ct Values), and (b) is a schematic diagram of the delta R before and after the TAPP-TFPy-COF is fixed for the bare gold electrode AE ct Schematic of the relationship between the values and the concentration of TAPP-TFPy-COF suspension, (c) preparation of aptamer sensor based on DES aptamer solutions of different concentrations and detection of DeltaR obtained by DES ct Value (TAPP-TFPy-COF DeltaR before and after DES aptamer immobilization) ct Value and aptamer sensor to detect ΔR before and after DES ct Values), and (d) is the ΔR before and after TAPP-TFPy-COF immobilization of DES aptamer ct A relation diagram of the value and DES aptamer solution concentration, (e) an EIS Nyquist curve diagram obtained by detecting the aptamer sensor and the DES under different binding time, and (f) a DeltaR before and after detecting the aptamer sensor and the DES under different binding time ct A value diagram;
fig. 7: (a) Schematic of EIS nyquist curves obtained for the aptamer sensor prepared in example 2 to detect DES solutions of different concentrations, (b) aptamer sensor prepared in example 2 to detect Δr before and after DES solutions of different concentrations ct Schematic of the values (inset: ΔR) ct A linear fit graph as a logarithmic function of DES concentration), (c) a schematic of the selective test results of the aptamer sensor, (d) a schematic of the reproducible test results of the aptamer sensor, (e) a schematic of the stability test results of the aptamer sensor, and (f) a schematic of the reproducible test results of the aptamer sensor.
Detailed Description
The technical scheme of the invention is further described below with reference to specific embodiments.
Materials used in the examples of the present invention: the DES nucleic acid aptamer is provided by Shanghai Biotechnology engineering Co., ltd, and has a sequence of 5'-ggC gAT ggg gTA ggg ggT gTg gAg ggg CCg gAC ggA ggg g-3';5,10,15, 20-tetra (4-aminophenyl) porphyrin (TAPP) and 1,3,6, 8-tetra (4-formylphenyl) pyrene (TFPy) are supplied by regular script Tree chemical technology Co., ltd (Shanghai, china); n, N-dimethylacetamide and o-dichlorobenzene were supplied by Shanghai A Ding Shenghua technologies Co., ltd. (Shanghai, china); acetone and Tetrahydrofuran (THF) are supplied by henna, chemicals limited, henna, china; glacial acetic acid (acetic acid) is provided by Tianjin Fuyu fine chemical industry Co., ltd (Tianjin, china); all chemical reagents were analytical reagent grade and were used without further purification.
The water used in the examples and experimental examples of the present invention was deionized water (resistivity at 25 ℃ C. Was 18.2. Omega. Cm).
The preparation methods of the phosphate buffer used in the examples and experimental examples of the present invention are as follows: will be 0.242g KH 2 PO 4 、1.445g Na 2 HPO 4 ·12H 2 O, 0.2g KCl and 8.003g NaCl were dissolved in deionized water to give phosphate buffer (PBS, 0.1mol/L, pH=7.4); phosphate buffer (PBS, 0.1mol/L, ph=7.4) was diluted 100-fold with deionized water to give 10mmol/L of PBS solution.
The preparation method of the DES aptamer solution used in the embodiment and experimental example of the invention is as follows: phosphate buffer solution (PBS, 0.1mol/L, ph=7.4) was added to DES aptamer stock solution to prepare DES aptamer concentration solutions of 1, 5,10, 50, 100 and 200nmol/L.
The preparation method of the DES solution used in the experimental example of the invention is as follows: phosphate buffer solution (PBS, 0.1mol/L, ph=7.4) was added to DES powder, and after ultrasonic shaking, DES solutions at a concentration of 1mg/mL were obtained, followed by dilution with phosphate buffer solution (PBS, 0.1mol/L, ph=7.4) to obtain DES solutions at concentrations of 0.001, 0.01, 0.1, 1, 10, 100 and 1000 pg/mL.
The preparation methods of the interferent solution and the mixture solution of DES and interferent used in the experimental example of the present invention are as follows: respectively adding different organic pollutants (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and terramycin) into phosphate buffer solution (PBS, 0.1mol/L, pH=7.4) to prepare enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and terramycin test solutions with the concentration of 1000 pg/mL; crCl is added to 3 、CuCl 2 And AgNO 3 Respectively adding into phosphate buffer solution (PBS, 0.1mol/L, pH=7.4) to prepare heavy metal ions (Cr) with the concentration of 1000pg/mL 3+ 、Cu 2+ Or Ag + ) A test solution; DES, organic contaminants (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and oxytetracycline) and CrCl 3 、CuCl 2 And AgNO 3 Adding phosphate buffer solution (PBS, 0.1mol/L, pH=7.4) to prepare DES concentration of 10pg/mL, organic pollutant (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and oxytetracycline) and heavy metal ion (Cr) 3+ 、Cu 2+ Or Ag + ) The concentrations of (C) were 1000pg/mL of the mixture test solution.
The electrolyte solutions used in the examples and experimental examples of the present invention were prepared by the method of preparing 1.65. 1.65g K 3 Fe(CN) 6 、2.11g K 4 Fe(CN) 6 ·3H 2 O and 7.5g KCl were dissolved in 1.0L of phosphate buffer (PBS, 0.1mol/L, pH=7.4).
1. Specific examples of aptamer sensors of the invention are as follows:
example 1
The aptamer sensor of the embodiment comprises a gold electrode matrix and a covalent organic framework material modified on the surface of the gold electrode matrix, wherein a nucleic acid aptamer for targeted detection of diethylstilbestrol is adsorbed on the covalent organic framework material.
The preparation method of the covalent organic framework material comprises the following steps:
67mg (0.1 mmol) of 5,10,15, 20-tetrakis (4-aminophenyl) porphyrin (TAPP) and 61mg (0.1 mmol) of 1,3,6, 8-tetrakis (4-formylphenyl) pyrene (TFPy) were charged into a 15mL Schlenk tube, 4mL of a mixed solvent (the mixed solvent consisting of N, N-dimethylacetamide and o-dichlorobenzene in a volume ratio of 3:1) was further added to the Schlenk tube, and the mixture was sonicated for 30 minutes to obtain a green solution, and 0.2mL of acetic acid as a catalyst was added to the obtained green solution, at N 2 Under protection, schlenk tubes were flash frozen in 77K liquid nitrogen, then degassed and sealed by three freeze-pump-thaw cycles. Heating to room temperature, heating Schlenk tube to 120deg.C, standing for 7 days, filtering the reaction system to obtain a greenish brown precipitate, and filteringWashing the solid with tetrahydrofuran for 3 times, washing with acetone for 3 times, and finally drying the washed solid in vacuum at 100 ℃ for 18 hours to obtain the covalent organic framework material, namely TAPP-TFPy-COF.
2. Specific examples of the preparation method of the aptamer sensor of the invention are as follows:
example 2
The preparation method of the aptamer sensor of the present embodiment is the preparation method of the aptamer sensor of embodiment 1, comprising the steps of:
(1) The working electrode is a bare gold electrode with the diameter of 3.0mm produced by Gaoss Union instruments, firstly, the bare gold electrode is polished by alumina slurry with the particle diameter of 0.3 mu m and 0.05 mu m in sequence, then the polished bare gold electrode is rinsed by ultrapure water for 2min, then the bare gold electrode rinsed by ultrapure water is rinsed by piranha solution and ethanol in sequence, the rinsing time of the piranha solution and the rinsing time of the ethanol are 15min, finally deionized water is used for cleaning, the cleaned bare gold electrode is dried in a nitrogen environment, the dried gold electrode is electrochemically activated after being circulated in sulfuric acid solution with the potential of 0.5mol/L by-0.2V to 1.6V, and then the pretreated bare gold electrode is obtained by rinsing with deionized water and drying under nitrogen, and is marked as AE; the piranha solution consists of concentrated sulfuric acid and hydrogen peroxide in the volume ratio of 7:3, wherein the mass fraction of the concentrated sulfuric acid is 98.08%, and the mass concentration of the hydrogen peroxide is 30%.
(2) The covalent organic framework material (TAPP-TFPy-COF) prepared in example 1 was dispersed in deionized water to give a TAPP-TFPy-COF suspension at a concentration of 1mg/mL (1 mg TAPP-TFPy-COF per 1mL deionized water). Then, 5 mu L TAPP-TFPy-COF suspension drops are placed on the surface of the pretreated bare gold electrode, and the surface of the gold electrode fixed with TAPP-TFPy-COF is obtained after drying for 6 hours at room temperature, the gold electrode is marked as TAPP-TFPy-COF/AE, and the area of the gold electrode coated with covalent organic framework material is 0.19cm 2 The coating amount of the TAPP-TFPy-COF material on the gold electrode was 26.3. Mu.g/cm 2 . Then, TAPP-TFPy-COF/AE was incubated in 100nmol/L DES aptamer solution (temperature: 4 ℃) for 1h (the incubation was performed by contacting the TAPP-TFPy-COF immobilized gold electrode with the aptamer solution to allow TAPP-TFPy-CO to passF, adsorbing and fixing the aptamer and reaching an adsorption equilibrium state) to obtain a gold electrode with the DES aptamer fixed on the surface, namely an aptamer sensor, wherein the mark is Apt/TAPP-TFPy-COF/AE.
Experimental example 1 structural characterization
1. Infrared spectrum
Characterization of the structures of TAPP, TFPy and TAPP-TFPy-COF (32 scans, resolution 4 cm) using Fourier transform Infrared Spectroscopy (FT-IR) of Bruker TENSOR27 Spectroscopy (Germany) -1 ) The results are shown in FIG. 1 a. The result shows that 3060cm in the infrared spectrogram of TFPy -1 And 1218cm -1 The absorption bands at the positions are respectively attributed to the stretching vibration and bending vibration of the C-H bond in the benzene ring; the TFPy infrared spectrum is located at 1687cm -1 The nearby peak corresponds to the stretching vibration of c=o in the aldehyde group; 1600cm in the infrared spectrum of TAPP-TFPy-COF -1 The band observed here comes from the stretching vibration of c=n, confirming the presence of imine bonds, which are shown by the chemical reaction between TAPP and TFPy.
X-ray diffraction (XRD)
The structure of TAPP-TFPy-COF was studied using an x-ray diffractometer (XRD, D/MAX-2500V/PC, rigaku, japan) with Cu K alpha radiation (λ= 0.15406 nm), and the results are shown in FIG. 1 b. As can be seen from fig. 1b, diffraction peaks at 2θ=9.17 °, 18.12 °, 20.08 °, 23.27 ° and 27.28 ° can be observed in the XRD pattern of the TAPP-TFPy-COF, indicating that the TAPP-TFPy-COF has a stacked structure and semi-crystalline properties.
3. Nuclear magnetic pattern
By passing through 13 C CP-MAS NMR analysis of the structure of TAPP-TFPy-COF prepared in example 1 was conducted as shown in FIG. 1C. As can be seen from FIG. 1C, characteristic peaks around 131 and 119ppm are derived from TAPP and TFPy building blocks, and the nuclear magnetic resonance signal at 149ppm is a characteristic peak of-C=N, confirming the presence of imine bonds in the backbone.
X-ray photoelectron spectroscopy (XPS)
The x-ray photoelectron spectroscopy (XPS) of TAPP-TFPy-COF was performed using an AXIS HIS 165 spectrometer (Kratos Analytical, manchester, U.K.) with a monochromatic Alkαx-ray source (1486.71 e K αv photons) and the results are shown in FIG. 2.
In the C1s XPS profile of TAPP-TFPy-COF, 6 peaks at Binding Energies (BEs) 283.6, 284.2, 284.8, 285.9, 288.2 and 290.9eV are assigned to c=c, graphite C, C-C, C-N, N-c=o and pi-pi bonds, respectively. Wherein, the existence of C=C, graphite C and pi-pi bond proves that the similar graphene structure of TAPP-TFPy-COF can greatly improve electrochemical activity and improve binding affinity to nucleic acid body chains through pi-pi accumulation.
In the N1s XPS spectrum of TAPP-TFPy-COF, two peaks with binding energies of 397.8eV and 399.2eV correspond to pyridine N, and a peak with binding energy of 401.6eV corresponds to graphite type N. These N atom containing groups not only promote the immobilization of DES aptamers on TAPP-TFPy-COF networks, but also promote electron transfer.
In the C1s XPS profile of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer immobilization, four peaks at binding energies 284.3, 285.1, 286.2 and 287.5eV correspond to C-C, C-N, C-O and C=O, respectively. In addition, the two peaks at binding energies 292.7eV and 295.5eV are derived from residual K in PBS solution + 。
In the N1s XPS spectrum of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer immobilization, two peaks corresponding to pyrrole N and a peak corresponding to graphite N appear. In the P2P XPS spectrum of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer immobilization, a large amount of P2P signals appear at binding energies of 132.9eV and 133.9eV, corresponding to P2P in the phosphate group on the DES aptamer chain 3/2 And P2P 1/2 . These results indicate successful immobilization of DES aptamer to the TAPP-TFPy-COF network.
Experimental example 2 characterization of morphology
The resultant samples were subjected to surface topography analysis using a JSM-6490LV field emission scanning electron microscope (FE-SEM) and a JEOL JEM-2100 high resolution transmission electron microscope (HR-TEM), and the results are shown in FIG. 3. The results show that the particle diameter of TAPP-TFPy-COF is about 100nm, and the TAPP-TFPy-COF has a multi-layer lamellar structure formed by pi-pi stacking and hydrogen bond interaction aggregation.
Experimental example 3 sensing Performance
By passing throughElectrochemical techniques the sensing performance of the aptamer sensor prepared in example 2 was evaluated, and the electrochemical measurements were as follows: electrochemical Impedance Spectroscopy (EIS) analysis was performed by a conventional three-electrode cell measurement system using a CHI 760E (CH Instruments inc., shanghai) electrochemical workstation, a platinum wire electrode as a counter electrode, a silver/silver chloride (Ag/AgCl) electrode as a reference electrode, a gold electrode as a working electrode, and recording EIS curves (EIS parameters: potential 0.21V) with frequencies ranging from 0.01Hz to 100kHz and amplitudes of 5 mV. EIS spectra were analyzed using the Scribner Associates Incorporated company Zview2 software which uses a nonlinear least squares fit to determine the component parameters in the equivalent circuit. The equivalent circuit is formed by a solution resistor (R s ) Charge transfer resistor (R) ct ) Phasing Element (CPE) and Warburg impedance (Wo). Each test was repeated at least three times.
The blocking effect of the aptamer sensor prepared in example 2 on BSA when tested for DES (DES solution concentration of 10pg/mL, aptamer sensor binding time to DES solution of 60 min) was tested by EIS technique. To eliminate non-specific adsorption, the aptamer sensor prepared in example 2 (Apt/TAPP-TFPy-COF/AE) was adsorbed with BSA, labeled BSA/Apt/TAPP-TFPy-COF/AE, and then DES was detected, and EIS nyquist curves and C-V curves obtained by constructing the aptamer sensor and detecting DES are shown in fig. 4. The result shows that Apt/TAPP-TFPy-COF/AE adsorbs R after BSA ct With a value of 872. Omega. And R of Apt/TAPP-TFPy-COF/AE ct The values were close indicating that no significant amount of BSA was adsorbed to the electrode Apt/TAPP-TFPy-COF/AE. Therefore, only slight nonspecific adsorption can occur between DES and TAPP-TFPy-COF biological platforms, and the use of blocking agents such as BSA can be avoided. These results demonstrate that the aptamer sensor prepared in example 2 can still bind specifically to DES without the need for a blocker.
The sensing performance of the aptamer sensor prepared in example 2 and the detection of DES (DES solution concentration 10pg/mL, and binding time of the aptamer sensor to DES solution 60 min) was studied by EIS and CV techniques, and the results are shown in fig. 5. Wherein FIG. 5a is the construction and detection D of an aptamer sensorEIS Nyquist diagram obtained by ES process, and semicircle diameter in Nyquist diagram corresponds to charge transfer resistance (R ct ) It reflects the electron transfer kinetics of the electrode surface redox probe, R can be obtained by fitting an EIS Nyquist plot using an equivalent circuit ct FIG. 5b is a schematic representation of the C-V curve obtained from the construction of an aptamer sensor and the detection of DES.
The results indicate that the bare gold electrode AE shows very small R ct The value was 84.8Ω, showing excellent electrochemical activity of the bare gold electrode AE. After the bare gold electrode AE fixes TAPP-TFPy-COF, R of TAPP-TFPy-COF/AE ct The value is 493.4Ω, which is probably because TAPP-TFPy-COF has poor electrochemical conductivity compared to AE, thus impeding electron transfer at the electrode/electrolyte interface. These results indicate that the TAPP-TFPy-COF prepared in example 1 has good electrical conductivity. The porphyrin structure on the TAPP-TFPy-COF network can greatly enhance the electron transfer capability and can be used as a good biological platform of the biosensor. After the TAPP-TFPy-COF is immobilized with DES aptamer, the obtained R of Apt/TAPP-TFPy-COF/AE ct The value is further increased to 851.4 omega, R ct The significant increase in value is attributed to the adsorbed aptamer. In aqueous solution, the negatively charged phosphate groups formed by the DES aptamer are combined with [ Fe (CN) 6] 3-/4- Has mutual repulsive interaction, thereby preventing the transfer of electrons from the electrolyte solution to the Apt/TAPP-TFPy-COF/AE electrode, thereby increasing R ct Values. When detecting DES using an aptamer sensor, R of electrode (DES/Apt/TAPP-TFPy-COF/AE) ct The value is increasing continuously to 1.44kΩ. This is due to the specific interaction between DES and DES aptamer chains, which can form G-quadruplexes, and the insulating complex formed by the aptamer and DES binding further prevents electron transfer.
As the peak current densities of TAPP-TFPy-COF, DES aptamer and DES on the gold electrode decrease and the peak potential difference increases, the TAPP-TFPy-COF/AE, apt/TAPP-TFPy-COF/AE and DES/Apt/TAPP-TFPy-COF/AE electrodes compared with AE, these results confirm the TAPP-TFPy-COF material, TAPP-TFPy-COF immobilized aptamer and aptamer-adsorbed D on the gold electrode surfaceES can block electron transfer at the interface. In addition, due to the high sensitivity of the EIS detection method, the current density variation value obtained by CV technology detection is much smaller than R obtained by EIS measurement ct Change in value. In addition, because of the large pore size of the TAPP-TFPy-COF, a large number of aptamer chains can be immobilized on the surface of the TAPP-TFPy-COF with a porous nano structure, and can penetrate into the TAPP-TFPy-COF network, so that the active site of the TAPP-TFPy-COF can be fully occupied.
Experimental example 4 optimization of aptamer sensor preparation and detection conditions
In order to obtain the best sensing performance of the prepared aptamer sensor and the detection of DES, the conditions for preparing the aptamer sensor and the detection of DES are optimized. The experimental conditions were as follows: TAPP-TFPy-COF suspensions at concentrations of 0.1, 0.2, 0.5, 0.8, 1 and 1.5mg/mL, DES aptamers at concentrations of 1, 5,10, 50, 100 and 200nmol/L, DES solutions combined with aptamer sensors for times of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110min, and DES solutions at concentrations of 10pg/mL.
Firstly, TAPP-TFPy-COF suspensions with different concentrations are used for preparing an aptamer sensor, then the prepared aptamer sensor is used for detecting DES (the concentration of DES aptamer solution used in preparation is 100nmol/L, the combination time of the aptamer sensor and the DES solution is 60 min), and the obtained DeltaR is detected ct The values are shown in FIGS. 6a and 6b, which show that TAPP-TFPy-COF/AE has a ΔR ct Value (R) ctTAPP-TFPy-COF/AE -R ctAE ) DeltaR of Apt/TAPP-TFPy-COF/AE ct Value (R) ctApt/TAPP-TFPy-COF/AE -R ctTAPP-TFPy-COF/AE ) Increasing with increasing concentration of TAPP-TFPy-COF suspension, when the concentration of TAPP-TFPy-COF suspension is greater than 1mg/mL, ΔR of TAPP-TFPy-COF/AE ct Value and ΔR of Apt/TAPP-TFPy-COF/AE ct The values all tended to stabilize. Thus, the optimal concentration of TAPP-TFPy-COF suspension is 1mg/mL.
Then, the DES was detected using an aptamer sensor prepared by incubating solutions of DES aptamer at different concentrations (the concentration of TAPP-TFPy-COF suspension used in preparation was 1mg/mL, aptamer at the time of detection)The combination time of the sensor and the DES solution is 60 min), and the delta R obtained by detection ct The values are shown in FIGS. 6c and 6d, which show that the ΔR of Apt/TAPP-TFPy-COF/AE ct Value (R) ctApt/TAPP-TFPy-COF/AE -R ctTAPP-TFPy-COF/AE ) DeltaR of DES/Apt/TAPP-TFPy-COF/AE ct Value (R) ctDES/Apt/TAPP-TFPy-COF/AE -R ctApt/TAPP-TFPy-COF/AE ) Both increased with increasing DES aptamer concentrations in the range of 1-100 nmol/L. When DES aptamer concentration is more than 100nmol/L, apt/TAPP-TFPy-COF/AE delta R ct Value DeltaR of DES/Apt/TAPP-TFPy-COF/AE ct The values all reached equilibrium, indicating that the interaction between the aptamer chain and DES was saturated. Thus, the optimal concentration of DES aptamer solution is 100nmol/L.
Finally, the aptamer sensor was tested for EIS Nquist curve and ΔR obtained at different binding times with DES solutions (TAPP-TFPy-COF suspension concentration of 1mg/mL for preparation, DES aptamer concentration of 100 nmol/L) ct The values, as shown in FIGS. 6e and 6f, indicate that the DES/Apt/TAPP-TFPy-COF/AE ΔR ct Value (R) ctDES/Apt/TAPP-TFPy-COF/AE -R ctApt/TAPP-TFPy-COF/AE ) Increasing with increasing binding time. When the binding time is more than 60min, the DES/Apt/TAPP-TFPy-COF/AE has a delta R ct The values reach equilibrium, indicating saturation of binding between DES and aptamer, prolonged binding time, ΔR ct The change of the value is small, and when the binding time is more than 80min, the DES/Apt/TAPP-TFPy-COF/AE delta R ct The slight decrease in value may be due to DES falling off the electrode, so the optimal binding time for the aptamer sensor to DES was 60min.
Experimental example 5 sensitivity
Under optimal detection conditions, different concentrations (0.001, 0.01, 0.1, 1, 10, 100 and 1000 pg/mL) of DES solutions were detected using the prepared aptamer sensor, wherein the semicircle radius in the nyquist plot was positively correlated with the concentration of DES, which can be explained by the G-quadruplex formed during the detection. The DES detection times of the same concentration are 3 times, and the detected EIS Nyquist curve and R ct Change value (DeltaR) ct ) Curves as shown in FIG. 7a andshown at 7 b. The results showed that R before and after DES was detected in the range of DES concentration from 1fg/mL to 1ng/mL ct Change value (DeltaR) ct ) Log to DES concentration (LogCon DES ) And the slope of the correlation is expressed by m. According to the IUPAC method, in the range of DES concentration from 1fg/mL to 1ng/mL, the calculated LOD is 0.37fg/mL, and the calculation formula of the LOD is as follows: lod=3sb/m, where Sb represents the standard deviation and m is Δr ct Slope of the linear fit curve to the logarithm of DES concentration. The aptamer sensor prepared in example 2 has a lower LOD value than other reported sensors for DES detection constructed using electrocatalytic and photoelectrochemical methods, table 1 shows the sensitivity of DES detection for different detection methods.
According to the relevant report, DES can be electrochemically oxidized and thus can be determined by electrochemical techniques. However, these sensors have low detection sensitivity for DES due to the slow electron transfer rate of DES. In contrast, the aptamer sensor prepared in example 2 overcomes the drawbacks of the conventional electrochemical sensor for detecting DES, and has extremely low LOD, fast response speed, and wide linear assay range of DES. The excellent sensing performance of the aptamer sensor prepared in example 2 is attributable to the following factors: (i) The synthesized TAPP-TFPy-COF can anchor a large number of aptamers through complex interactions (pi-pi stacking, electrostatic interactions, hydrogen bonds or Van der Waals forces), can serve as a sensitive biological platform, and can enhance electrochemical response when detecting targets; (ii) Because the TAPP-TFPy-COF has a porous nano structure and a larger pore diameter, the aptamer is promoted to be combined on the surface and the inside of the TAPP-TFPy-COF material, so that the detection sensitivity is improved; (iii) The occupation of a large number of aptamers on TAPP-TFPy-COF can avoid non-specific adsorption between DES and TAPP-TFPy-COF matrix. Thus, TAPP-TFPy-COF can serve as a sensitive platform for anchoring a large number of DES aptamer chains, thereby exhibiting superior detection performance for DES.
TABLE 1 sensitivity of detection of DES by different detection methods
Reference is made to:
document 1: r. Zhang, X. j. Li, A. l. Sun, S. q. Song, and X. z. Shi, "A highly selective fluorescence nanosensor based on the dual-function molecularly imprinted layer coated quantum dots for the sensitive detection of diethylstilbestrol/cypermethrin in fish and seawater," Food Control, vol.132, p.108438,2022/02/01/2022.
Document 2: r. Manzoor et al, "Ultrasensitive competitive electrochemiluminescence immunosensor based on luminol-AuNPs@Mo 2 C and upconversion nanoparticles for detection of diethylstilbestrol,"Microchemical Journal,vol.158,p.105283,2020/11/01/2020。
Document 3: X.Dong, G.Zhao, L.Liu, X.Li, Q.Wei, and W.Cao, "Ultrasensitive competitive method-based electrochemiluminescence immunosensor for diethylstilbestrol detection based on Ru (bpy) 3 2+ as luminophor encapsulated in metal–organic frameworks UiO-67,"Biosensors and Bioelectronics,vol.110,pp.201-206,2018/07/01/2018。
Document 4: liu et al, "Electrochemical immunosensor based on mesoporous nanocomposites and HRP-functionalized nanoparticles bioconjugates for sensitivity enhanced detection of diethylstilbestrol," Sensors and Actuators B: chemical, vol.166-167, pp.562-568,2012/05/20/2012.
Document 5: T.Wu et al, "A competitive photoelectrochemical immunosensor for the detection of diethylstilbestrol based on an Au/UiO-66 (NH) 2 )/CdS matrix and a direct Z-scheme Melem/CdTe heterojunction as labels,"Biosensors and Bioelectronics,vol.117,pp.575-582,2018/10/15/2018。
Experimental example 6 Selectivity
By detecting other organic contaminants in the wastewater that may co-exist with DES, such as Enrofloxacin (ENR), salbutamol (SAL), zearalenone (ZEA), aflatoxin (AFT), deoxynivalenol (DON), oxytetracycline (OTC) and heavy metal ions (Cr) 3+ 、Cu 2+ Or Ag + ) The development of the selectivity of aptamer sensors was investigated. Detecting 1000pg/mL of a test solution containing organic pollutants (ENR, SAL, ZEA, AFT, DON or OTC) and heavy metal ions (Cr) at a detection time of 60min 3+ 、Cu 2+ Or Ag + ) The concentration of (C) was 1000pg/mL, the concentration of DES was 10pg/mL, or the concentration of DES was 10pg/mL, and the concentration of heavy metal ion (Cr) 3+ 、Cu 2+ Or Ag + ) And organic contaminants (ENR, SAL, ZEA, AFT, DON and OTC) at a concentration of 1000pg/mL, respectively ct The values are shown in fig. 7 c. The results show that the aptamer sensor prepared in example 2 has a significant EIS response when detecting DES, while the EIS response when detecting other organic contaminants or heavy metal ions is low, which is negligible. Furthermore, when the aptamer sensor prepared in example 2 was used to detect a mixed test solution consisting of DES, organic pollutants and heavy metal ions, the EIS response caused was only 4.9% when DES was detected alone, and these results confirmed that the prepared aptamer sensor had good selectivity in a complex environment.
Experimental example 7 reproducibility
Reproducibility of the aptamer sensor was evaluated by the electrochemical response result obtained by immersing 5 aptamer sensors prepared by the same preparation method as that of the aptamer sensor prepared in example 2 in DES solution having a concentration of 10pg/mL for 60min, and the result is shown in fig. 7 d. The results showed that the 5 aptamer sensors prepared were small in RSD for DES detection, which was only 3.25%, indicating that the aptamer sensor prepared in example 2 had good reproducibility.
Experimental example 8 stability
The stability of the sensor was evaluated by detecting electrochemical response after DES solution at a concentration of 10pg/mL once daily for 15 consecutive days using the sensor prepared in example 2. The specific operation process is as follows: in the first day of test, detecting DES by using a sensor under the condition that the detection time is 60min to obtain R before and after the sensor is fixed to the DES ct The value is calculated to obtain delta R ct Value, then place the electrode in phosphoric acidSalt buffer (PBS, 0.01mol/L, ph=7.4), refrigerated in a refrigerator at 4 ℃; the next day of testing, the electrode was directly removed, the measurement was performed at room temperature, and R was recorded ct The value of DeltaR is calculated ct Value (R obtained by measurement after one day of DES-immobilized sensor placement) ct Value and R before sensor fixed DES at first day test ct Difference in values), repeating the operation until day 15, and finally obtaining DeltaR of 15 days for detecting DES ct Values, results are shown in FIG. 7 e. The results show that ΔR obtained by the test ct The value is kept stable, the RSD is 2.75%, and the prepared aptamer sensor has strong fixing effect on DES and good stability when used for DES detection.
Experimental example 9. Regenerability
The reproducibility of the aptamer sensor was evaluated by detecting the electrochemical response obtained with the regenerated aptamer sensor with DES solution at a concentration of 10pg/mL. The regeneration process is as follows: firstly, an aptamer sensor for detecting DES is soaked in NaOH solution with the concentration of 0.05mol/L for 2-3min, and the aptamer sensor soaked in the NaOH solution is thoroughly washed by water, so that the regenerated aptamer sensor is obtained. After DES was detected using the regenerated aptamer sensor, the above regeneration process was repeated, DES was then detected, and the regeneration process was performed a total of 5 times, and EIS responses obtained using the original aptamer sensor and the regenerated aptamer sensor to detect DES were recorded, and the results are shown in fig. 7 f. The results showed that after 5 times of regeneration treatment, the aptamer sensor of the regeneration treatment detected DES obtained an EIS response close to that obtained by DES using the original aptamer sensor, demonstrating that the aptamer sensor prepared in example 2 had good reproducibility.
Experimental example 10 practicality
In order to further investigate the application prospect of the aptamer sensor prepared in example 2, it was applied to the detection of DES in a variety of real samples (human serum, milk and frozen shrimp).
The real sample is processed as follows:
(1) Human serum was obtained from beijing soley biotechnology limited. Before use, the mixture is filtered by a 3kDa dialysis bag to remove possible interfering compounds, then the mixture is placed at room temperature for 0.5h, and centrifuged at 2000r/min for 10min, and the separated supernatant serum is stored in an environment of-20 ℃ for later use.
(2) Adding 0.2mL of NaOH solution with the concentration of 0.1mol/L and 0.8mL of acetonitrile into milk, uniformly mixing, performing ultrasonic treatment for 30min, centrifuging at the room temperature at the rotation speed of 5000rpm for 5min, collecting supernatant, diluting 50 times with 10mmol/L of PBS solution, and preserving in the environment of 4 ℃ for later use.
(4) The frozen shrimps are smashed and ground and then placed in a 15mL centrifuge tube, then the frozen shrimps are vigorously vibrated for 1h by ultrasound, the supernatant is taken after centrifugation for 5min at a rotating speed of 1000rpm, the supernatant is dried in an environment of 40 ℃, then the solid residues obtained by drying are redissolved in 1.0mL of 50% methanol solution, and the obtained solution is added into PBS solution with the concentration of 10mmol/L for further use.
DES was added to each of the treated authentic samples (human serum, milk, frozen shrimp), and then possible interferents were removed by filtration using a 3kDa dialysis bag to obtain solutions containing 4 authentic samples of different concentrations (0.001, 0.01, 0.1, 1, 10, 100 and 1000 pg/mL) of DES, and then the aptamer sensor prepared in example 2 was immersed in the authentic sample for 60min to obtain DeltaR for detection of DES ct The value, then, the DES concentration obtained by the detection is calculated from the correction curve between the EIS response and the logarithm of the DES concentration, and compared with the actual value. The actual amount added (concentration of DES actually added in the actual sample), concentration of DES detected (amount detected), calculated recovery and RSD are listed in table 1. The result shows that the recovery rate of DES in human serum detected by the aptamer sensor prepared in the embodiment 2 is 96.2-112.9%, and the RSD is 0.92-2.09%; the recovery rate of DES in milk detected by the aptamer sensor prepared in the embodiment 2 is 92.87% -118.5%, and RSD is less than 3.11%; the aptamer sensor prepared in example 2 has a recovery rate of DES of 91.8% -113.4% and an RSD of less than 2.69%. The results show that the aptamer sensor prepared in the embodiment 2 can sensitively detect DES in different samples, and has a wide application prospect.
Table 2 test results of the aptamer sensor prepared in example 2 for detecting real samples
/>
Claims (8)
1. An aptamer sensor is characterized by comprising an electrode matrix and a covalent organic framework material modified on the surface of the electrode matrix, wherein a nucleic acid aptamer for targeted detection of diethylstilbestrol is adsorbed on the covalent organic framework material; the covalent organic framework material is prepared from 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene by adopting a solvothermal method through Schiff base reaction; the catalyst adopted by the Schiff base reaction is acetic acid; the solvent adopted in the Schiff base reaction consists of N, N-dimethylacetamide and o-dichlorobenzene; the volume ratio of the N, N-dimethylacetamide to the o-dichlorobenzene is (2.8-3.2): 1.
2. The aptamer sensor of claim 1, wherein the molar ratio of 5,10,15, 20-tetrakis (4-aminophenyl) porphyrin to 1,3,6, 8-tetrakis (4-formylphenyl) pyrene is (0.8-1.2): 0.8-1.2.
3. The aptamer sensor of claim 1 or2, wherein the solvothermal method has a soak temperature of 120-125 ℃; the heat preservation time of the solvothermal method is 144-168 hours.
4. A method of preparing an aptamer sensor according to any one of claims 1 to 3, comprising the steps of: firstly, loading a covalent organic framework material onto an electrode matrix to obtain a modified electrode; the modified electrode is then incubated in a nucleic acid aptamer solution for targeted detection of diethylstilbestrol.
5. The method of preparing an aptamer sensor according to claim 4, wherein the loading is by coating a suspension of covalent organic framework material onto an electrode substrate, followed by a drying process.
6. The method of preparing an aptamer sensor of claim 5, wherein the concentration of the suspension of covalent organic framework material is 0.8-1.5mg/mL.
7. The method for preparing an aptamer sensor according to any one of claims 4 to 6, wherein the concentration of the aptamer solution for targeted detection of diethylstilbestrol is 100-200nmol/L.
8. The method of preparing an aptamer sensor according to any one of claims 4 to 6, wherein the incubation temperature is 0 to 4 ℃; the incubation time is 60-80min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111531807.4A CN114113265B (en) | 2021-12-14 | 2021-12-14 | Aptamer sensor and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111531807.4A CN114113265B (en) | 2021-12-14 | 2021-12-14 | Aptamer sensor and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114113265A CN114113265A (en) | 2022-03-01 |
| CN114113265B true CN114113265B (en) | 2023-08-08 |
Family
ID=80364962
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111531807.4A Active CN114113265B (en) | 2021-12-14 | 2021-12-14 | Aptamer sensor and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114113265B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114755278A (en) * | 2022-04-19 | 2022-07-15 | 常州大学 | Ppy-MIP/ZrO2@ C/NPG/GCE molecularly imprinted electrochemical sensor, and preparation method and application thereof |
Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998047002A2 (en) * | 1997-04-11 | 1998-10-22 | Advanced Medicine, Inc. | Polyvalent presenter combinatorial libraries and their uses |
| WO2001006016A2 (en) * | 1999-07-20 | 2001-01-25 | Clinical Micro Sensors, Inc. | Amplification of nucleic acids with electronic detection |
| WO2006083269A2 (en) * | 2004-05-14 | 2006-08-10 | Florida Atlantic University | Luminescent nanosensors |
| CN103228663A (en) * | 2010-09-27 | 2013-07-31 | 加利福尼亚大学董事会 | Conductive open frameworks |
| CN105778897A (en) * | 2016-04-11 | 2016-07-20 | 湖南大学 | PH sensitive fluorochrome and preparation method and application thereof |
| CN106018514A (en) * | 2016-07-05 | 2016-10-12 | 济南大学 | Preparation method of photoelectrochemical diethylstilbestrol sensor based on copper-doped nano photoelectric material |
| CN108362750A (en) * | 2018-03-07 | 2018-08-03 | 扬州大学 | A kind of preparation method for adulterating covalent organic framework composite electrode based on gold nanoparticle |
| CN109851807A (en) * | 2019-01-11 | 2019-06-07 | 郑州赫诺瑞信息科技有限公司 | Py-M-COF and its electrochemical sensor and application |
| CN110426434A (en) * | 2019-09-02 | 2019-11-08 | 西北师范大学 | A kind of building and its application based on copper porphyrin base covalent organic frame material electrochemical sensor |
| CN111175369A (en) * | 2020-02-24 | 2020-05-19 | 陕西师范大学 | MALDI-TOF MS matrix for small molecule detection and application thereof |
| CN111239213A (en) * | 2020-03-23 | 2020-06-05 | 江南大学 | Covalent organic framework material in-situ modified electrode and electrochemical biosensor |
| CN112114018A (en) * | 2020-09-08 | 2020-12-22 | 常州大学 | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof |
| CN112126236A (en) * | 2020-10-13 | 2020-12-25 | 济南大学 | Porphyrin covalent organic framework/graphene aerogel composite material and electrochemical sensor and application thereof |
| CN112156758A (en) * | 2020-09-15 | 2021-01-01 | 清华大学 | Porous material and preparation method and application thereof |
| CN112321839A (en) * | 2020-10-30 | 2021-02-05 | 郑州轻工业大学 | Bimetal covalent organic framework material, preparation method thereof and aptamer sensor |
| CN113185660A (en) * | 2021-04-14 | 2021-07-30 | 南通大学 | Covalent organic framework based on pillar arene and preparation method and application thereof |
| CN113504274A (en) * | 2021-07-20 | 2021-10-15 | 郑州轻工业大学 | Covalent organic framework material, preparation method and application thereof, aptamer sensor and preparation method thereof |
| CN113774447A (en) * | 2021-10-14 | 2021-12-10 | 福州大学 | Porphyrin-based covalent organic framework solid-phase microextraction coating prepared by one-step electrodeposition and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1733056B1 (en) * | 2004-03-31 | 2013-05-22 | The General Hospital Corporation | Method to determine responsiveness of cancer to epidermal growth factor receptor targeting treatments |
| FR2943421B1 (en) * | 2009-03-18 | 2012-11-30 | Commissariat Energie Atomique | ELECTRICAL DETECTION AND QUANTIFICATION OF MERCURIC DERIVATIVES |
| US11634446B2 (en) * | 2019-04-22 | 2023-04-25 | Trustees Of Dartmouth College | Two-dimensional stimuli-responsive covalent organic frameworks with high intrinsic conductivity |
| US11659758B2 (en) * | 2019-07-05 | 2023-05-23 | Semiconductor Energy Laboratory Co., Ltd. | Display unit, display module, and electronic device |
-
2021
- 2021-12-14 CN CN202111531807.4A patent/CN114113265B/en active Active
Patent Citations (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998047002A2 (en) * | 1997-04-11 | 1998-10-22 | Advanced Medicine, Inc. | Polyvalent presenter combinatorial libraries and their uses |
| WO2001006016A2 (en) * | 1999-07-20 | 2001-01-25 | Clinical Micro Sensors, Inc. | Amplification of nucleic acids with electronic detection |
| WO2006083269A2 (en) * | 2004-05-14 | 2006-08-10 | Florida Atlantic University | Luminescent nanosensors |
| CN103228663A (en) * | 2010-09-27 | 2013-07-31 | 加利福尼亚大学董事会 | Conductive open frameworks |
| CN105778897A (en) * | 2016-04-11 | 2016-07-20 | 湖南大学 | PH sensitive fluorochrome and preparation method and application thereof |
| CN106018514A (en) * | 2016-07-05 | 2016-10-12 | 济南大学 | Preparation method of photoelectrochemical diethylstilbestrol sensor based on copper-doped nano photoelectric material |
| CN108362750A (en) * | 2018-03-07 | 2018-08-03 | 扬州大学 | A kind of preparation method for adulterating covalent organic framework composite electrode based on gold nanoparticle |
| CN109851807A (en) * | 2019-01-11 | 2019-06-07 | 郑州赫诺瑞信息科技有限公司 | Py-M-COF and its electrochemical sensor and application |
| CN110426434A (en) * | 2019-09-02 | 2019-11-08 | 西北师范大学 | A kind of building and its application based on copper porphyrin base covalent organic frame material electrochemical sensor |
| CN111175369A (en) * | 2020-02-24 | 2020-05-19 | 陕西师范大学 | MALDI-TOF MS matrix for small molecule detection and application thereof |
| CN111239213A (en) * | 2020-03-23 | 2020-06-05 | 江南大学 | Covalent organic framework material in-situ modified electrode and electrochemical biosensor |
| CN112114018A (en) * | 2020-09-08 | 2020-12-22 | 常州大学 | Sensor for detecting diethylstilbestrol by electrochemiluminescence method based on resonance energy transfer and preparation method and application thereof |
| CN112156758A (en) * | 2020-09-15 | 2021-01-01 | 清华大学 | Porous material and preparation method and application thereof |
| CN112126236A (en) * | 2020-10-13 | 2020-12-25 | 济南大学 | Porphyrin covalent organic framework/graphene aerogel composite material and electrochemical sensor and application thereof |
| CN112321839A (en) * | 2020-10-30 | 2021-02-05 | 郑州轻工业大学 | Bimetal covalent organic framework material, preparation method thereof and aptamer sensor |
| CN113185660A (en) * | 2021-04-14 | 2021-07-30 | 南通大学 | Covalent organic framework based on pillar arene and preparation method and application thereof |
| CN113504274A (en) * | 2021-07-20 | 2021-10-15 | 郑州轻工业大学 | Covalent organic framework material, preparation method and application thereof, aptamer sensor and preparation method thereof |
| CN113774447A (en) * | 2021-10-14 | 2021-12-10 | 福州大学 | Porphyrin-based covalent organic framework solid-phase microextraction coating prepared by one-step electrodeposition and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| "共价有机框架材料催化研究进展";刘建国等;《化学学报》;第78卷(第4期);第311-325页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114113265A (en) | 2022-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Novel nanoarchitecture of Co-MOF-on-TPN-COF hybrid: Ultralowly sensitive bioplatform of electrochemical aptasensor toward ampicillin | |
| Zhang et al. | Covalent organic framework as a novel electrochemical platform for highly sensitive and stable detection of lead | |
| Ghanei-Motlagh et al. | A novel voltammetric sensor for sensitive detection of mercury (II) ions using glassy carbon electrode modified with graphene-based ion imprinted polymer | |
| Fu et al. | Electropolymerized surface ion imprinting films on a gold nanoparticles/single-wall carbon nanotube nanohybrids modified glassy carbon electrode for electrochemical detection of trace mercury (II) in water | |
| Baghayeri et al. | Fabrication of a facile electrochemical biosensor for hydrogen peroxide using efficient catalysis of hemoglobin on the porous Pd@ Fe3O4-MWCNT nanocomposite | |
| Gupta et al. | A novel sensitive Cu (II) and Cd (II) nanosensor platform: graphene oxide terminated p-aminophenyl modified glassy carbon surface | |
| Ru et al. | GaOOH-modified metal-organic frameworks UiO-66-NH2: Selective and sensitive sensing four heavy-metal ions in real wastewater by electrochemical method | |
| Yuan et al. | Design and fabrication of an electrochemical sensing platform based on a porous organic polymer for ultrasensitive ampicillin detection | |
| Wen et al. | Electrochemical sensor for arsenite detection using graphene oxide assisted generation of prussian blue nanoparticles as enhanced signal label | |
| CN111234243B (en) | Metal organic framework material, preparation method thereof and immunosensor | |
| CN109851807B (en) | Py-M-COF and electrochemical sensor and application thereof | |
| Yi et al. | Sensitive electrochemical sensor based on poly (L-glutamic acid)/graphene oxide composite material for simultaneous detection of heavy metal ions | |
| Liu et al. | A high–selectivity electrochemical sensor for ultra-trace lead (II) detection based on a nanocomposite consisting of nitrogen-doped graphene/gold nanoparticles functionalized with ETBD and Fe3O4@ TiO2 core–shell nanoparticles | |
| Feng et al. | A label-free electrochemical immunosensor for rapid detection of salmonella in milk by using CoFe-MOFs-graphene modified electrode | |
| Li et al. | Intense charge transfer surface based on graphene and thymine–Hg (II)–thymine base pairs for detection of Hg2+ | |
| Zhu et al. | Non-enzymatic xanthine sensor of heteropolyacids doped ferrocene and reduced graphene oxide via one-step electrodeposition combined with layer-by-layer self-assembly technology | |
| CN111537577B (en) | Metal-organic framework graphene analogue and preparation method thereof, aptamer sensor and preparation method thereof | |
| Zhang et al. | Direct growth of two-dimensional phthalocyanine-based COF on Cu-MOF to construct a photoelectrochemical-electrochemical dual-mode biosensing platform for high-efficiency determination of Cr (iii) | |
| CN114113265B (en) | Aptamer sensor and preparation method thereof | |
| Tashkhourian et al. | Ascorbic acid determination based on electrocatalytic behavior of metal-organic framework MIL-101-(Cr) at modified carbon-paste electrode | |
| CN113552199A (en) | Based on FeS2Molecular imprinting electrochemical sensor of/C/MQDs/GCE modified electrode and preparation method thereof | |
| Dang et al. | Determination of norfloxacin using a tetraoxocalix [2] arene [2]-triazine covalently functionalized multi-walled carbon nanotubes modified electrode | |
| Yuan et al. | Electrochemical assay for As (III) by combination of highly thiol-rich trithiocyanuric acid and conductive reduced graphene oxide nanocomposites | |
| Huang et al. | Cytochrome c-multiwalled carbon nanotube and cobalt metal organic framework/gold nanoparticle immobilized electrochemical biosensor for nitrite detection | |
| Chen et al. | Impedimetric aptasensor based on porphyrin-based covalent-organic framework for determination of diethylstilbestrol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |