CN114113265A - Aptamer sensor and preparation method thereof - Google Patents

Aptamer sensor and preparation method thereof Download PDF

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CN114113265A
CN114113265A CN202111531807.4A CN202111531807A CN114113265A CN 114113265 A CN114113265 A CN 114113265A CN 202111531807 A CN202111531807 A CN 202111531807A CN 114113265 A CN114113265 A CN 114113265A
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des
aptamer
tfpy
tapp
cof
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CN114113265B (en
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崔静
张治红
方少明
何领好
王明花
阚伦
吴百威
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Zhengzhou University of Light Industry
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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 substrate and a covalent organic framework material modified on the surface of the electrode substrate, 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 by reacting 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene through Schiff base by adopting a solvothermal method. When the aptamer sensor is used for detecting DES, the sensitivity is high, and the sensitivity, the reproducibility, the stability, the reproducibility and the applicability acceptable in complex environments are good.

Description

Aptamer sensor and preparation method thereof
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 medicine application, the pharmacological and therapeutic effects of DES are the same as those of natural estrogens. Compared with natural estrogen, DES is more stable and stays in the body for a longer time. Since DES is dark and difficult to degrade, human health is severely compromised. Accordingly, the us, japan, the european union and china prohibit the use of DES by food animals. Nevertheless, DES abuse is widespread and found in many countries in both the environment and aquatic life. Therefore, the development of a highly sensitive and reliable method for determining DES in contaminated food is of great significance to the guarantee of human health. Currently, there are a number of 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 fluorescence chemiluminescence, Surface Enhanced Raman Spectroscopy (SERS), quartz crystal microbalances, paper sensors, immunochromatography, electrochemical immunosensor, Electrochemiluminescence (ECL), photoelectrochemical techniques, and the like. However, these methods require special equipment and a large number of separate analysis procedures, resulting in more complex, time-consuming and laborious screening procedures. Among different technologies, electrochemical methods have the advantages of fast response, low cost, low detection limit, easy miniaturization, small sample usage amount and the like. However, some direct electrochemical methods tend to be less selective 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. In addition, DNA aptamers that bind specifically to DES can also be used to assay DES. Compared with immunosensors, aptamer sensors constructed by using DNA aptamers often have the advantages of high sensitivity, good stability, low cost and the like.
To date, 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 framework materials, such as metal organic framework Materials (MOFs) and covalent organic framework materials (COFs), are attracting attention due to their high specific surface area, customizable structure and tunable pore size.
Most MOFs-based DES sensors rely on the electrocatalytic properties of the MOFs-loaded material on electrodes for DES, however, most electrode materials typically exhibit effective electrocatalytic capacity for other small molecules, such as H2O2、H2S, uric acid or dopamine, thereby reducing the selectivity of DES detection, increasing the steps of electrode material optimization, prolonging the detection period, increasing the detection time and the detection cost, and the prepared biosensor has lower sensitivity when detecting DES.
Disclosure of Invention
The invention aims to provide an aptamer sensor, which is used for solving the problem that the existing sensor for detecting DES is low in sensitivity.
Another object of the present invention is to provide a method for preparing an aptamer sensor.
In order to achieve the above object, the technical solution adopted by the aptamer sensor of the invention is as follows:
an aptamer sensor comprises an electrode substrate and a covalent organic framework material modified on the surface of the electrode substrate, 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 by reacting 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene through Schiff base by adopting a solvothermal method.
The covalent organic framework material is used as an electrode material for constructing an aptamer sensor for targeted detection of diethylstilbestrol, 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 a solvothermal method through Schiff base reaction, and the electrode material 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 on the surface and within the covalent organic framework materials through complex interactions. When the aptamer sensor is used for detecting DES, the sensitivity is high, and the sensitivity, the reproducibility, the stability, the reproducibility and the applicability acceptable in complex environments are good.
Preferably, the molar ratio of the 5,10,15, 20-tetra (4-aminophenyl) porphyrin to the 1,3,6, 8-tetra (4-formylphenyl) pyrene is (0.8-1.2) to (0.8-1.2). Further, the molar ratio of the 5,10,15, 20-tetra (4-aminophenyl) porphyrin to the 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 process 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 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 N2。
Preferably, the temperature for the solvothermal method is 120-125 ℃. Further, the holding temperature of the solvothermal method is 120 ℃. Preferably, the holding time of the solvothermal method is 144-168 h. Further, the holding time of the solvothermal method is 168 h.
Preferably, the solvent used for 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 completion of the schiff base reaction, the system after the schiff base reaction is subjected to solid-liquid separation, 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 solid obtained by solid-liquid separation is washed with tetrahydrofuran for 3 times and then with acetone for 3 times.
Preferably, the drying is carried out by drying the washed solid under vacuum at 100 ℃ for 18 h.
The preparation method of the aptamer sensor adopts the technical scheme that:
a preparation method of the aptamer sensor comprises the following steps: firstly, loading a covalent organic framework material on an electrode substrate to obtain a modified electrode; the modified electrode was 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 carried out by coating a suspension of the covalent organic framework material onto the electrode substrate and then carrying out a drying treatment.
Preferably, the suspension of covalent organic framework material has a concentration of 0.8-1.5 mg/mL. Further preferably, the suspension of covalent organic framework material has a concentration of 1 mg/mL.
Preferably, the suspension of covalent organic framework material consists essentially of water and covalent organic framework material. Water acts as a dispersant, allowing better dispersion of the covalent organic framework material.
Preferably, the covalent organic framework material is coated on the electrode substrate in an amount of 21-39.4. mu.g/cm2。
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 modified electrode is incubated in the aptamer solution for the following steps: and (3) immersing the modified electrode into a nucleic acid aptamer solution to obtain the aptamer sensor.
Preferably, the incubation temperature is 0-4 ℃; the incubation time is 60-80 min. Further, the incubation temperature is 4 ℃; the incubation time was 1 h.
Preferably, the concentration of the aptamer solution for targeted detection of diethylstilbestrol is 100-200 nmol/L. Further preferably, the concentration of the aptamer solution for targeted detection of diethylstilbestrol is 100 nmol/L.
Preferably, the aptamer solution for targeted detection of diethylstilbestrol consists essentially of water, phosphate and an aptamer for targeted detection of diethylstilbestrol. Preferably, the aptamer solution for targeted detection of diethylstilbestrol further comprises an alkali metal chloride. Preferably, the phosphate is KH2PO4and/Na2HPO4·12H2And O. Preferably, the alkali chloride is KCl and/or NaCl.
Drawings
FIG. 1: (a) fourier Transform Infrared (FTIR) spectra of different materials (TAPP, TFPy and TAPP-TFPy-COF), (b) XRD pattern of TAPP-TFPy-COF, and (c) XRD pattern of TAPP-TFPy-COF13C CP-MAS NMR spectra;
FIG. 2: (a) a high resolution XPS spectrum of C1s for TAPP-TFPy-COF, (b) a high resolution XPS spectrum of N1s for TAPP-TFPy-COF, (C) a high resolution XPS spectrum of C1s for TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after fixing DES aptamer, (d) a high resolution XPS spectrum of N1s for TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after fixing DES aptamer; (e) high resolution XPS spectra of P2P for TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after immobilization of DES aptamer;
FIG. 3: (a) the image is a low-power Scanning Electron Microscope (SEM) image of TAPP-TFPy-COF, (b) the image is a high-power Scanning Electron Microscope (SEM) image of TAPP-TFPy-COF, (c) the image is a low-power Transmission Electron Microscope (TEM) image of TAPP-TFPy-COF, and (d) the image is a high-power Transmission Electron Microscope (TEM) image of TAPP-TFPy-COF;
FIG. 4: (a) an EIS Nquist curve schematic diagram obtained by the construction of the aptamer sensor and the detection of the DES process when the blocking effect of BSA is tested, and a C-V curve schematic diagram obtained by the construction of the aptamer sensor and the detection of the DES process when the blocking effect of BSA is tested;
FIG. 5: (a) a schematic diagram of an EIS nyquist curve obtained in the processes of preparing an aptamer sensor and detecting DES in example 2, and (b) a schematic diagram of a C-V curve obtained in the processes of preparing an aptamer sensor and detecting DES in example 2;
FIG. 6: (a) delta R obtained during preparation of aptamer sensors using TAPP-TFPy-COF suspensions of varying concentrationsctValue (Δ R before and after TAPP-TFPy-COF fixation of bare gold electrode AE)ctValue and Δ R before and after TAPP-TFPy-COF fixation of DES aptamerctValues), (b) Δ R before and after immobilization of TAPP-TFPy-COF for bare gold electrode AEctValue versus TAPP-TFPy-COF suspension concentration (c) preparation of aptamer sensors based on DES aptamer solutions of different concentrations and detection of DeltaR from DESctValue (Δ R before and after TAPP-TFPy-COF fixation of DES aptamerctValue and aptamer sensor detection of Δ R before and after DESctValues) of (d) Δ R before and after fixation of DES aptamer for TAPP-TFPy-COFctA relation diagram of the value and the concentration of DES aptamer solution, (e) an EIS Nquist curve diagram obtained by detecting an aptamer sensor and DES at different combination time, (f) a Delta R before and after detecting DES at different combination time by the aptamer sensor and DESctA value diagram;
FIG. 7: (a) an EIS Nquist curve diagram obtained by detecting DES solutions with different concentrations by the aptamer sensor prepared in example 2, (b) Δ R before and after detecting DES solutions with different concentrations by the aptamer sensor prepared in example 2ctValue diagram (inset: Δ R)ctAs a linear fit plot of DES concentration log functions), (c) as a plot of the results of the selectivity test for the aptamer sensor, (d) as a plot of the results of the reproducibility test for the aptamer sensor, (e) as a plot of the results of the stability test for the aptamer sensor, and (f) as a plot of the results of the reproducibility test for the aptamer sensor.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples.
Materials used in the examples of the present invention: DES aptamers were provided by Shanghai Biotechnology, Inc., with sequence 5'-ggC gAT ggg gTA ggg ggT gTg gAg ggg CCg gAC ggA ggg g-3'; 5,10,15, 20-tetrakis (4-aminophenyl) porphyrin (TAPP) and 1,3,6, 8-tetrakis (4-formylphenyl) pyrene (TFPy) were provided by Takara chemical technology, Inc. (Shanghai, China); n, N-dimethylacetamide and o-dichlorobenzene are provided by shanghai alatin biochemical technology ltd (shanghai, china); acetone and Tetrahydrofuran (THF) were supplied by the chemical reagent of the nhua chemical, loyang, of the south of china; glacial acetic acid (acetic acid) was provided by Tianjin Fuyu Fine chemical Co., Ltd. (Tianjin, China); all chemical reagents were analytical reagent grade and 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 method of the phosphate buffer solution used in the examples and experimental examples of the present invention is as follows: mixing 0.242g KH2PO4、1.445g Na2HPO4·12H2O, 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 obtain a 10mmol/L PBS solution.
The preparation method of the DES aptamer solution used in the embodiment and the experimental example of the invention is as follows: phosphate buffer solution (PBS,0.1mol/L, pH 7.4) was added to the DES aptamer stock solution to prepare solutions of DES aptamers at concentrations of 1, 5,10, 50, 100, and 200 nmol/L.
The preparation method of the DES solution used in the experimental examples of the present invention is as follows: phosphate buffer solution (PBS,0.1mol/L, pH 7.4) was added to the DES powder, and after sonication, a DES solution with a concentration of 1mg/mL was obtained, which was then diluted with phosphate buffer solution (PBS,0.1mol/L, pH 7.4) to obtain solutions with DES 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 examples of the present invention were as follows: adding different organic pollutants (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol, and oxytetracycline) into phosphate buffer solution (PBS,0.1mol/L, pH 7.4) respectively, and making into preparationEnrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and oxytetracycline test solution with the concentration of 1000 pg/mL; adding CrCl3、CuCl2And AgNO3The resulting solutions were added to phosphate buffer solutions (PBS,0.1mol/L, pH 7.4) to prepare heavy metal ions (Cr) each having a concentration of 1000pg/mL3+、Cu2+Or Ag+) A test solution; DES, organic pollutants (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and oxytetracycline) and CrCl3、CuCl2And AgNO3Adding into phosphate buffer solution (PBS,0.1mol/L, pH 7.4), and making into DES with concentration of 10pg/mL, organic pollutants (enrofloxacin, salbutamol, zearalenone, aflatoxin, deoxynivalenol and oxytetracycline), and heavy metal ions (Cr, N3+、Cu2+Or Ag+) All concentrations of (1) are 1000pg/mL of the mixture test solution.
The electrolyte solutions used in the examples and experimental examples of the present invention were prepared by mixing 1.65g K3Fe(CN)6、2.11g K4Fe(CN)6·3H2O and 7.5g KCl were dissolved in 1.0L phosphate buffer (PBS,0.1mol/L, pH 7.4).
First, a specific embodiment of the aptamer sensor of the invention is as follows:
example 1
The aptamer sensor comprises a gold electrode substrate and a covalent organic framework material modified on the surface of the gold electrode substrate, 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 (0.1mmol) of 5,10,15, 20-tetrakis (4-aminophenyl) porphyrin (TAPP) and 61mg (0.1mmol) 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 is composed of N, N-dimethylacetamide and o-dichlorobenzene, the volume ratio of N, N-dimethylacetamide and o-dichlorobenzene is 3:1) was added into the Schlenk tube, and the mixture was subjected to ultrasonic treatment for 30min to obtain a green solution,to the resulting green solution was added 0.2mL of catalytic acetic acid in N2Under protection, Schlenk tubes were snap frozen in 77K liquid nitrogen, then degassed and sealed by three freeze-pump-thaw cycles. And (2) heating the Schlenk tube to 120 ℃ after the temperature is raised to room temperature, standing for 7 days, filtering a system after the reaction is finished, wherein the solid obtained by filtering is a green-brown precipitate, washing the solid obtained by filtering for 3 times by using tetrahydrofuran, then washing for 3 times by using acetone, and finally drying the washed solid in vacuum at 100 ℃ for 18 hours to obtain a covalent organic framework material which is marked as TAPP-TFPy-COF.
Secondly, the specific embodiment of the preparation method of the aptamer sensor of the invention is as follows:
example 2
The method for producing the aptamer sensor of the present embodiment is the method for producing the aptamer sensor of embodiment 1, and includes the steps of:
(1) the working electrode is a bare gold electrode with the diameter of 3.0mm, which is produced by Gaoss Union instruments, and is polished by alumina slurry with the particle size of 0.3 mu m and 0.05 mu m in sequence, the polished bare gold electrode is rinsed by ultrapure water for 2min, the bare gold electrode rinsed by the ultrapure water is rinsed by piranha solution and ethanol in sequence, the rinsing time of the piranha solution and the ethanol is 15min, the bare gold electrode is cleaned by deionized water, the cleaned bare gold electrode is dried in a nitrogen environment, the dried gold electrode is subjected to electrochemical activation after passing through-0.2V-1.6V potential circulation in 0.5mol/L sulfuric acid solution, and then is rinsed by deionized water and dried under nitrogen to obtain a pretreated bare gold electrode, and the electrode is marked as AE; wherein the piranha solution consists of concentrated sulfuric acid and hydrogen peroxide in a volume ratio of 7:3, 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 backbone material (TAPP-TFPy-COF) prepared in example 1 was dispersed in deionized water to give a suspension of TAPP-TFPy-COF at a concentration of 1mg/mL (1 mg TAPP-TFPy-COF per 1mL deionized water). Then, 5 mu L of TAPP-TFPy-COF suspension is dripped on the surface of the pretreated bare gold electrode, and the gold electrode with the TAPP-TFPy-COF fixed on the surface is obtained after drying for 6h at room temperatureThe electrode is marked as TAPP-TFPy-COF/AE, and the area of the covalent organic framework material coated on the gold electrode is 0.19cm2The coating amount of the TAPP-TFPy-COF material on the gold electrode is 26.3 mu g/cm2. Then, the TAPP-TFPy-COF/AE is incubated for 1h in a DES aptamer solution (the temperature is 4 ℃) of 100nmol/L (the incubation is to contact a gold electrode fixed with the TAPP-TFPy-COF with the aptamer solution, so that the TAPP-TFPy-COF adsorbs the immobilized aptamer and reaches an adsorption equilibrium state), and the gold electrode fixed with the DES aptamer on the surface, namely an aptamer sensor, is obtained and is marked as Apt/TAPP-TFPy-COF/AE.
Experimental example 1 structural characterization
1. Infrared spectroscopy
The structures of TAPP, TFPy and TAPP-TFPy-COF were characterized by Fourier transform Infrared Spectroscopy (FT-IR) using a Bruker TENSOR27 spectrometer (Germany) (32 scans with a resolution of 4 cm)-1) The results are shown in FIG. 1 a. The result shows that 3060cm in the infrared spectrogram of TFPy-1And 1218cm-1The absorption band at (a) is respectively attributed to stretching vibration and bending vibration of the C-H bond in the benzene ring; the TFPy infrared spectrum is positioned at 1687cm-1The nearby peaks correspond to the stretching vibration of C ═ O in the aldehyde group; 1600cm in infrared spectrogram of TAPP-TFPy-COF-1The bands observed here are derived from the stretching vibration of C ═ N, confirming the presence of imine bonds, which indicates the formation of imine bonds by 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 Ka radiation (λ ═ 0.15406nm) and the results are shown in FIG. 1 b. As can be seen from FIG. 1b, diffraction peaks at 2 θ of 9.17 °, 18.12 °, 20.08 °, 23.27 ° and 27.28 ° can be observed in the XRD pattern of TAPP-TFPy-COF, indicating that TAPP-TFPy-COF has a stacked structure and semi-crystalline properties.
3. Nuclear magnetic map
By passing13The structure of TAPP-TFPy-COF prepared in example 1 was analyzed by C CP-MAS NMR as shown in FIG. 1C. As can be seen from FIG. 1c, the characteristic peaks around 131 and 119ppm originate from the TAPP and TFPy building blocks, 149ppmThe nuclear magnetic resonance signal is a characteristic peak of-C ═ N, confirming the presence of imine bonds in the main chain.
X-ray photoelectron spectroscopy (XPS)
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 Al K α x-ray source (1486.71e K α v photons), and the results are shown in fig. 2.
In the C1s XPS spectrum of TAPP-TFPy-COF, 6 peaks with Binding Energies (BEs) of 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. The existence of C ═ C, graphite C and pi-pi bonds proves that the graphene-like structure of TAPP-TFPy-COF can greatly improve the electrochemical activity and improve the binding affinity to a nucleic acid body chain 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 a binding energy of 401.6eV corresponds to graphite type N. These N-containing groups not only facilitate immobilization of DES aptamers on TAPP-TFPy-COF networks, but also facilitate electron transfer.
In the C1s XPS spectrum of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer immobilization, four peaks with binding energies of 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 292.7eV and 295.5eV of binding energy are derived from the residual K in PBS solution+。
Two peaks corresponding to pyrrole N and graphite type N appear in the N1s XPS spectrum of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer is immobilized. In P2P XPS spectra of TAPP-TFPy-COF (Apt/TAPP-TFPy-COF/AE) after DES aptamer immobilization, a large number of P2P signals were observed at binding energies of 132.9eV and 133.9eV, corresponding to P2P in the phosphate group on the DES aptamer chain3/2And P2P1/2. These results indicate that DES aptamers were successfully immobilized onto the network of TAPP-TFPy-COF.
Experimental example 2 morphology characterization
The synthesized sample was subjected to surface topography analysis using JSM-6490LV field emission scanning electron microscope (FE-SEM) and JEOL JEM-2100 high resolution transmission electron microscope (HR-TEM), and the results are shown in FIG. 3. The result shows that the particle diameter of the TAPP-TFPy-COF is about 100nm, and the TAPP-TFPy-COF has a multilayer sheet structure formed by aggregation through pi-pi stacking and hydrogen bond interaction.
Experimental example 3 sensory Properties
The sensing performance of the aptamer sensor prepared in example 2 was evaluated by an electrochemical technique, and the electrochemical measurement method was as follows: electrochemical Impedance Spectroscopy (EIS) analysis was performed 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, and a gold electrode as a working electrode, by means of a conventional three-electrode cell measurement system, and an EIS curve (EIS parameter: potential of 0.21V) was recorded with a frequency range of 0.01Hz to 100kHz and an amplitude of 5 mV. EIS spectra were analyzed using the Zview2 software from Scribner Associates Incorporated, which uses a non-linear least squares fit to determine the element parameters in the equivalent circuit. The equivalent circuit is composed of solution resistance (R)s) Charge transfer resistance (R)ct) Phased Element (CPE) and Warburg impedance (Wo). Each test was repeated at least three times.
The aptamer sensor prepared in example 2 was tested for its preparation and for blocking of BSA when DES (DES solution concentration of 10pg/mL, and binding time of the aptamer sensor to the DES solution of 60min) was detected by EIS technique. In order to eliminate non-specific adsorption, BSA was adsorbed on the aptamer sensor (Apt/TAPP-TFPy-COF/AE) prepared in example 2, labeled as BSA/Apt/TAPP-TFPy-COF/AE, and then DES was detected, and the EIS Nquist curve and C-V curve obtained by the construction of the aptamer sensor and the detection of DES were shown in FIG. 4. The result shows that the R of Apt/TAPP-TFPy-COF/AE after BSA adsorptionctValue 872. omega. with Apt/TAPP-TFPy-COF/AE RctThe values are close, indicating that no large amount of BSA is 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 and the like can be avoided. These results demonstrate that the aptamer sensor prepared in example 2 does not require a resistorIn the case of a breaker, specific binding to DES is still possible.
The preparation process of the aptamer sensor prepared in example 2 and the sensing performance when DES (the concentration of the DES solution is 10pg/mL, and the binding time of the aptamer sensor and the DES solution is 60min) were investigated by EIS and CV techniques, and the results are shown in fig. 5. Wherein, FIG. 5a is a schematic diagram of an EIS Nquist curve obtained from the construction and detection DES process of the aptamer sensor, and the semi-circle diameter in the Nyquist diagram corresponds to the charge transfer resistance (R)ct) Which reflects the electron transfer kinetics of the electrode surface redox probe, R can be obtained by fitting an EIS Nyquist plot using an equivalent circuitctFigure 5b is a schematic diagram of the C-V curve obtained from the construction of the aptamer sensor and the process of detecting DES.
The results show that the bare gold electrode AE shows very small RctThe value, 84.8 Ω, shows excellent electrochemical activity of the bare gold electrode AE. When the bare gold electrode AE is fixed on TAPP-TFPy-COF, R of TAPP-TFPy-COF/AEctThe value of 493.4 Ω, probably because TAPP-TFPy-COF has a poor electrochemical conductivity compared to AE, thus hindering electron transfer at the electrode/electrolyte interface. These results show that TAPP-TFPy-COF prepared in example 1 has good 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 a biosensor. When the DES aptamer is fixed by the TAPP-TFPy-COF, the R of the obtained Apt/TAPP-TFPy-COF/AEctThe value is further increased to 851.4 omega, RctThe significant increase in value is attributed to the adsorbed aptamer. In aqueous solution, DES aptamer forms negatively charged phosphate groups with [ Fe (CN)6]3-/4-Mutual repulsion exists between the two, and then the transfer of electrons from an electrolyte solution to an Apt/TAPP-TFPy-COF/AE electrode is blocked, so that the R is increasedctThe value is obtained. R of electrode (DES/Apt/TAPP-TFPy-COF/AE) when detecting DES using aptamer sensorctThe value increases continuously to 1.44k omega. This is due to the specific binding interaction between DES and DES aptamer chains, which can form G-quadruplexes, and the insulating complex formed by the binding of the aptamer and DES further prevents electron transfer.
With the loading of TAPP-TFPy-COF, DES aptamer and DES on the gold electrode, compared with AE, the peak current density of TAPP-TFPy-COF/AE, Apt/TAPP-TFPy-COF/AE and DES/Apt/TAPP-TFPy-COF/AE is continuously reduced, and the peak potential difference is continuously increased. In addition, because of the high sensitivity of the EIS detection method, the current density change value obtained by the CV technology detection is far smaller than the R obtained by the EIS measurementctThe value of the value change. In addition, due to the large pore diameter of the TAPP-TFPy-COF, a large number of aptamer chains can be fixed on the surface of the TAPP-TFPy-COF with a porous nano structure and can also penetrate into the interior of a TAPP-TFPy-COF network, so that the active sites of the TAPP-TFPy-COF can be completely 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 DES detection, the conditions for preparing the aptamer sensor and detecting the DES are optimized. The experimental conditions were as follows: the concentrations of TAPP-TFPy-COF suspensions were 0.1, 0.2, 0.5, 0.8, 1 and 1.5mg/mL, the concentrations of DES aptamers were 1, 5,10, 50, 100 and 200nmol/L, the binding times of DES solutions to the aptamer sensors were 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110min, and the concentrations of DES solutions were 10 pg/mL.
Firstly preparing aptamer sensors by using TAPP-TFPy-COF suspensions with different concentrations, detecting DES (the concentration of DES aptamer solution used in the preparation is 100nmol/L, the combination time of the aptamer sensors and DES solution is 60min) by using the prepared aptamer sensors, and detecting the obtained delta RctThe values are shown in FIGS. 6a and 6b, and the results show that the Δ R of TAPP-TFPy-COF/AEctValue (R)ctTAPP-TFPy-COF/AE-RctAE) And Δ R of Apt/TAPP-TFPy-COF/AEctValue (R)ctApt/TAPP-TFPy-COF/AE-RctTAPP-TFPy-COF/AE) Increasing with the increase of the concentration of the TAPP-TFPy-COF suspension, when the concentration of the TAPP-TFPy-COF suspension is more than 1mg/mL, the TAPP-TFPDelta R of y-COF/AEctValues and Δ R of Apt/TAPP-TFPy-COF/AEctThe values all tend to be stable. Therefore, the optimal concentration of TAPP-TFPy-COF suspension is 1 mg/mL.
Then, an aptamer sensor prepared by incubation of DES aptamer solutions with different concentrations is used for detecting DES (the concentration of TAPP-TFPy-COF suspension used in the preparation is 1mg/mL, and the binding time of the aptamer sensor and the DES solution in detection is 60min), and the obtained delta R is detectedctThe values are shown in FIGS. 6c and 6d, and the results indicate that the Δ R of Apt/TAPP-TFPy-COF/AEctValue (R)ctApt/TAPP-TFPy-COF/AE-RctTAPP-TFPy-COF/AE) And Delta R of DES/Apt/TAPP-TFPy-COF/AEctValue (R)ctDES/Apt/TAPP-TFPy-COF/AE-RctApt/TAPP-TFPy-COF/AE) Both increase with increasing DES aptamer concentrations in the range of 1-100 nmol/L. When the DES aptamer concentration is more than 100nmol/L, the delta R of Apt/TAPP-TFPy-COF/AEctValues and Δ R of DES/Apt/TAPP-TFPy-COF/AEctThe values are all in equilibrium, indicating that the binding interaction between the aptamer chain and the DES is saturated. Therefore, the optimal concentration of the DES aptamer solution is 100 nmol/L.
Finally, the EIS Nquist curve and the delta R obtained by testing the aptamer sensor and DES solution (the concentration of TAPP-TFPy-COF suspension used in the preparation is 1mg/mL, and the concentration of DES aptamer is 100nmol/L) under different binding timectThe results, shown in FIGS. 6e and 6f, indicate Δ R for DES/Apt/TAPP-TFPy-COF/AEctValue (R)ctDES/Apt/TAPP-TFPy-COF/AE-RctApt/TAPP-TFPy-COF/AE) Increasing with longer binding times. When the binding time is more than 60min, the delta R of DES/Apt/TAPP-TFPy-COF/AEctThe values reached equilibrium, indicating that the binding between DES and aptamer was saturated, extended binding time, Δ RctThe change of the value is small, when the combination time is more than 80min, the delta R of DES/Apt/TAPP-TFPy-COF/AEctThe values dropped slightly, probably due to DES coming off the electrodes, so the optimal binding time of the aptamer sensor to DES was 60 min.
Experimental example 5 sensitivity
Under optimal detection conditions, using the prepared aptamerThe sensors detect DES solutions at different concentrations (0.001, 0.01, 0.1, 1, 10, 100 and 1000pg/mL), with the semi-circle radius in the nyquist plot being positively correlated with the concentration of DES, which can be explained by the G-quadruplexes formed during the detection process. The DES detection times of the same concentration are 3 times, and an EIS Nquist curve and R obtained by detectionctChange value of (Δ R)ct) The curves are shown in fig. 7a and 7 b. The results show that R before and after DES is detected in the range of DES concentration from 1fg/mL to 1ng/mLctChange value of (Δ R)ct) Logarithm of concentration to DES (LogCon)DES) And the slope is expressed by m in linear correlation. According to the IUPAC method, in the range of DES concentration from 1fg/mL to 1ng/mL, the calculated LOD is 0.37fg/mL, the formula for LOD is: LOD is 3Sb/m, where Sb represents the standard deviation and m is Δ RctSlope of a 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 detecting DES constructed using electrocatalytic and photoelectrochemical methods, and table 1 shows the sensitivity of different detection methods to detect DES.
According to the related reports, DES can be electrochemically oxidized, and thus can be measured by electrochemical techniques. However, these sensors have low detection sensitivity to DES due to the slow electron transfer speed of DES. In contrast, the aptamer sensor prepared in example 2 overcomes the defects of the traditional electrochemical sensor for detecting DES, and has the advantages of extremely low LOD, high response speed and wide range of linear determination of DES. The excellent sensing performance of the aptamer sensor prepared in example 2 can be attributed to the following factors: (i) the synthesized TAPP-TFPy-COF can anchor a large amount of aptamers through complex interaction (pi-pi accumulation, electrostatic interaction, hydrogen bond or van der Waals force), can be used as a sensitive biological platform, and can enhance electrochemical response when a target is detected; (ii) the TAPP-TFPy-COF has a porous nano structure and a larger pore diameter, so that the aptamer is promoted to be combined on the surface and inside of the TAPP-TFPy-COF material, and the detection sensitivity is improved; (iii) the large amount of aptamer occupied on the TAPP-TFPy-COF can avoid non-specific adsorption between DES and TAPP-TFPy-COF matrix. Therefore, TAPP-TFPy-COF can be used as a sensitive platform for anchoring a large number of DES aptamer chains, and further shows excellent detection performance on DES.
TABLE 1 sensitivity of different detection methods for detecting DES
Figure BDA0003411065700000121
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document 1: r. r.Zhang, X.j.Li, A. -l.Sun, S. -q.Song, and X. -z.Shi, "A high selectivity fluorescence nano sensor based on the dual-function modulated layers coated with quantum dots for the sensitive detection of diethyl tilben/cyclic in fish and sea," Food Control, vol.132, p.108438, 2022/02/01/2022.
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Document 3: X.Dong, G.ZHao, L.Liu, X.Li, Q.Wei, and W.Cao, "ultrasensive comparative method-based electrocheminence immunoassay for diethyl tiltest detection based 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。
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EXAMPLE 6 Selectivity
By detecting other organic pollutants possibly coexisting with DES in the wastewater, such as Enrofloxacin (ENR), Salbutamol (SAL), Zearalenone (ZEA), Aflatoxin (AFT), Deoxynivalenol (DON), Oxytetracycline (OTC) and heavy metal ions (Cr)3+、Cu2+Or Ag+) The selectivity of developing aptamer sensors was investigated. Under the condition that the detection time is 60min, detecting a test solution with the concentration of organic pollutants (ENR, SAL, ZEA, AFT, DON or OTC) being 1000pg/mL and heavy metal ions (Cr3+、Cu2+Or Ag+) The concentration of (A) is 1000pg/mL, the concentration of DES is 10pg/mL, or both the concentration of DES is 10pg/mL and the concentration of heavy metal ions (Cr) is 1000pg/mL3+、Cu2+Or Ag+) And Δ R obtained from a mixed test solution of organic contaminants (ENR, SAL, ZEA, AFT, DON and OTC) at a concentration of 1000pg/mLctThe values are shown in figure 7 c. The result shows that the EIS response of the aptamer sensor prepared in example 2 is more obvious when detecting DES, and the EIS response of the aptamer sensor prepared in example 2 when detecting other organic pollutants or heavy metal ions is lower and can be ignored. In addition, when a mixed test solution consisting of DES, organic pollutants and heavy metal ions was tested using the aptamer sensor prepared in example 2, the EIS response was only 4.9% of that when DES was tested alone, and these results confirmed that the prepared aptamer sensor had good selectivity in a complex environment.
Experimental example 7 reproducibility
The 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 a DES solution having a concentration of 10pg/mL for 60min, and the result is shown in fig. 7 d. The results showed that the RSD of 5 prepared aptamer sensors for DES detection was small, only 3.25%, indicating that the aptamer sensor prepared in example 2 has good reproducibility.
EXAMPLE 8 stability
By using the implementationThe sensor prepared in example 2 evaluated the stability of the sensor by measuring the electrochemical response of a DES solution at a concentration of 10pg/mL once a day for 15 consecutive days. The specific operation process is as follows: during the first day of test, the DES is detected by using the sensor under the condition that the detection time is 60min, and the R before and after the DES is fixed by the sensor is obtainedctValue of Δ R is calculatedctThe electrodes were then placed in phosphate buffered saline (PBS, 0.01mol/L, pH 7.4) and refrigerated in a refrigerator at 4 ℃; the next day of testing, the electrodes were taken out directly, measurements were made at room temperature and R was recordedctValue of Δ R obtained by calculationctValue (R measured after one day of sensor fixed with DESctValue and R before fixed DES of sensor during first day testctDifference in value), repeating the operation until day 15, and finally obtaining the Delta R of the DES detected for 15 daysctThe results are shown in FIG. 7 e. The results show that the Δ R obtained by the testctThe value is kept stable, and the RSD is 2.75%, which shows that the prepared aptamer sensor has stronger fixing effect on DES and has good stability when used for DES detection.
EXAMPLE 9 renewability
The reproducibility of the aptamer sensor was evaluated by measuring the electrochemical response obtained by DES solution at a concentration of 10pg/mL using the regenerated aptamer sensor. The regeneration treatment process is as follows: firstly, soaking the aptamer sensor subjected to DES detection for 2-3min by using a NaOH solution with the concentration of 0.05mol/L, and thoroughly washing the aptamer sensor soaked by the NaOH solution by using water to obtain a regenerated aptamer sensor. After DES was detected using the regenerated aptamer sensor, the above regeneration process was repeated, and then DES was detected again, so that the regeneration process was performed 5 times in total, and the EIS response obtained by detecting DES using the original aptamer sensor and the regenerated aptamer sensor was recorded, with the results shown in fig. 7 f. The results show that after 5 times of regeneration treatment, the EIS response obtained by DES detection by the regenerated aptamer sensor is close to that obtained by DES detection by the original aptamer sensor, and prove that the aptamer sensor prepared in example 2 has good reproducibility.
Experimental example 10 practical application
To further investigate the potential of the aptamer sensor prepared in example 2 for use in the detection of DES in a variety of authentic samples (human serum, milk and frozen shrimp).
The real sample treatment process is as follows:
(1) human serum was obtained from Beijing Sorley Biotechnology, Inc. Before use, filtering with 3kDa dialysis bag to remove possible interfering compounds, standing at room temperature for 0.5h, centrifuging at 2000r/min for 10min, and storing the separated supernatant in-20 deg.C environment.
(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, finally centrifuging at the rotation speed of 5000rpm for 5min at room temperature, collecting supernatant, diluting by 50 times with 10mmol/L of PBS solution, and storing at the temperature of 4 ℃ for later use.
(4) Crushing and grinding frozen shrimps, placing the crushed and ground frozen shrimps into a 15mL centrifuge tube, then carrying out ultrasonic violent shaking for 1h, then centrifuging the shrimps for 5min at the rotating speed of 1000rpm, taking supernatant, drying the supernatant in an environment at 40 ℃, then re-dissolving the dried solid residue into 1.0mL of 50% methanol solution, and adding the obtained solution into 10mmol/L PBS solution for further use.
DES was added to each of the treated real samples (human serum, milk, frozen shrimp), followed by filtration using a 3kDa dialysis bag to remove possible interferents, resulting in a solution of 4 real samples containing DES at different concentrations (0.001, 0.01, 0.1, 1, 10, 100 and 1000pg/mL), and then the aptamer sensor prepared in example 2 was immersed in the real samples for 60min to obtain Δ R for detection of DESctThe value is then calculated from the calibration curve between the EIS response and the logarithm of the DES concentration, and the DES concentration detected is compared to the actual value. The actual amount added (the concentration of DES actually added in the real sample), the concentration of DES detected (the amount detected), the calculated recovery rate and RSD are shown in table 1. The result shows that the recovery rate of DES in human serum detected by the aptamer sensor prepared in example 2 is 96.2-112.9%, and RSD is 0.92-2.09%; EXAMPLE 2 preparation ofThe recovery rate of DES in milk detected by the prepared aptamer sensor is 92.87% -118.5%, and RSD is less than 3.11%; the aptamer sensor prepared in example 2 detects that the recovery rate of DES in frozen shrimps is 91.8% -113.4%, and the RSD is less than 2.69%. The result shows that the aptamer sensor prepared in the embodiment 2 can sensitively detect DES in different samples, and shows wide application prospect.
Table 2 test results of the aptamer sensor prepared in example 2 for detecting real samples
Figure BDA0003411065700000151
Figure BDA0003411065700000161

Claims (10)

1. An aptamer sensor is characterized by comprising an electrode substrate and a covalent organic framework material modified on the surface of the electrode substrate, 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 by reacting 5,10,15, 20-tetra (4-aminophenyl) porphyrin and 1,3,6, 8-tetra (4-formylphenyl) pyrene through Schiff base by adopting a solvothermal method.
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 according to claim 1 or2, wherein the catalyst for the schiff base reaction is acetic acid.
4. The aptamer sensor according to claim 1 or2, wherein the temperature of the solvothermal method is 120-125 ℃; the heat preservation time of the solvothermal method is 144-168 h.
5. The aptamer sensor according to claim 1 or2, wherein the solvent used for 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.
6. The method of preparing an aptamer sensor according to any of claims 1 to 5, comprising the steps of: firstly, loading a covalent organic framework material on an electrode substrate to obtain a modified electrode; the modified electrode is then incubated in a solution of an aptamer for targeted detection of diethylstilbestrol.
7. The method of claim 6, wherein the loading is by coating a suspension of the covalent organic framework material onto the electrode substrate followed by a drying process.
8. The method of preparing an aptamer sensor according to claim 7, wherein the concentration of the suspension of the covalent organic framework material is 0.8-1.5 mg/mL.
9. The method for preparing an aptamer sensor according to any one of claims 6 to 8, wherein the concentration of the aptamer solution for targeted detection of diethylstilbestrol is 100nmol/L and 200 nmol/L.
10. The method of preparing an aptamer sensor according to any one of claims 6 to 8, wherein the incubation temperature is 0 to 4 ℃; the incubation time is 60-80 min.
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