CN113484386B - Preparation method and application of metal phthalocyanine nano material, aptamer sensor and preparation method thereof - Google Patents

Preparation method and application of metal phthalocyanine nano material, aptamer sensor and preparation method thereof Download PDF

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CN113484386B
CN113484386B CN202110559968.8A CN202110559968A CN113484386B CN 113484386 B CN113484386 B CN 113484386B CN 202110559968 A CN202110559968 A CN 202110559968A CN 113484386 B CN113484386 B CN 113484386B
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metal
aptamer
salt
polycofepc
enr
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CN113484386A (en
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杜淼
王明花
户敏
朱磊
徐妙然
栗俊广
张治红
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Zhengzhou University of Light Industry
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Zhengzhou University of Light Industry
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Abstract

The invention relates to a preparation method and application of a metal phthalocyanine nano material, an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensing. The metal phthalocyanine nanometer material is prepared from pyromellitic dianhydride, urea and NH 4 Cl, ammonium molybdate, cobalt and/or ferric salt are obtained through heating reaction, and the prepared metal polymalocyanine nano material has a two-dimensional conjugated porous nano structure, rapid charge transfer capability and mixed metal valence (Fe) 2+ /Fe 3+ And/or Co 2+ /Co 3+ ) And the nitrogen-rich function improves the electrochemical performance of the material and the immobilization of the aptamer. The prepared aptamer sensor shows very high electrochemical response when used for detecting enrofloxacinAnd extremely low detection limit, high selectivity, remarkable stability and reproducibility, and has great application prospect in the aspects of environmental monitoring and food safety.

Description

Preparation method and application of metal phthalocyanine nano material, aptamer sensor and preparation method thereof
Technical Field
The invention relates to a preparation method and application of a metal phthalocyanine nano material, an aptamer sensor and a preparation method thereof, and belongs to the technical field of electrochemical sensing.
Background
Enrofloxacin (ENR) is often used in the livestock industry as a common antibiotic for treating bacterial infections and promoting the growth of poultry, livestock or fish. However, abuse or misuse of antibiotics can result in antibiotic residues in meat, eggs, fish and milk, thereby contaminating the food or drinking water source consumed by humans and severely jeopardizing human health. Therefore, sensitive, selective detection of ENR residues in food and environmental systems is necessary. Some conventional techniques, such as capillary electrophoresis (high performance) and liquid chromatography-tandem mass spectrometry, have been used to analyze ENR content. Although the analysis methods have higher detection sensitivity and accuracy, the wide application of the techniques is greatly limited due to complex sample pretreatment, complex data statistics and difficult data analysis, and the requirements of ENR rapid detection cannot be met.
Disclosure of Invention
The invention aims to provide a preparation method of a metal phthalocyanine nanomaterial, which is used for rapidly detecting ENR.
The second object of the present invention is to provide an application of the metal phthalocyanine nanomaterial prepared by the preparation method as an electrode material for an aptamer sensor.
A third object of the present invention is to provide an aptamer sensor.
The fourth object of the invention is to provide a method for preparing an aptamer sensor.
In order to achieve the above purpose, the technical scheme of the preparation method of the metal phthalocyanine nanometer material of the invention is as follows:
a method for preparing a metal polymorpha (polyMPc) nanomaterial, comprising: pyromellitic dianhydride, urea and NH 4 Heating Cl, ammonium molybdate and metal salt to react to obtain metal polymalocyanine; the metal salt is selected from one or two of cobalt salt and ferric salt.
The metal phthalocyanine nano-sheet prepared by the invention has a two-dimensional conjugated porous nano-structure, rapid charge transfer capability and mixed metal valence state (Fe) 2+ /Fe 3+ And/or Co 2+ /Co 3+ ) And the synergistic effect (polyCoFePc) and nitrogen-rich function between cobalt and iron clusters, can improve the electrochemical performance of the material and the immobilization effect of the nucleic acid aptamer in the electrode material for the aptamer sensor, and can be used for rapidly detecting ENR.
Preferably, the temperature of the heating reaction is 200-260 ℃ and the time is 2-6 h.
More preferably, the temperature of the heating reaction is 220 ℃, the heating time is 3 hours, and the heating rate is 5 ℃ min -1 。
Preferably, the molar ratio of the metal elements in the pyromellitic dianhydride, the urea and the metal salt is 4:8-32:1-3.
Preferably, the metal salt consists of cobalt salt and iron salt; the molar ratio of cobalt element in the cobalt salt to iron element in the ferric salt is 1-2:1.
Preferably, the molar ratio of pyromellitic dianhydride to ammonium molybdate is 20-40:1.
The cobalt salt is CoCl 2 ·6H 2 O, the ferric salt is FeCl 3 ·6H 2 O。
The technical scheme of the application of the metal phthalocyanine nano material as the electrode material for the aptamer sensor is as follows:
the application of the prepared metal phthalocyanine nanometer material as an electrode material for an aptamer sensor.
Among the numerous detection methods, electrochemical aptamer sensors are widely used for environmental monitoring and food safety detection due to their non-labeling, low cost and other characteristics. Phthalocyanines (Pc) are aromatic heterocycles consisting of four nitrogen-bridged isoindoles and can therefore be used as building blocks for different metal-organic frameworks (MOFs), covalent Organic Frameworks (COFs) and polymers. The two-dimensional conjugated metal polymorphic (polyMPc) nanomaterial takes benzene rings as connecting units, and has wide application prospects in the fields of electromagnetism, catalysis and the like. The two-dimensional conjugated polyPc is used for constructing an electrochemical biosensor because of strong bonding effect between local d-electrons in Transition Metal (TM) atoms and delocalized p-electrons on a polyPc framework.
The technical scheme of the aptamer sensor is as follows:
an aptamer sensor comprises an electrode matrix, a metal phthalocyanine nanomaterial, and a nucleic acid aptamer, wherein the metal phthalocyanine nanomaterial is modified on the surface of the electrode matrix and is obtained by the preparation method, and the nucleic acid aptamer is anchored on the metal phthalocyanine nanomaterial and used for targeted detection of enrofloxacin.
In order to explore an ultrasensitive layer for detecting the ENR by an electrochemical technology, the invention takes a metal cobalt and/or iron polymalocyanine network as a bracket for anchoring an ENR targeting nucleic acid aptamer chain, thereby realizing the detection of the ENR for the first time.
Compared with the existing ENR detection technology, the aptamer sensor based on the polyCoFePc can be directly used as a sensor platform, has very high electrochemical response and very low detection limit when being used for detecting the ENR, and has high selectivity, remarkable stability and reproducibility.
The preparation method of the aptamer sensor comprises the following steps:
a method of making an aptamer sensor, comprising: pretreating an electrode to obtain an electrode matrix; modifying the suspension of the metal phthalocyanine nano material obtained by the preparation method on an electrode matrix to obtain a modified electrode; inoculating the modified electrode in a solution of a nucleic acid aptamer for targeted detection of enrofloxacin.
The preparation method of the aptamer sensor has the advantages of simple and efficient technical scheme and good stability and reproducibility.
Preferably, the concentration of the suspension of the metal phthalocyanine nano-material is 0.1-2 mg.mL -1 。
Preferably, the concentration of the aptamer solution for targeted detection of enrofloxacin is between 10 and 500nM.
Preferably, the electrode is a bare gold electrode.
Preferably, the electrode pretreatment process is as follows: the electrodes are respectively polished, washed and dried, electrochemically activated and washed and dried.
Preferably, the suspension of the metal phthalocyanine nanometer material is modified to an electrode by the following steps: the metal phthalocyanine nanomaterial is dispersed in deionized water to give a uniform suspension, which is coated onto the pretreated bare electrode surface and then dried overnight at room temperature.
Preferably, the process of inoculating the modified electrode in a solution of a nucleic acid aptamer for targeted detection of enrofloxacin is: immersing the modified electrode into a nucleic acid aptamer solution for targeted detection of enrofloxacin to obtain the aptamer sensor.
Drawings
Fig. 1: (a) is an X-ray diffraction pattern, (b) is a fourier transform infrared spectrogram, (c) is a raman spectrogram, (d) is an X-ray photoelectron spectrogram, wherein (i) is a polycompc, (ii) is a polycompc, and (iii) is a polycompc;
Fig. 2: (a) a high-resolution XPS spectrum curve of Co 2p of polycofxpc, (b) a high-resolution XPS spectrum curve of Fe 2p of polycofxpc, (C) a high-resolution XPS spectrum curve of C1s of polycofxpc, (d) a high-resolution XPS spectrum curve of N1s of polycofxpc (M stands for metallic cobalt, iron);
fig. 3: (a) a high-resolution XPS spectrum curve of Co 2p of polycompc, (b) a high-resolution XPS spectrum curve of Fe 2p of polyFePc, (C) a high-resolution XPS spectrum curve of C1s of polycompc, (d) a high-resolution XPS spectrum curve of N1s of polycompc, (e) a high-resolution XPS spectrum curve of C1s of polyFePc, and (f) a high-resolution XPS spectrum curve of N1s of polyFePc;
fig. 4: (a) a high-resolution XPS spectrum curve of C1s of Apt/PolyCoFePc, (b) a high-resolution XPS spectrum curve of N1s of Apt/PolyCoFePc (M stands for metal cobalt and iron), (C) a high-resolution XPS spectrum curve of Co 2P of Apt/PolyCoFePc, (d) a high-resolution XPS spectrum curve of Fe 2P of Apt/PolyCoFePc, and (e) a high-resolution XPS spectrum curve of P2P of Apt/PolyCoFePc;
fig. 5: (a) is a low-magnification SEM image of the polyCoFePc, (b) is a high-magnification SEM image of the polyCoFePc, (c) is a low-magnification TEM image of the polyCoFePc, (d) is a high-magnification TEM image of the polyCoFePc, (e) is a high-resolution TEM image of the polyCoFePc (the annular selected region is an electron diffraction (SAED) image), and (f) is a HAADF-STEM image of the polyCoFePc and a corresponding element map image;
Fig. 6: (a) is a low-magnification SEM image of the polyCoPc, (b) is a high-magnification SEM image of the polyCoPc, (c) is a low-magnification TEM image of the polyCoPc, (d) is a high-magnification TEM image of the polyCoPc, (e) is a high-resolution TEM image of the polyCoPc, (f) is a HAADF-STEM image of the polyCoPc and a corresponding element map image;
fig. 7: (a) is a low-power SEM image of polyFePc, (b) is a high-power SEM image of polyFePc, (c) is a low-power TEM image of polyFePc, (d) is a high-power TEM image of polyFePc, (e) is a high-resolution TEM image of polyFePc, (f) is a HAADF-STEM image of polyFePc and a corresponding element map image;
fig. 8: (a) Is a nitrogen adsorption-desorption isotherm, (b) is a pore size distribution curve, wherein (i) is polycompc, (ii) is polyFePc, and (iii) is polycofxpc;
fig. 9: (a) EIS Nyquist plot for an aptamer sensor based on polyCoPc, (b) EIS Nyquist plot for an aptamer sensor based on polyFePc, (c) EIS Nyquist plot for an aptamer sensor based on polyCoFePc, (d) polyCoPc, polyFePc and ENR adaptive sensor manufacturing process of polyCoFePc at each stage R ct A change in value;
fig. 10: (a) For the influence of the concentration of the polyCoFePc suspension on the aptamer sensor manufacture and ENR detection, Δr obtained from each step was used ct The value represents (b) the effect of aptamer concentration on the ENR assay, using ΔR obtained from the ENR assay ct The values represent (c) for the effect of different binding times on the detection of ENR, the detection of ENR using an aptamer sensor based on polyCoFePc (0.1 fg. ML -1 ) ΔR of (2) ct A value representation;
fig. 11: (a) Detection of different concentrations (0, 0.1 fg. ML for aptamer sensor -1 、1fg·mL -1 、10fg·mL -1 、100fg·mL -1 、1pg·mL -1 、10pg·mL -1 And 100 pg.mL -1 ) EIS Nyquist plot of ENR of (b) is ΔR ct And the concentration of ENR (inset: deltaR) ct A linear fit as a logarithmic function of ENR concentration, where the error line is the standard deviation of n=3), (c) detection of various interferents for a polyCoFePC-based aptamer sensor (STR, OFLX, DOX, OTC, LMFH, CPFX, PNC, ag + 、Pb 2+ 、NO 3- And Hg of 2+ )(10fg·mL -1 ) And ENR (10 fg. ML) -1 ) Is a mixture of (C) and ENR (0.1 fg. ML) -1 ) ΔR of (2) ct The value (d) is the value of the aptamer sensor (0.1 fg. ML -1 ) (n=3) stability for 15 days, (e) is in the presence of 5mM [ Fe (CN) 6 ] 3-/4- In 0.1M PBS (ph=7.4), 0.14M NaCl and 0.1M KCl, ENR (0.1 fg·ml) was detected using five preparation identical polyCoFePc-based aptamer sensors -1 ) ΔR of (2) ct Values.
Detailed Description
Embodiments of the present invention will be further described with reference to the accompanying drawings.
The materials used in the invention are as follows: coCl 2 ·6H 2 O and FeCl 3 ·6H 2 O is supplied by Comamic chemical reagent Co., ltd, pyromellitic dianhydride, urea, ammonium chloride, ammonium molybdate are purchased from Shanghai Ala Ding Huaxue reagent Co., ltd, streptomycin (STR), ofloxacin (OFLX), doxycycline (DOX), oxytetracycline (OTC), lomefloxacin hydrochloride(LMFH), ciprofloxacin (CPFX), penicillin (PNC), enrofloxacin (ENR) were purchased from Solarbio life sciences Co. All solutions were prepared with ultrapure water (. Gtoreq.18.2 M.OMEGA.cm). The Enrofloxacin (ENR) targeting aptamer is synthesized by Shanghai Biotechnology Inc. of China and has a sequence of 5- 'CCC ATC AGG GGG CTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-3'.
Preparation of phosphate buffer solution: will be 0.242g KH 2 PO 4 ,1.445g Na 2 HPO 4 ·12H 2 Phosphate buffer (PBS, 0.1m, ph=7.4) was prepared by dissolving 0.200g of potassium chloride and 8.003g of sodium chloride in 1.0L of ultrapure water.
Preparation of electrolyte solution: will be 1.6g K 3 Fe(CN) 6 ，2.1g K 4 Fe(CN) 6 And 7.5g of KCl was added to the phosphate buffer solution PBS prepared above to obtain an electrolyte solution.
Preparation of interfering substances and aptamer solutions: the interference substance and aptamer solutions with different concentrations are prepared by PBS and stored at 4 ℃ for standby.
1. The specific examples of the preparation method of the metal phthalocyanine nanometer material of the invention are as follows:
example 1
The preparation method of the metal phthalocyanine nanometer material comprises the following specific steps:
1g of pyromellitic dianhydride, 2.1g of urea and 0.5g of NH 4 Cl, 25mg ammonium molybdate ((NH) 4 )6Mo 7 O 24 ·4H 2 O)、0.370g CoCl 2 ·6H 2 O and 0.370g FeCl 3 ·6H 2 O was thoroughly mixed in a 500mL ceramic crucible. Subsequently, the ceramic crucible was placed in a muffle furnace and heated at 220℃for 3 hours at a heating rate of 5℃min -1 . After cooling to room temperature, washing with water and ethanol respectively 3 times, and vacuum drying at 60 ℃ for 8 hours, a dark blue solid product called polyCoFePc is obtained.
Example 2
The preparation method of the metal phthalocyanine nanometer material comprises the following specific steps:
the metal of this embodimentThe difference between the polymorpha nanomaterial and the preparation step of the metal polymorpha nanomaterial of example 1 is only that FeCl is not added 3 ·6H 2 O, the metal phthalocyanine nanomaterial obtained in this example is called polycompc.
Example 3
The preparation method of the metal phthalocyanine nanometer material comprises the following specific steps:
the metal polymalocyanine nanomaterial of this example differs from the metal polymalocyanine nanomaterial of example 1 in the preparation steps only in that no CoCl is added 2 ·6H 2 O, the metal phthalocyanine nanomaterial obtained in this example is called polyFePc.
2. The metal phthalocyanine nanometer material prepared by the method is applied to an electrode material for an aptamer sensor.
The metal phthalocyanine nano-material prepared in the embodiment 1-3 is used as an electrode material to be modified on an electrode matrix, and the electrode matrix is gold.
3. Specific examples of aptamer sensors of the invention are as follows:
inoculating the electrode matrix modified by the prepared suspension of the metal phthalocyanine nanometer material in a nucleic acid aptamer solution to obtain the aptamer sensor.
4. Specific examples of the preparation method of the aptamer sensor of the invention are as follows:
example 4
The preparation method of the aptamer sensor of the embodiment comprises the following specific steps:
(1) Pretreatment of gold electrodes
Bare gold electrodes of 3mm diameter were polished with 0.05 μm alumina powder and then with H in a volume ratio of 3:7, respectively 2 O 2 And H 2 SO 4 Is washed with ethanol and water for 10 minutes and dried at room temperature under nitrogen atmosphere. Then bare gold electrode is arranged at 0.5M H 2 SO 4 The solution was electrochemically activated in a cycle between-0.2 and 1.6V, then rinsed with water and dried under nitrogen.
(2) Modified electrode
Dispersing 2.0mg of the polyCoFePc powder in 2.0mL of deionized water to give a concentration of 1 mg.ml -1 Is a uniform suspension of (a). Subsequently, 10.0 μl of the polyCoFePc suspension was coated onto the pretreated bare electrode (AE) surface, followed by drying overnight at room temperature, rinsing with deionized water several times, and removing the excess polyCoFePc material to give a modified electrode, labeled polyCoFePc/AE.
(3) Aptamer sensor
The modified electrode, polyCoFePc/AE, was immersed in a nucleic acid aptamer solution (100 nM) for 1h, followed by three washes with deionized water to obtain an aptamer sensor labeled Apt/polyCoFePc/AE.
Example 5
The preparation method of the aptamer sensor of the embodiment comprises the following specific steps:
(1) Pretreatment of gold electrodes
Bare gold electrodes of 3mm diameter were polished with 0.05 μm alumina powder and then with H in a volume ratio of 3:7, respectively 2 O 2 And H 2 SO 4 Is washed with ethanol and water for 10 minutes and dried at room temperature under nitrogen atmosphere. Then bare gold electrode is arranged at 0.5M H 2 SO 4 The solution was electrochemically activated in a cycle between-0.2 and 1.6V, then rinsed with water and dried under nitrogen.
(2) Modified electrode
Dispersing 2.0mg of the polycompc powder in 2.0mL of deionized water to give a concentration of 1 mg.ml -1 Is a uniform suspension of (a). Subsequently, 10.0 μl of a polycompc suspension was applied to the surface of the pre-treated bare electrode (AE), followed by drying overnight at room temperature to give a modified electrode, labeled polycompc/AE, which was rinsed several times with deionized water to remove excess polycompc material.
(3) Aptamer sensor
The modified electrode polycompc/AE was immersed in a nucleic acid aptamer solution (100 nM) for 1h, followed by three washes with deionized water to obtain an aptamer sensor labeled Apt/polycompc/AE.
Example 6
The preparation method of the aptamer sensor of the embodiment comprises the following specific steps:
(1) Pretreatment of gold electrodes
Bare gold electrodes of 3mm diameter were polished with 0.05 μm alumina powder and then with H in a volume ratio of 3:7, respectively 2 O 2 And H 2 SO 4 Is washed with ethanol and water for 10 minutes and dried at room temperature under nitrogen atmosphere. Then bare gold electrode is arranged at 0.5M H 2 SO 4 The solution was electrochemically activated in a cycle between-0.2 and 1.6V, then rinsed with water and dried under nitrogen.
(2) Modified electrode
Dispersing 2.0mg of polyFePc powder in 2.0mL of deionized water to obtain a concentration of 1 mg.mL -1 Is a uniform suspension of (a). Subsequently, 10.0 μl of a polyFePc suspension was applied to the surface of the pre-treated bare electrode (AE), followed by drying overnight at room temperature to give a modified electrode, labeled polyFePc/AE, which was rinsed several times with deionized water to remove excess polyFePc material.
(3) Aptamer sensor
The modified electrode polyFePc/AE was immersed in a nucleic acid aptamer solution (100 nM) for 1h, followed by three washes with deionized water to obtain an aptamer sensor labeled Apt/polyFePc/AE.
Experimental example
Experimental example 1 structural characterization
X-ray powder diffraction (PXRD)
The metal polymalocyanine nanomaterials obtained in example 1, example 2 and example 3 were characterized by X-ray powder diffraction (PXRD) and the results obtained are shown in fig. 1 a.
In fig. 1a, (i) is the X-ray powder diffraction curve of polycompc, (ii) is the X-ray powder diffraction curve of polyFePc, and (iii) is the X-ray powder diffraction curve of polycofxpc, as can be seen, the three samples have similar diffraction peaks, indicating the presence of crystal domains in the polymer, and can be described as two pi-pi stacked crystal forms of metallophthalocyanine, including a uniformly aligned alpha form and a beta staggered form.
2. Infrared spectrum
The metal phthalocyanine nanomaterial obtained in example 1, example 2 and example 3 was characterized by infrared spectroscopy, and the obtained results are shown in fig. 1 b.
In FIG. 1b, (i) is an infrared spectrum curve of a polyCoPc, (ii) is an infrared spectrum curve of a polyFePc, and (iii) is an infrared spectrum curve of a polyCoFe Pc, and it can be seen that the infrared spectra of the three samples are consistent with the reported poly phthalocyanine nanoplatelets. 1606cm -1 ，1510cm -1 And 1332cm -1 The position is a phthalocyanine skeleton vibration absorption peak of 1107cm -1 And 1315cm -1 The strong absorption peaks at the positions are respectively a flexural vibration absorption peak of an pyrrole ring and a telescopic vibration absorption peak of C=C, and 900cm -1 Is the vibration absorption peak of the metal ligand.
3. Raman spectrum
The metal polymalocyanine nanomaterials obtained in example 1, example 2 and example 3 were characterized by raman spectroscopy, and the results obtained are shown in fig. 1 c.
In FIG. 1c, (i) is a Raman spectrum curve of polyCoPc, (ii) is a Raman spectrum curve of polyFePc, and (iii) is a Raman spectrum curve of polyCoFe Pc, as can be seen, three metal polymalocyanine nanoplatelets show similar A 1g ,B 1g and B 2g Peak shape, corresponds to the in-plane vibration of the phthalocyanine macrocycle structure.
X-ray photoelectron spectroscopy (XPS)
The metal polymalocyanine nanomaterials obtained in example 1, example 2 and example 3 were characterized by X-ray photoelectron spectroscopy (XPS) and the results obtained are shown in fig. 1 d.
In FIG. 1d, (i) is the X-ray photoelectron spectrum of polyCoPc, (ii) is the X-ray photoelectron spectrum of polyFePc, and (iii) is the X-ray photoelectron spectrum of polyCoFePc, as can be seen, the XPS measurement scans for the presence of the elements C1s, N1 s, and O1s corresponding to Co. In polyCoFePc, weak Co 2p and Fe 2p signals are present.
The composition and chemical environment of each element in the bimetal polyCoFePc nanoplatelets were further analyzed and the results are shown in fig. 2.
In fig. 2, (a) is a high-resolution XPS spectrum curve of Co 2p of polyCoFePc, (b) is a high-resolution XPS spectrum curve of Fe 2p of polyCoFePc, (C) is a high-resolution XPS spectrum curve of C1s of polyCoFePc, and (d) is a high-resolution XPS spectrum curve of N1s of polyCoFePc.
As can be seen from FIG. (a), the high resolution XPS spectrum of Co 2p is divided into two sets of peaks, with Binding Energies (BEs) of 780.8eV and 795.5eV, corresponding to Co 2p3/2 and Co 2p1/2, respectively. Among the Co 2p3/2S group peaks, peaks at 779.8eV and 781.3eV correspond to Co, respectively 3+ And Co 2+ Binding Energy (BEs); peaks with binding energies (Bes) of 783.4 and 786.3eV are assigned to respective satellite peaks. Co 2p1/2 has only one main peak with a Binding Energy (BEs) of 795.5eV, corresponding to Co 3+ 。
As can be seen from FIG. (b), the peaks at Binding Energies (BEs) 709.5, 711.8, 715.6 and 718.5eV correspond to Fe 2p3/2, wherein the first two peaks are due to Fe respectively 2+ And Fe (Fe) 3+ The latter two peaks correspond to respective satellites. Two peaks with Binding Energy (BEs) of 722.3 and 724.2eV correspond to Fe 2p1/2 2+ And Fe (Fe) 3+ The other two peaks at Binding Energies (BEs) 727.8 and 733.9eV correspond to the respective satellite peaks. Thus, the polyCoFePc nanosheet material contains Co 2+ /Co 3+ And Fe (Fe) 2+ /Fe 3+ The mixed metal valence state provides a very convenient condition for electron transfer under electrochemical conditions and aids in the anchoring of the aptamer chain between the ion and the oligonucleotide.
From graph (C), the C1s XPS spectrum curve has three main peaks: C-C/c=c (284.3 eV), C-N (285.3 eV), pi-pi * (288.5 eV), and two auxiliary peaks c=n (286.9 eV) and N-c=o (290.8 eV). Wherein a high content of C-N indicates the formation of a phthalocyanine ring, while pi-pi * Implying the presence of a conjugated ring. N-c=o is present due to oxygen contamination during XPS measurement. As can be seen from panel (d), the N1s XPS spectrum includes two parts, C-N (398.4) and M-N (399.9 eV), M representing metallic cobalt or iron.
In addition, high resolution XPS spectra of Co 2p in the polyCoPc material and Fe 2p in the polyFePc material, and C1s and N1s of each material were analyzed, as shown in FIG. 3.
In fig. 3, (a) is a high-resolution XPS spectrum curve of Co 2p of polyepc, (b) is a high-resolution XPS spectrum curve of Fe 2p of polyepc, (C) is a high-resolution XPS spectrum curve of C1s of polyepc, (d) is a high-resolution XPS spectrum curve of N1s of polyepc, (e) is a high-resolution XPS spectrum curve of C1s of polyepc, and (f) is a high-resolution XPS spectrum curve of N1s of polyepc. As can be seen from FIG. (a), similar to the polyCoFePc, the Co 2p XPS spectrum in the polyCoPc includes Co 2+ And Co 3+ And mixed valence state. As can be seen from FIG. (b), XPS spectrum of Fe 2p in polyFePc is divided into Fe 2+ And Fe (Fe) 3+ . From figures (C) to (f), it can be seen that the C1 and N1s spectra in the polycompc and polyFePc materials are similar to those of the bimetallic polycofxpc.
To examine whether the aptamer chains were firmly immobilized on the polyCoFePc nanoplates, XPS spectra were used to characterize the modified material Apt/polyCoFePc, as shown in fig. 4.
In fig. 4, (a) is a high-resolution XPS spectrum curve of Apt/PolyCoFePc for C1s, (b) is a high-resolution XPS spectrum curve of Apt/PolyCoFePc for N1s, (C) is a high-resolution XPS spectrum curve of Apt/PolyCoFePc for Co 2P, (d) is a high-resolution XPS spectrum curve of Apt/PolyCoFePc for Fe 2P, and (e) is a high-resolution XPS spectrum curve of Apt/PolyCoFePc for P2P. In the Co 2p,C 1s,and N1s XPS spectrum, there was no significant change in both compared to the polyCoFePc. The weak peak at Binding Energy (BE) 293.1eV comes from the K of the PBS buffer + . Notably, the Fe 2p signal is negligible, mainly because it is masked by the immobilized aptamer chain. Furthermore, the clear P2P XPS signal appears due to the phosphate group of the aptamer chain, which is divided into P2P 3/2 And P2P 1/2 Two parts. The above conclusion shows that the aptamer chains are immobilized on the polyCoFePc nanoplates, providing conditions for the subsequent electrochemical applications.
Experimental example 2 characterization of morphology
Sem and TEM images
The polyCoFePc material obtained in example 1 was characterized by SEM and TEM as shown in fig. 5.
In fig. 5, (a) is a low-power SEM image of polycofipc, (b) is a high-power SEM image of polycofipc, (c) is a low-power TEM image of polycofipc, (d) is a high-power TEM image of polycofipc, (e) is a high-resolution TEM image of polycofipc, and (f) is a HAADF-STEM image of polycofipc and a corresponding element map image. From fig. (a), it can be seen that the polyCoFePc consists of irregular nanoparticles, agglomerated in solid blocks. As is clear from fig. (b) to (d), the polyCoFePc phthalocyanine polymer exhibits a layered morphology, and these nanoparticles are formed by layering a plurality of nano-platelets. As can be seen from the graph (e), the lattice spacing of the polyCoFePc is about 0.34nm, corresponding to the graphitic carbon 002 lattice plane, and the annular selected region electron diffraction (SAED) in the graph indicates poor crystallinity of the polyCoFePc nanoplatelets, and the elemental mapping image indicates that the elements Co, fe, C and N are uniformly distributed throughout the material.
The polycompc material obtained in example 2 was characterized by SEM and TEM, as shown in fig. 6.
In fig. 6, (a) is a low-magnification SEM image of polycompc, (b) is a high-magnification SEM image of polycompc, (c) is a low-magnification TEM image of polycompc, (d) is a high-magnification TEM image of polycompc, (e) is a high-resolution TEM image of polycompc, and (f) is a HAADF-STEM image of polycompc and a corresponding element map image. From fig. (a) and (b), it is known that the polycompc material is composed of a large number of nanoparticles, takes an irregular shape, and has some cracks on the surface of the material. From fig. (c) and (d), it is known that the polycompc has a similar nanoplatelet structure, and a large number of nanoplatelets are assembled together. From the graph (e), the edges of the polycompc nanoplates were composed of 3 to 5 layers, and from the graph (f), the distribution of Co, C, N, and O elements was relatively uniform in the region selected by the polycompc.
The polyFePc material obtained in example 3 was characterized by SEM and TEM as shown in FIG. 7.
In fig. 7, (a) is a low-power SEM image of polyFePc, (b) is a high-power SEM image of polyFePc, (c) is a low-power TEM image of polyFePc, (d) is a high-power TEM image of polyFePc, (e) is a high-resolution TEM image of polyFePc, and (f) is a HAADF-STEM image of polyFePc and a corresponding element map image. From figures (a) to (e), it can be seen that the polyFePc material exhibits similar morphology and structure, in comparison with the polyFePc material in which the nanoplatelets are smaller and stacked together, and mainly exhibits a porous nanostructure. The Fe, C, N and O elements in the selected area of the polyFePc are distributed uniformly.
2. Adsorption isotherm and pore size distribution
The adsorption isotherms and pore size distribution of the metallophthalocyanine nanomaterials obtained in example 1, example 2 and example 3 were respectively tested, and the obtained results are shown in fig. 8. In fig. 8a, (i) is a nitrogen adsorption-desorption isotherm of polycompc, (ii) is a nitrogen adsorption-desorption isotherm of polyFePc, and (iii) is a nitrogen adsorption-desorption isotherm of polyCoFe Pc; in fig. 8b, (i) is the pore size distribution curve of polycompc, (ii) is the pore size distribution curve of polyFePc, and (iii) is the pore size distribution curve of polyCoFe Pc. The specific surface area and pore size obtained by the test are listed in table 1. As can be seen from Table 1, among the three metal polymalocyanine nanomaterials, the polyCoFePc nanoplatelets have the largest specific surface area of 162.5m 2 ·g -1 The average pore diameter is at a medium level, 17.2nm. The large specific surface area and the pore diameter can greatly improve the fixing efficiency of the aptamer, and stabilize the quadruplex formed by the aptamer and the ENR.
Table 1 polyCoPc,polyFePc BET specific surface area and pore size of the PolyCoFePc materials
Experimental example 3 electrochemical performance of aptamer sensor
1. Test conditions
Electrochemical sensing performance of polyCoPc, polyFePc and polyCoFePc was studied by Electrochemical Impedance Spectroscopy (EIS) technique for synthetic metal polymalocyanine networks with two-dimensional conjugated porous nanostructures and nitrogen-functionalized chemical components. Specifically, the bare electrode, the electrode modified by the metallophthalocyanine network, the electrode fixed by the nucleic acid aptamer chain and the prepared aptamer transfer in examples 4-6 are respectively measured by using a traditional three-electrode measuring system The sensor detects ENR (ENR concentration is 1 fg.mL -1 The binding time of the sensor and ENR was 40 minutes), and the EIS nyquist diagram was obtained. The three-electrode measurement system comprises a gold electrode with a diameter of 3mm as a working electrode, an Ag/AgCl (saturated KCl) electrode as a reference electrode, and a platinum sheet as a counter electrode. EIS Nyquist diagram containing 5mM [ Fe (CN) 6] 3-/4- Obtained in 0.1M PBS (ph=7.4), 0.14M NaCl and 0.1M KCl, at a potential of 0.21v, in a frequency range of 100khz to 0.1hz.
2. Test results
The results of electrochemical sensing properties of the synthesized polyCoPc, polyFePc and polyCoFePc are shown in fig. 9. In FIG. 9, (a) is an EIS Nyquist plot of an aptamer sensor based on polyCoPc, (b) is an EIS Nyquist plot of an aptamer sensor based on polyFePc, (c) is an EIS Nyquist plot of an aptamer sensor based on polyCoFePc, (d) is an ENR detection sensor manufacturing and ENR detection process based on polyCoPc, polyFePc and polyCoFePc at various stages R ct A change in value. R in each step of manufacturing aptamer sensor and ENR detection process based on polyCoPc, polyFePc and polyCoFePc nano material ct The values are summarized in table 2.
TABLE 2R for each step in the manufacture of aptamer sensor and the detection of ENR based on polyCoPc, polyFePc and polyCoFePc ct Value of
As can be seen from Table 2, the bare electrode has a small Rct value (30.9Ω), showing excellent electrochemical performance. However, the Rct values of the electrodes modified with polyCoPc, polyFePc and polyCoFePc increased to 1222, 351.6 and 597.5 Ω, respectively. These results indicate that the metal polymalocyanine blocks electron transfer, exhibits poor electrochemical activity and slow electron transfer ability in the bare electrode.
In three samples, the polycofxpc polymer showed a compromise Rct value compared to the polycofxpc and polycofxpc polymers. In addition, the Rct value of the polyFePc polymer is minimum,indicating that the electron transfer rate is relatively fast, mainly due to Fe 2+ /Fe 3+ And mixing ion pairs. After immobilization of the nucleic acid aptamer chains on different metal phthalocyanine polymer networks (polyMPc), the Rct value of the aptamer sensor (Apt/polyMPc/AE) prepared from the metal phthalocyanine polymer network was further significantly increased.
Since the change in Rct (Δrct=rct, after-Rct, before and after aptamer immobilization) positively correlates with the immobilization amount of aptamer chains, it was possible to explore the anchoring efficiency of the aptamer on different sensitive layers of polyCoPc, polyFePc and polyCoFePc. Obviously, the ΔRct of the polyCoFePc/AE is maximum, which is 273.8Ω, which is higher than the Apt/polyCoPc/AE (85.1Ω) and the Apt/polyFePc/AE (160.1Ω). In contrast, the polyCoFePc network shows high binding interactions to the aptamer chains, thus showing superior sensing performance for ENR. The Rct values of these electrodes increased continuously as different aptamer sensors were incubated with ENR solutions. It is well known that due to specific binding (FIG. 9 d) a G-quadruplex complex can be formed between the aptamer chain and the ENR, which reduces the electrons entering the electrode surface, resulting in an increase of the Rct value. Among the three aptamer sensors, the polyCoFePc-based sensor showed the greatest Δrct value of 580.8 Ω, indicating its superior sensing ability for ENR, indicating that polyCoFePc has a high biocompatibility for the aptamer chain. Therefore, the bimetallic polyCoFePc network is suitable as a sensitive layer of an ENR electrochemical aptamer sensor.
Experimental example 4 optimization of parameters and conditions for electrochemical detection of ENR
In order to obtain optimal sensing performance for detecting ENR using the constructed polyCoFePc network, some construction parameters or measurement parameters, such as the concentration of the polyCoFePc suspension used for modifying the electrode, the concentration of the nucleic acid aptamer, and the binding time of ENR to the nucleic acid aptamer, have to be considered. For optimal sensing performance, experimental conditions were optimized, including polyCoFePc suspensions (0.1, 0.2, 0.5, 1 and 2 mg-mL -1 ) Concentration of aptamer (10, 20, 50, 100, 200 and 500 nM) and ENR (concentration of 1 fg. Mu.mL) -1 ) Binding to aptamerAnd (3) the room(s). For all solutions, e.g. aptamer and ENR, PBS (ph= 7.4,0.1M) was used as dilution solution, containing 5mm [ fe (CN) 6 ] 3-/4- Electrochemical measurements were performed with 0.1M PBS (ph=7.4) as electrolyte, 0.14M NaCl and 0.1M KCl.
Since the variation of Rct (Δrct=rct, after-Rct, before) positively correlates with the fixed amount of polyCoFePc, the fixed amount of aptamer chain and the fixed amount of ENR, Δr obtained by each step ct Values are used to illustrate the effect of the concentration of the polyCoFePc suspension, the concentration of aptamer and the different binding times on aptamer sensor manufacture and ENR detection, and the results are shown in fig. 10. In FIG. 10, (a) is the effect of the concentration of the polycofePc suspension on the aptamer sensor manufacture and the ENR detection, using the ΔR obtained from each step ct The value represents (b) the effect of aptamer concentration on the ENR assay, using ΔR obtained from the ENR assay ct The value represents. (c) For the effect of different binding times on the detection of ENR, ΔR from the detection of ENR is used ct The value represents.
1. Optimal PolyCoFePc suspension dosage
FIG. 10a summarizes the ΔR of all aptamer sensors obtained at each step ct Value of ΔR of polyCoFePc/AE ct The value increased from 0.1 to 2 mg.mL with the concentration of polyCoFePc -1 And increases. Similar results are also obtained for the immobilization of aptamer and detection of ENR. This result indicates that the thick polyCoFePc network layer severely suppresses electron transfer. In addition, as the thickness of the polyCoFePc network layer increases, more aptamer molecules can adsorb on the polyCoFePc sensitive layer, further leading to specific recognition of more ENRs. When the concentration of the polyCoFePc is more than 1 mg.mL -1 At the time, ΔR of polyCoFePc/AE ct The value is not increased any more, and 1 mg.mL is used -1 The results of aptamer sensors developed from the polyCoFePc suspension were similar. In fact, too thick a polyCoFePc is easily removed from the electrode surface due to weak interactions and is no longer stable in aqueous solutions. Therefore, the concentration was selected to be 1 mg/mL -1 The electrochemical aptamer sensor is developed by taking the polyCoFePc nanomaterial suspension as the drug concentration of the bare electrode.
2. Optimal aptamer concentration
With 1 mg.mL -1 The polyCoFePc nanomaterial suspension treated polyCoFePc/AE electrodes were incubated with different concentrations of aptamer (10, 20, 50, 100, 200, and 100 nM) to prepare different aptamer sensors. As shown in FIG. 10b, immobilization of aptamer on a layer of polycofePc resulted in ΔR ct The value increases as the aptamer concentration increases from 10nM to 100 nM. The result shows that the polyCoFePC layer can anchor the aptamer chain with higher concentration, thereby further improving the detection efficiency of ENR. When the aptamer concentration is greater than 100nM, ΔR is obtained ct Equilibrium was reached indicating saturation of adsorption. Because of the negative charge of the aptamer strands immobilized on the polyCoFePC layer, their strong repulsive interaction with the aptamer strands in aqueous solution prevents them from further adsorption, ultimately resulting in only a certain amount of the aptamer strands being immobilized. Thus, a nucleic acid aptamer solution at a concentration of 100nM was used to develop a polyCoFePc-based aptamer sensor.
3. Optimal binding time of ENR to aptamer strand
FIG. 10c summarizes the ΔR obtained at different binding times of ENR to aptamer strands ct The value, which increases significantly at the very beginning (0-40 minutes), rises slowly to plateau after 40 minutes. This finding suggests that binding of ENR to aptamer occurs mainly within 40min and this is the optimal binding time. Thus, 1 mg/mL -1 The polyCoFePc suspension and 100nM nucleic acid aptamer solution were used as optimal conditions for constructing the aptamer sensor, and the aptamer sensor was incubated in the ENR solution for 40min as optimal conditions for detecting ENR.
Experimental example 5 quantitative analysis of ENR by an aptamer sensor based on PolyCoFePc
Under the optimum experimental parameters, to contain 5mM [ Fe (CN) 6 ] 3-/4- 0.1M PBS (ph=7.4) of 0.14M NaCl and 0.1M KCl as electrolytes, respectively soaking the aptamer sensor with ENR of different concentrations, so as to perform quantitative analysis using the aptamer sensor based on polyCoFePc, and deriving the aptamer transfer according to IUPAC standardLimit of detection (LOD) of the sensor. Then, the correction curve is obtained by taking the logarithm of the EIS response function and the ENR concentration. As a result, FIG. 11 (a-b) shows that FIG. 11a shows that the aptamer sensor detects different concentrations (0, 0.1 fg. ML -1 、1fg·mL -1 、10fg·mL -1 、100fg·mL -1 、1pg·mL -1 、10pg·mL -1 And 100 pg.mL -1 ) EIS nyquist plot of ENR of (c). FIG. 11b shows ΔR ct And the concentration of ENR (inset: deltaR) ct A linear fit plot as a function of ENR concentration logarithm, where the error line is the standard deviation of n=3). As can be seen from FIG. 11a, as the ENR concentration increases from 0 to 100pg mL -1 The EIS response is significantly enhanced. After that, when the detection concentration is more than 10 pg.mL -1 At ENR of (c), the EIS response will slowly settle. As can be seen from FIG. 11b, the ΔR obtained ct The values increase in sequence from 300.69 Ω to 1741.7 Ω. The resulting DeltaR ct Shows good linear relation with the logarithm of the concentration of ENR, and the fitting linear regression equation is delta R ct (kΩ)＝0.079LogCon ENR +0.12, square correlation coefficient (R 2 ) 0.9921 (fig. 11b inset). According to the LOD equation (3S/S, where S is the standard deviation of the blank response solution and S is the slope of the calibration curve), the concentration at ENR is 0.1 fg.mL -1 To 100 pg.mL -1 Within the range of (2), it can be deduced that LOD is 0.06 fg.mL -1 。
The detection of ENR by the aptamer sensor of the invention is compared with the detection of ENR by the prior art, and the results are shown in table 3. The polyCoFePc based aptamer sensor has a much lower LOD than the ENR sensor of the prior art. Considering the advantages of the chemical structure and nanostructure of the polyCoFePc nanoplates, and the construction steps viable with current sensing methods, the low LOD of the aptamer sensor of the invention is mainly due to the following aspects:
(i) The fully exfoliated and interlaminar stacked 2D structure of the prepared polyCoFePc nanoplatelets can greatly increase the accessibility of redox to electrochemically active surfaces and amplify the electrochemical response. In addition, M-N 4 The exposure of the active site also increases the availability and thus promotes the interaction between the metal cluster and the oligonucleotideIs a complex interaction with a substrate.
(ii) Since the polyCoFePc has highly conjugated phthalocyanine ring, nano lamellar structure and rich M-N 4 More aptamer chains will be immobilized centrally by complex interactions, such as stacking, coordination bonds between metal ions and nitrogen on the aptamer chain, hydrogen bonding, etc. van der waals forces, resulting in high detection sensitivity.
(iii) The full coverage of the aptamer chain on the polyCoFePc nanosheets not only can improve the sensing performance of ENR, but also can enable the sensing system to have good blocking effect on some possible interferences in a complex environment. Therefore, a retarder is not needed in the construction process of the polyCoFePc, so that the development steps of the sensor are simplified, and the detection efficiency is ensured.
(iv) The prepared polyCoFePc matrix can adsorb more aptamers by utilizing the synergistic effect between cobalt and iron clusters, can stabilize the quadruplex formed by the aptamers and ENR, and has higher detection capability.
TABLE 3 comparison of the invention with the prior art for ENR detection
Reference is made to:
document 1: H.Lee, S.Lee, D.Kwon, C.Yim, S.Jeon, microbial respiration-based detection of enrofloxacin in milk using capillary-tube indicators, sensor. Act. B-chem.244 (2017) 559-564.
Document 2: X.Guo, L.Zhang, Z.Wang, Y.Sun, Q.Liu, W.Dong, A.Hao, fluorescent carbon dots based sensing system for detection of enrofloxacin in water solutions, spectrochim. Acta A219 (2019) 15-22.
Document 3: F.Y.Yard1m,Z./>Electroanalytical determination of enrofloxacin based on the enhancement effect of the anionic surfactant at anodically pretreated boron-doped diamond electrode,Diam.Relat.Mater.84(2018)95-102。
document 4: W.Chen, R.Zhou, X.Yao, K.Zhao, A.Deng, J.Li, sensitive detection of enrofloxacin using an electrochemiluminescence immunosensor based on gold-functionalized C60 and Au@BSA nanoflower, new J.chem.42 (2018) 14142-14148.
Document 5: X.Huang, Z.P.Aguilar, H.Li, W.Lai, H.Wei, H.Xu, Y.Xiong Fluorescent Ru (phen) 3 2+ -doped silica nanoparticles-based ICTS sensor for quantitative detection of enrofloxacin residues in chicken meat,Anal.Chem.85(2013)5120-5128。
Document 6: S.Li, Y.Zhang, W.Wen, W.Sheng, J.Wang, S.Wang, J.Wang, A high-sensitivity thermal analysis immunochromatographic sensor based on au nanoparticle-enhanced two-dimensional black phosphorus photothermal-sensing materials, biosensors and Bioelectronics 133 (2019) 223-229.
Document 7: X.Liu, J.Ren, L.Su, X.Gao, Y.Tang, T.Ma, L.Zhu, J.Li, novel hybrid probe based on double recognition of aptamer-molecularly imprinted polymer grafted on upconversion nanoparticles for enrofloxacin sensing, biosens. Bioelectron.87 (2017) 203-208.
Document 8: Y.Song, M.Xu, X.Liu, Z.Li, C.Wang, Q.Jia, Z.Zhang, M.Du, A label-free enrofloxacin electrochemical aptasensor constructed by a semiconducting CoNi-based metal-organic framework (MOF), electrochim. Acta 368 (2021) 137609.
Experimental example 6 Selectivity, stability and reproducibility of aptamer sensor
Various interfering substances may coexist with ENR, including Streptomycin (STR), ofloxacin (OFLX), doxycycline (DOX), terramycin (OTC), lomefloxacin hydrochloride (LMFH), ciprofloxacin (CPFX), penicillin (PNC), ag + 、Pb 2+ 、NO 3- 、Hg 2+ And mixtures thereof with ENR, to evaluate the selectivity of the developed aptamer sensor, a constructed biological sensorThe sensor detects ENR, various interfering substances, and mixtures of ENR and various interfering substances. To contain 5mM [ Fe (CN) 6 ] 3-/4- 0.1M PBS (pH=7.4) containing 0.14M NaCl and 0.1M KCl as electrolyte, and the concentration of the interfering substance in the mixture was higher than that of ENR (0.1 fg. ML) -1 ) 100 times higher than that of 10 fg.mL -1 。
In addition, the same electrochemical aptamer sensor pair ENR (concentration of 0.1fg·ml -1 ) The stability of which was tested. Specifically, during the first day of testing, a sensor is used for detecting a target object ENR to obtain Rct values before and after the sensor is fixed to ENR, and delta R is calculated ct The electrodes were then placed in a PBS solution and refrigerated in a refrigerator at 4 ℃; the next day of test, the electrode is directly taken out, the measurement is carried out at room temperature, the Rct value is recorded, and the delta R is obtained through calculation ct Values (Rct value before sensor fixation ENR with respect to first day test), the operation was repeated until day 15.
To contain 5mM [ Fe (CN) 6 ] 3-/4- 0.1M PBS (pH=7.4) containing 0.14M NaCl and 0.1M KCl was used as an electrolyte, and ENR was detected by measuring five kinds of the same freshly prepared Apt/polyCoFePc/AE (0.1 fg. ML) -1 ) ΔR of (2) ct The reproducibility of the aptamer sensor was investigated.
The experimental results are shown in FIG. 11 (c-e), FIG. 11c shows the detection of various interferents by an aptamer sensor based on polyCoFePC (STR, OFLX, DOX, OTC, LMFH, CPFX, PNC, ag + 、Pb 2+ 、NO 3- And Hg of 2+ ) (the concentrations were 10 fg. ML -1 ) And the ENR (concentration of 10 fg.mL) -1 ) Is a mixture of (A) and ENR (concentration of 0.1 fg. Mu.mL -1 ) ΔR of (2) ct FIG. 11d shows the values of an aptamer sensor (0.1 fg. ML for ENR detection -1 N=3) stability for 15 days, fig. 11e is in the presence of 5mm [ fe (CN) 6 ] 3-/4- In 0.1M PBS (ph=7.4), 0.14M NaCl and 0.1M KCl, ENR was detected using five identically constructed polyCoFePc-based aptamer sensors (concentration of 0.1fg·ml -1 ) ΔR of (2) ct Values.
As can be seen from the view of figure 11c,the detection of interfering substances exhibits a negligible EIS response, whereas the EIS response changes substantially when the ENR is detected. Furthermore, analysis of the mixed solution with the developed aptamer sensor showed a response rate of 120% for ENR detection. These results demonstrate that the aptamer sensor of the invention has very high selectivity and interference immunity. As can be seen from fig. 11d, no significantly changed EIS response was observed (rsd=0.56%) indicating that the aptamer sensor of the invention has good stability. As can be seen from FIG. 11e, the detection of ENR (concentration of 0.1 fg. Ml using 5 independent aptamer sensors -1 ) And EIS test was performed with RSD values below 1.06%, indicating good reproducibility.
In summary, the present invention uses highly conjugated polyCoFePc nanoplates as a platform for anchoring ENR targeting nucleic acid aptamers, when contacted with highly conjugated, graphene-like and defective bimetallic phthalocyanine polymer nanoplates, a large number of nucleic acid aptamers are anchored to the polyCoFePc surface by complex interactions such as stacking, hydrogen bonding, van der waals forces or coordination between metal and oligonucleotide chains. The polyCoFePc has a higher binding affinity for the aptamer sensor and thus a better sensing performance for ENR than the monometal polycpc material. The aptamer sensor based on the polyCoFePc has extremely low LOD, good stability and good reproducibility, and when corresponding nucleic acid aptamers are anchored, the invention can be further expanded to the preparation of other types of aptamer sensors, thereby showing great application prospects in the aspects of environmental monitoring and food safety.

Claims (10)

1. The application of the metal phthalocyanine nano material as an electrode material for an aptamer sensor is characterized in that the metal phthalocyanine nano material is prepared by a method comprising the following steps: pyromellitic dianhydride, urea and NH 4 Heating Cl, ammonium molybdate and metal salt to react to obtain metal polymalocyanine; the metal salt is selected from one or two of cobalt salt and ferric salt; the temperature of the heating reaction is 200-260 ℃ and the time is 2-6 h, and the molar ratio of the metal elements in the pyromellitic dianhydride, the urea and the metal salt is 4:8-32:1～3。
2. The use according to claim 1, wherein the metal salt consists of a cobalt salt and an iron salt; the molar ratio of cobalt element in the cobalt salt to iron element in the ferric salt is 1-2:1.
3. The use according to claim 1, wherein the molar ratio of pyromellitic dianhydride to ammonium molybdate is 20 to 40:1.
4. The use according to claim 1, wherein the cobalt salt is CoCl 2 ·6H 2 O, the ferric salt is FeCl 3 ·6H 2 O。
5. An aptamer sensor is characterized by comprising an electrode matrix, a metal polymorphine nanomaterial modified on the surface of the electrode matrix and a nucleic acid aptamer anchored on the metal polymorphine nanomaterial and used for targeted detection of enrofloxacin; the metal phthalocyanine nano material is prepared by a method comprising the following steps: pyromellitic dianhydride, urea and NH 4 Heating Cl, ammonium molybdate and metal salt to react to obtain metal polymalocyanine; the metal salt is selected from one or two of cobalt salt and ferric salt; the temperature of the heating reaction is 200-260 ℃ and the time is 2-6 h, and the molar ratio of the metal elements in the pyromellitic dianhydride, the urea and the metal salt is 4:8-32:1-3.
6. The aptamer sensor of claim 5, wherein the metal salt consists of a cobalt salt and an iron salt; the molar ratio of cobalt element in the cobalt salt to iron element in the ferric salt is 1-2:1.
7. The aptamer sensor of claim 5 wherein the molar ratio of pyromellitic dianhydride to ammonium molybdate is 20 to 40:1.
8. The adapter of claim 5A ligand sensor is characterized in that the cobalt salt is CoCl 2 ·6H 2 O, the ferric salt is FeCl 3 ·6H 2 O。
9. A method of preparing an aptamer sensor according to any one of claims 5 to 8, comprising: pretreating an electrode to obtain an electrode matrix; modifying the suspension of the metal phthalocyanine nano material on an electrode matrix to obtain a modified electrode; inoculating the modified electrode in a solution of a nucleic acid aptamer for targeted detection of enrofloxacin.
10. The method for preparing an aptamer sensor according to claim 9, wherein the concentration of the metal polymalocyanine nanomaterial suspension is 0.1-2 mg-mL -1 。
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