CN110514713B - Preparation method and application of aptamer biosensor based on ferrocene as electron donor - Google Patents

Preparation method and application of aptamer biosensor based on ferrocene as electron donor Download PDF

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CN110514713B
CN110514713B CN201811239957.6A CN201811239957A CN110514713B CN 110514713 B CN110514713 B CN 110514713B CN 201811239957 A CN201811239957 A CN 201811239957A CN 110514713 B CN110514713 B CN 110514713B
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aptamer
ferrocene
graphene oxide
solution
chitosan
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CN110514713A (en
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郭业民
符家韵
史孝杰
孙霞
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Shandong University of Technology
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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
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Abstract

A preparation method and application of an aptamer biosensor based on ferrocene as an electron donor are disclosed, and the preparation method comprises the following steps: adding a chitosan solution into a graphene oxide solution for activating carboxyl, and then modifying the mixed solution on the surface of a screen-printed carbon electrode; adding a specific base pair at the tail end of an aptamer to design and synthesize a hairpin structure, respectively modifying amino and ferrocene at two ends, and effectively fixing an aptamer chain on the surface of a graphene oxide-chitosan/screen printing carbon electrode through formation of an amido bond; the specific recognition of the aptamer and the target induces the conformation change of the aptamer, so that the current signal generated by the ferrocene in the phosphate buffer solution is changed, and the target is monitored. The invention can realize the ultra-sensitive detection of four organophosphorus pesticides including profenofos, phorate, isocarbophos and omethoate, has short detection time and low detection limit, and provides a new technology for detecting organophosphorus pesticides and harmful substances in other fields.

Description

Preparation method and application of aptamer biosensor based on ferrocene as electron donor
Technical Field
The invention relates to a preparation method and application of an aptamer biosensor based on ferrocene as an electron donor, and belongs to the technical field of electrochemical biosensor detection.
Background
Organophosphorus pesticides are phosphorus-containing organic compounds, and are widely used in agriculture for controlling plant diseases and insect pests and weeds due to the advantages of high efficiency, low toxicity and low residue. The use of these pesticides can help prevent crop losses. However, excessive organophosphorus pesticide left on agricultural products can enter human bodies through food chains, and is combined with acetylcholinesterase to cause nerve dysfunction, so that paralysis and even death are caused, and the human health is greatly damaged. Profenofos, phorate, isocarbophos and omethoate are widely used organophosphorus pesticides, and overproof events often occur in vegetable products. Therefore, it is necessary to construct a rapid, sensitive, safe and reliable pesticide residue detection and analysis method.
Over the past several decades, various effective methods for detecting organophosphorus pesticide residues have been established, such as gas chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, colorimetric assay, fluorescent assay, and the like. However, most of them are time-consuming and labor-consuming, expensive in instruments and equipment, high in technical requirements of operators, and not suitable for rapid analysis on site. To improve these problems, electrochemical aptamer sensors are widely used due to their advantages of good selectivity, high sensitivity, and low price. In the application process of the electrochemical aptamer sensor, the generation and amplification of electrochemical signals are key factors for restricting the performances of the sensor such as sensitivity and the like.
Graphene oxide is a well-known excellent conductive nanomaterial, and the combination of graphene oxide and a chitosan solution provides good biocompatibility and stability for a biological recognition molecule while amplifying an electrochemical signal. Ferrocene, an organic transition metal compound, is widely used as an electrochemical signaling probe due to its excellent reversible redox properties and good chemical stability. In recent years, it has been reported that electrons can be supplied in a phosphate buffer solution, and thus the electrochemical signal generated therefrom has an ultra-high sensitivity. Therefore, the material is expected to be used for developing an ultra-sensitive electrochemical aptamer sensor and detecting organophosphorus pesticide.
Disclosure of Invention
The invention aims to provide an aptamer biosensor which is high in sensitivity, simple to operate and good in selectivity and is used for detecting four organophosphorus pesticides including profenofos, phorate, isocarbophos and omethoate.
The technical scheme of the invention is as follows: fixing a ferrocene-labeled hairpin aptamer on the surface of a screen-printed carbon electrode of which the surface is modified with a graphene oxide-chitosan composite film to form an aptamer biosensor; the method comprises the steps of pretreatment of an electrode, preparation of a graphene oxide-chitosan solution, design and marking of an aptamer hairpin structure and detection of an organophosphorus pesticide, and comprises the following specific steps:
(1) pretreatment of the electrode: completely immersing a working area of a screen printing carbon electrode into 0.5M sulfuric acid solution, and carrying out 5-circle cyclic voltammetry scanning within a potential range of-1.0 to + 1.0V to obtain an electrode with stable performance;
(2) preparing a graphene oxide-chitosan solution: adding EDC and NHS into the graphene oxide solution, incubating and activating carboxyl on the graphene oxide at room temperature, centrifugally cleaning, and adding a chitosan solution to prepare a 1 mg/mL graphene oxide-chitosan solution;
(3) designing and marking the structure of the adapter hairpin: adding base pair CAAGCT complementary to the first few base pairs (AGCTTG) of the 5 'end to the 3' end of the aptamer (5'-AGCTTGCTGCAGCGA TTCTTGATCGCCACAGAGCT-3'), and labeling amino group (-NH) at both ends2) And ferrocene (Fc), a newly synthesized aptamer chain (NH)2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGC TCAAGCT-Fc) is effective for forming hairpin structures;
(4) detection of organophosphorus pesticide: dripping organophosphorus pesticide standard substances with different concentrations on the prepared biosensor, and incubating for a certain time at room temperature to ensure that the aptamer and the pesticide are fully combined; the specific binding of the target pesticide and the aptamer opens a hairpin structure of the aptamer to cause the electron donor ferrocene to be far away from the surface of the electrode, and when electrochemical differential pulse voltammetry detection is performed in phosphate buffer solution with the pH of 7.4, the current peak value appearing at about +0.7V is reduced, so that the concentration of a target object is monitored; the selected aptamer is a broad-spectrum aptamer and has good detection capability on profenofos, phorate, isocarbophos and omethoate.
The preparation principle of the invention is as follows: the graphene oxide-chitosan nano composite material modified on the surface of the screen printing carbon electrode can amplify electrochemical signals, and a large amount of carboxyl groups on the graphene oxide can be combined with amino groups modified on the aptamer to be beneficial to fixing the aptamer. The synthetic hairpin aptamers were designed to contain two fragments: one is an aptamer sequence specifically recognizing organophosphorus pesticides, and the other is an oligonucleotide sequence which is added by self design and is used for forming a stem-loop structure. Respectively modifying amino and ferrocene at the 5 'end and the 3' end of the hairpin aptamer sequence, and fixing the hairpin aptamer on the surface of the screen printing carbon electrode modified by graphene oxide-chitosan through the combination of amino and carboxyl. Under the condition that no target exists, the aptamer maintains a hairpin structure, and the ferrocene is tightly attached to the surface of the electrode, so that a current signal generated by the electron donor ferrocene under the action of certain voltage is highlighted. When the target molecule is present, the aptamer hairpin structure unfolds, resulting in a reduction in electrochemical signal. The electric signal intensity gradually decreases with the increase of the target concentration, so that the concentration of the organophosphorus pesticide target can be measured.
The invention has the beneficial effects that: the invention provides a preparation method and application of an aptamer biosensor based on ferrocene as an electron donor, and the aptamer biosensor is used for detecting four organophosphorus pesticides. The strategy of using ferrocene as an electric signal probe enables the signal of the sensor to be very sensitive, and in addition, the sensor is simple in preparation method, has higher stability and specificity, and can be used for detecting organophosphorus pesticides in vegetable samples. Therefore, the invention provides a novel analysis and detection means with high sensitivity and accuracy for detecting the residue of the small molecule harmful substances.
Drawings
FIG. 1 Process for preparing aptamer biosensors.
FIG. 2 electrochemical characterization of aptamer biosensors.
FIG. 3 feasibility study of aptamer biosensor.
Figure 4 measures the DPV response and corresponding standard curve for different concentrations of profenofos.
Figure 5 measures DPV responses and corresponding standard curves for different concentrations of phorate.
Figure 6 measures DPV responses and corresponding standard curves for various concentrations of isocarbophos.
Figure 7 measures the DPV response and corresponding standard curve for various concentrations of omethoate.
Figure 8 the sensors detect the main parameters of different pesticides.
Figure 9 actual sample spiking recovery.
FIG. 10 specificity analysis of aptamer biosensors.
Detailed Description
The present invention is described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the present invention without limiting its scope.
Example 1:
a method for preparing an aptamer biosensor based on ferrocene as an electron donor comprises the following specific steps:
(1) design of hairpin aptamers (HP) and preparation of solutions thereof
Adding base pair CAAGCT complementary to the first few base pairs (AGCTTG) of the 5 'end to the 3' end of the aptamer (5'-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3'), and labeling amino group (-NH) at both ends2) And ferrocene (Fc), a newly synthesized aptamer chain (NH)2AGCTTGCTGC AGCGATTCTTGATCGCCACAGAGCTCAAGCT-Fc) was analyzed by DNAman software fitting and found to be effective in forming hairpin structures. In order to form the designed and synthesized aptamer into a hairpin structure, the aptamer stock solution was heated in a water bath kettle at 95 ℃ for 5min and then slowly cooled to room temperature. It was diluted to 2. mu.M stock solution with Tris-HCl pH 7.5 as solvent.
(2) Preparation of graphene oxide-chitosan (GO-CS) solution
Adding 5 mg chitosan powder into 25 mL of 1.0% acetic acid solution to prepare 0.2 wt% chitosan solution, and magnetically stirring for more than 8 hours to completely dissolve chitosan. Adding 0.15M EDC and 0.1M NHS into 1 mg/mL graphene oxide solution, incubating for 30 min at room temperature to activate carboxyl on the graphene oxide, then centrifuging by a centrifuge (8000 rpm, 15 min), adding water to wash for 3 times to remove residual EDC and NHS, and finally adding 1 mL 0.2 wt% chitosan solution into the obtained precipitate to prepare 1 mg/mL graphene oxide-chitosan solution.
(3) Pretreatment of electrodes
The Screen Printed Carbon Electrode (SPCE) working area was completely immersed in a 0.5M sulfuric acid solution and 5 cyclic voltammetric scans were performed at potentials ranging from-1.0 to + 1.0V to obtain stable electrochemical signals. And washing with ultrapure water for later use.
(4) Construction of aptamer biosensor
Diluting the 1 mg/mL graphene oxide-chitosan solution prepared in the step (2) to 0.2 mg/mL, dropwise adding 8 mu L of the nano composite solution into the SPCE working area pretreated in the step (3), and airing at room temperature; dropwise adding 8 mu L of 2 mu M aptamer solution on the graphene oxide-chitosan modified electrode, wherein amino on the aptamer and carboxyl of graphene oxide can form an amido bond, so that the aptamer is tightly fixed on the surface of the electrode; finally, the dried sensor was lightly rinsed with ultrapure water and placed in a 4 ℃ freezer for use. The preparation process of the aptamer biosensor is shown in fig. 1.
(5) Electrochemical characterization of aptamer biosensors
CV characterization was performed on the assembly process of the aptamer biosensor manufacturing process, as shown in fig. 2. The redox peak current of the graphene oxide-chitosan modified electrode was higher (fig. 2-b) compared to the bare screen printed carbon electrode (fig. 2-a), due to the stronger electrical conductivity of the composite. In addition, an increase in the DPV peak current and a shift in the peak potential also indicate that the electrode surface was successfully modified. With further modification of the hairpin, the current peaks increased significantly (FIG. 2-c), probably due to the proximity of ferrocene labeled on the hairpin to the electrode surface, resulting in a larger current signal. When the sensor was incubated with the target molecule profenofos, the peak current decreased significantly (fig. 2-d), probably because: the target object-aptamer compound formed by combining the aptamer and the target object blocks the electron transfer in the potassium ferricyanide solution; the target opens the aptamer hairpin structure, leaving ferrocene away from the electrode surface. The above experimental results demonstrate the successful construction of the aptamer biosensor.
(6) Feasibility study of aptamer biosensor
Fig. 3 illustrates the feasibility of the aptamer biosensor. When the bare electrode is immersed in phosphoric acidWhen the salt buffer solution is subjected to DPV measurement (potential range: +0.4 to + 0.9V) (FIG. 3-a), the current curve is found to be almost a straight line with no distinct peak. After the graphene oxide-chitosan is modified (fig. 3-b), a very weak electrochemical signal is obtained, which indicates that the graphene oxide-chitosan is helpful for amplifying the electrochemical signal. The resulting electrode was further modified with hairpin aptamers designed according to the invention (fig. 3-d), resulting in a very large current peak. However, when organophosphorus aptamers (Apt, NH) labeled only with amino groups are used2AGCTTGCTG CAGCGATTCTTGATCGCCACAGAGCT) was immobilized on the graphene oxide-chitosan modified electrode surface (fig. 3-c), the resulting peak signal was negligible. This indicates that the electrochemical signal generated by the system is generated by ferrocene under a certain potential. The addition of the target pesticide profenofos leads to a reduction in current (fig. 3-e), which shows that the combination of the target substance and the hairpin aptamer leads the ferrocene to be away from the surface of the electrode, and the ultrasensitive detection of the target substance can be realized according to the difference of electrochemical signals.
Example 2:
use of an aptamer biosensor based on ferrocene as electron donor:
(1) detection of organophosphorus pesticides in water
8 mu L of different organophosphorus pesticides with different concentrations are dripped on the aptamer biosensor and incubated in the air for 1 h. After being lightly rinsed with ultra pure water and blown dry with nitrogen, the DPV electrochemical signals were measured in a pH 7.4 phosphate buffer solution. The results are shown in FIGS. 4-7 (profenofos FIG. 4, phorate FIG. 5, isocarbophos FIG. 6, omethoate FIG. 7), with a good linear relationship between the DPV response signal and the logarithm of the concentrations of the four organophosphorus pesticides. Their linear regression equation, linear range, correlation coefficient and detection limit are summarized in fig. 8. The extremely low detection line of the sensor indicates that the aptamer biosensor prepared by the invention has extremely high sensitivity.
(2) Detection of organophosphorus pesticides in vegetable samples
To further investigate the utility of aptamer biosensors, recovery experiments were performed using standard addition methods. Briefly, vegetable samples (rape, cabbage, spinach, pakchoi) were sprayed with profenofos standard solutions of various concentrations (10 nM, 100 nM) and then pre-treated to extract residual pesticides. Subsequently, all the extracts were subjected to electrochemical detection using the aptamer biosensor prepared according to the present invention. Fig. 9 shows the results of the experiment, with recovery rates between 79.66% and 136.90%, indicating that the sensor can be used for detection analysis of actual samples.
Example 3:
performance testing of an aptamer biosensor based on ferrocene as electron donor:
(1) specificity analysis
The selectivity of the aptamer sensor is detected by using carbaryl, malathion, methamidophos, monocrotophos and chlorpyrifos and a mixture of carbaryl, malathion, methamidophos, monocrotophos and chlorpyrifos as interfering pesticides. As shown in FIG. 10 (a, b, c, d, e, f represent five interfering pesticides and their mixtures, respectively; g, h, i, j, k represent profenofos, phorate, isocarbophos and omethoate and their mixtures, respectively; l represents the nine pesticide mixtures; all pesticide concentrations are 10 nM), the electrochemical signal is greater in the absence of specific targets (profenofos, phorate, isocarbophos and omethoate), indicating that the aptamer cannot bind to these interfering pesticide molecules. In addition, the histogram k is not obviously different from the histogram l, and the aptamer biosensor prepared by the invention is proved to have good specificity and anti-interference capability again.
(2) Stability study
To study the stability of the aptamer biosensor, 10 prepared sensors were stored in a 4 ℃ refrigerator and their DPV signals were measured after a period of time and compared to the DPV signal of a freshly prepared sensor. The change in electrochemical signal after one week of storage was negligible. After 15 days of storage, the peak current remained around 91.35%, with a relative standard deviation of 7.06%. The biosensor has good stability.
SEQUENCE LISTING
<110> university of Shandong's science
<120> preparation method and application of aptamer biosensor based on ferrocene as electron donor
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 35
<212> DNA
<213> aptamer
<400> 1
agcttgctgc agcgattctt gatcgccaca gagct 35

Claims (6)

1. A preparation method of an aptamer biosensor based on ferrocene as an electron donor is characterized in that the preparation method comprises the steps of fixing a ferrocene-labeled hairpin aptamer on the surface of a screen-printed carbon electrode of which the surface is modified with a graphene oxide-chitosan composite film; the design synthesis and marking of the hairpin aptamer are that base pairs CAAGCT which are complementarily paired with AGCTTG of a plurality of base pairs in front of a 5 'end are added at the 3' end of the aptamer 5'-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCT-3', amino and ferrocene are respectively marked at two ends, and a newly synthesized aptamer chain NH2-AGCTTGCTGCAGCGATTCTTGATCGCCACAGAGCTCAAGCT-Fc can effectively form a hairpin structure; the aptamer is a broad-spectrum aptamer and has good specific binding capacity with profenofos, phorate, isocarbophos and omethoate.
2. The method of claim 1, wherein the screen-printed carbon electrode is pretreated by completely immersing the working area of the screen-printed carbon electrode in a 0.5M sulfuric acid solution and performing cyclic voltammetric scanning for 5 cycles at a potential ranging from-1.0 to + 1.0V to obtain an electrode with stable performance.
3. The method of claim 1, wherein the graphene oxide-chitosan mixed solution is prepared by adding EDC and NHS to a graphene oxide solution, incubating and activating carboxyl groups on graphene oxide at room temperature, centrifuging and washing, and adding a chitosan solution to prepare a 1 mg/mL graphene oxide-chitosan solution.
4. The method of claim 1, wherein the biosensor is used to determine the presence of an organophosphorus pesticide: dripping organophosphorus pesticide standard substances with different concentrations on the prepared biosensor, and incubating for a certain time at room temperature to ensure that the aptamer and the pesticide are fully combined; the sensor was then placed in a pH 7.4 phosphate buffered solution to perform electrochemical differential pulse voltammetry detection.
5. The method of claim 1, wherein the electrochemical signal of the biosensor is derived from ferrocene: under the action of voltage of about +0.7V, ferrocene in phosphate buffer solution can generate electrons to provide electrochemical signals.
6. The method of claim 1, wherein the different electrochemical signals resulting from the conformational change in the aptamer reflect the concentration of the pesticide: the specific binding of the target pesticide and the aptamer opens a hairpin structure of the aptamer, so that the ferrocene is far away from the surface of the electrode, the electrochemical signal generated by the electrode is reduced, and the concentration of the target substance is monitored.
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