CN114371203A - Sensing electrode suitable for in-situ detection of living body and preparation method and application thereof - Google Patents
Sensing electrode suitable for in-situ detection of living body and preparation method and application thereof Download PDFInfo
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- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
- A61B5/25—Bioelectric electrodes therefor
- A61B5/263—Bioelectric electrodes therefor characterised by the electrode materials
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- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/24—Detecting, measuring or recording bioelectric or biomagnetic signals of the body or parts thereof
- A61B5/25—Bioelectric electrodes therefor
- A61B5/279—Bioelectric electrodes therefor specially adapted for particular uses
- A61B5/294—Bioelectric electrodes therefor specially adapted for particular uses for nerve conduction study [NCS]
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- 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
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- 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
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- 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
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- 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
The invention discloses a sensing electrode suitable for in vivo detection, which comprises: the surface of the carbon fiber electrode is modified with a first hydrophobic group; an aptamer modified with a second hydrophobic group; the aptamer modified with the second hydrophobic group is self-assembled on the surface of the carbon fiber electrode modified with the first hydrophobic group through hydrophobic interaction. The sensing electrode has good stability under physiological conditions, and is not easy to interfere by sulfur active molecules in a receptor; meanwhile, the electrode has the advantages of both the carbon fiber electrode and the aptamer, can maintain the flexibility and the specificity recognition capability of the aptamer, and can carry out real-time detection on the neurotransmitter in a living body with high space-time resolution and high specificity; in addition, the sensing electrode is simple in preparation method, the nucleic acid sequence in the nucleic acid aptamer can be replaced according to requirements, and the sensing electrode has a wide application prospect.
Description
Technical Field
The present invention relates to the field of electrochemical detection. And more particularly, to a sensing electrode suitable for in vivo detection, and a preparation method and application thereof.
Background
Neurotransmitters are basic substances for maintaining physiological functions of the brain, and their content in the brain is closely related to numerous neurophysiological and disease pathological states. For example, dopamine is a representative monoamine neurotransmitter and plays an important role in the brain ' memory ' and ' reward ' systems, and abnormal dopamine levels have strong correlation with the occurrence and development of nervous system diseases such as parkinson's disease. The sensitive and rapid selective detection of neurotransmitters in the central nervous system of living animals is one of the important means for understanding brain development and disease diagnosis.
At present, an electrochemical sensing platform based on a microelectrode has the advantages of excellent space-time resolution, high sensitivity and the like, and is a main method for in-situ detection of neurotransmitter in vivo. The affinity ligand with high specificity molecular recognition performance is combined with an implantable electrochemical platform, and a powerful tool is provided for realizing high-selectivity molecular sensing in a complex nervous system.
Functional ligands, such as aptamers, are short oligonucleotide sequences of 20 to 80 bases obtained by in vitro screening technology separation, can bind to specific target molecules with high affinity, and are widely applied to the analysis and detection field as specific biological recognition elements. At present, the combination of the aptamer and the electrode interface is mostly realized by adopting a gold electrode as a substrate and forming a gold-sulfur bond through a sulfhydryl group at the tail end of the aptamer and the gold electrode. This modification method, although widely used in vitro diagnostics, is less useful in vivo detection of neurotransmitters. The main reasons are as follows: in order to reduce the wound and inflammatory reaction in vivo, the diameter of an electrode used for in vivo analysis is usually about 10 μm. However, the gold fiber electrode with the size is low in hardness, is very easy to bend in the process of being implanted into a body, and cannot be accurately implanted into a detection area; secondly, because various sulfur-containing biomolecules exist in the living environment, the biomolecules are easy to adsorb on the surface of the gold electrode, so that the reaction active area of the surface of the electrode is reduced, and finally the loss of the sensitivity and the stability of the electrode is caused; and thirdly, the traditional electrochemical detection method based on the aptamer usually adopts a differential pulse voltammetry method to realize high-sensitivity detection. However, the method has low stability in the long-term continuous detection process, and is not beneficial to in-situ analysis and detection of living bodies.
The carbon fiber electrode has excellent biological stability and is one of the ideal electrodes for in vivo neurochemical detection. However, how to organically combine the aptamer with the surface inert carbon fiber electrode surface is a major challenge to the electrode interface construction. The method commonly used at present is to carry out pretreatment on the surface of a carbon fiber electrode to make the carbon fiber electrode carry positive charges, and the nucleic acid aptamer is fixed on the surface of the carbon fiber electrode by utilizing the electrostatic interaction between the positive charges on the surface of the electrode and the negative charges of a nucleic acid phosphate skeleton. However, this binding is easily disrupted in high ionic strength environments and exhibits poor stability in solution at physiological conditions. Furthermore, direct interaction with the phosphate backbone may reduce the flexibility of conformational folding of the aptamer, thereby affecting its specific recognition capability.
Therefore, a new carbon fiber electrode modification strategy capable of efficiently and stably modifying nucleic acid on the surface of a carbon fiber electrode and maintaining the high specific recognition capability of a nucleic acid aptamer is needed to meet the requirement of high-selectivity in-vivo analysis of neurotransmitters.
Disclosure of Invention
The invention aims to provide a sensing electrode suitable for in vivo detection, wherein a carbon fiber electrode and an aptamer in the sensing electrode are modified with hydrophobic groups, and the carbon fiber electrode and the aptamer are self-assembled through hydrophobic interaction, so that the sensing electrode has good stability under physiological conditions, and the flexibility and the specificity recognition capability of the aptamer can be maintained, and the sensing electrode is used for in vivo detection.
Another object of the present invention is to provide a method for preparing a sensing electrode suitable for in vivo detection.
It is yet another object of the present invention to provide a method for detecting neurotransmitters in vivo.
In order to achieve the purpose, the invention adopts the following technical scheme:
a sensing electrode suitable for in vivo sensing, comprising:
the surface of the carbon fiber electrode is modified with a first hydrophobic group;
an aptamer modified with a second hydrophobic group;
the aptamer modified with the second hydrophobic group is self-assembled on the surface of the carbon fiber electrode modified with the first hydrophobic group through hydrophobic interaction.
The aptamer is self-assembled on the carbon fiber electrode through hydrophobic effect, the combination mode has good stability under in-vivo physiological conditions, and meanwhile, the acting force can not influence the conformation of the aptamer, so that the sensing electrode can keep stable and high-sensitivity specific recognition capability in vivo.
The nucleic acid sequence in the aptamer is not limited in the invention, and a person skilled in the art can select a suitable nucleic acid sequence with a specific recognition function according to needs.
Preferably, the contact angle between the carbon fiber electrode modified with the first hydrophobic group and water is 65-120 degrees, and the excellent hydrophobic property of the carbon fiber electrode enables the carbon fiber electrode to have larger hydrophobic acting force with the aptamer under physiological conditions, so that the distribution of the aptamer on the surface of the electrode is more stable, and the stability of the sensing electrode is better.
Preferably, the first hydrophobic group is an alkyl chain. A large amount of alkyl chains are modified on the carbon fiber electrode, compact hydrophobic layers can be formed by the alkyl chains and distributed on the surface of the carbon fiber electrode, meanwhile, the alkyl chains are soft and long in chain length, and when a large number of binding sites are improved for hydrophobic groups on the aptamer, large hydrophobic acting force can be provided, and the stability of the sensing electrode is improved.
Preferably, the second hydrophobic group is selected from cholesterol, tocopherol or phospholipid polyethylene glycol derivatives;
preferably, the number of modified second hydrophobic groups on the single aptamer is 1 to 3.
The invention provides a preparation method of a bare carbon fiber electrode, which comprises the following steps:
the carbon fiber and the conductive copper wire with proper length are closely bonded through the conductive silver adhesive, after the conductive silver adhesive is solidified, the copper wire bonded with the carbon fiber penetrates through the conical capillary with the open top end, so that the carbon fiber is exposed at the thin open end of the capillary, and the copper wire is exposed at the other end of the capillary. The capillary was sealed at both ends with epoxy, briefly soaked with acetone or acetonitrile to remove excess epoxy on the fibers, and then the electrodes were rinsed clean with deionized water. And finally, cutting the tip carbon fiber under a microscope to 300 microns in size to obtain the unmodified bare carbon fiber electrode.
Preferably, the cleaning process of the bare carbon fiber electrode comprises the steps of sequentially cleaning the bare carbon fiber electrode in acetone and 3.0M HNO3And (3) carrying out ultrasonic treatment in the solution and a 1.0M KOH solution for 3-5 min to remove surface impurities.
According to the invention, the bare carbon fiber electrode is modified by an alkyl chain after being activated, and one possible embodiment is that the activation of the bare carbon fiber electrode comprises the following steps: soaking the cleaned bare carbon fiber electrode in a 1.0M NaOH solution, and performing ampere method treatment for 80-100 s by applying a voltage of +1.5V to perform primary activation; then, a plurality of cycles of cyclic voltammograms are scanned at a scanning speed of 50mV/s in a voltage range of 0 to +1V until a stable cyclic voltammogram is obtained.
Preferably, the alkyl chain is modified on the surface of the bare carbon fiber electrode in the following way:
and (3) placing the activated bare carbon fiber electrode in an ethanol solution containing alkylamine, and performing cyclic voltammetry scanning for 5-10 circles within a voltage range of-0.2-1.6V at a scanning speed of 5 mV/s.
In a particular implementation, the specific number of cycles of cyclic voltammetric scan is based on obtaining a stable cyclic voltammogram.
Preferably, lithium perchlorate is taken as a supporting electrolyte in the ethanol solution of alkylamine;
preferably, the concentration of the lithium perchlorate is 0.1M;
preferably, the concentration of the alkylamine in the ethanol solution of the alkylamine is 5 mM;
preferably, the alkylamine is a primary amine compound, including but not limited to n-hexylamine, heptylamine, nonylamine and higher primary amines.
In a specific implementation process, after an alkyl chain is modified on the surface of the carbon fiber electrode, the electrode can be repeatedly washed by using ethanol and deionized water for multiple times, and then the electrode is sequentially soaked in the ethanol and the deionized water for 30min to remove substances adsorbed on the surface of the electrode, and finally the electrode is dried for later use.
In another aspect, the present invention provides a method for preparing the sensing electrode for in vivo detection, which includes the following steps:
soaking the carbon fiber electrode with the surface modified with the first hydrophobic group in a buffer solution containing a nucleic acid aptamer, and cleaning the electrode by using the buffer solution and deionized water after 8-16 h;
and sealing the cleaned electrode in 0.5-2mM amphipathic molecule solution containing a second hydrophobic group for 0.5-2h, and cleaning with deionized water to obtain the sensing electrode.
Preferably, the buffer solution is selected from a PBS solution;
preferably, the concentration of the aptamer in the aptamer-containing buffer is 500nM to 1. mu.M.
And soaking the carbon fiber electrode modified with the first hydrophobic group on the surface in a nucleic acid aptamer buffer solution and a solution containing a second hydrophobic group amphipathic molecule in sequence, wherein the second hydrophobic group is modified in the nucleic acid aptamer and then inserted into the gap of the first hydrophobic group, and thus the self-assembly process of the nucleic acid aptamer on the surface of the electrode is completed.
Preferably, the amphiphilic molecule containing a second hydrophobic group, such as cholesterol, tocopherol, diacyl lipid molecules, and the like, may be cholesterol-polyethylene glycol, distearoylphosphatidylethanolamine-polyethylene glycol, and the like.
Preferably, the prepared sensing electrode can be stored in a buffer solution and stored in a refrigerator at 4 ℃ for later use.
In another aspect, the present invention provides a method for in situ detection of neurotransmitters in a living body, comprising:
the concentration of the neurotransmitter is detected by detecting current response signals between the working electrode and the counter electrode by using the sensing electrode as a working electrode, a platinum wire as a counter electrode and an Ag/AgCl electrode as a reference electrode.
Taking the detection of the neurotransmitter dopamine as an example, as shown in fig. 1, the detection principle is as follows: the carbon fiber electrode is a living body implantable electrode, the surface of the carbon fiber electrode is modified with first hydrophobic group alkylamine through chemical oxidation reaction, the aptamer modified with second hydrophobic group cholesterol group has amphipathy, and self-assembly is completed on the surface of the carbon fiber electrode through hydrophobic effect, so that the sensing electrode is obtained. In the presence of a target substance dopamine, the modified aptamer on the carbon fiber electrode specifically recognizes and captures the dopamine, and then the dopamine is subjected to oxidation reaction on the surface of the electrode to generate an electrochemical redox peak. The chemical measurement is carried out on an electrochemical workstation, the modified carbon fiber electrode is used as a working electrode, a platinum wire is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, and the neurotransmitter concentration is detected by detecting current response signals between the working electrode and the counter electrode.
Preferably, the specific methods for detecting the neurotransmitter concentration are cyclic voltammetry and amperometry.
Further preferably, the cyclic voltammetry has a sweep potential in the range of-0.2 to +0.6V and a sweep rate of 50mV/s, and detects the electrochemical response of the neurotransmitter. The amperometric potential was +0.3V and the current response of the electrode to different concentrations of neurotransmitter and to various interferents was recorded.
The sensing electrode has the advantages of both a carbon fiber electrode and an aptamer, can detect the neurotransmitter dopamine with high space-time resolution and high specificity under the condition of a living body, and has stable performance, simple and easy preparation method and wide application potential.
The invention has the following beneficial effects:
the carbon fiber electrode and the aptamer in the sensing electrode are modified with hydrophobic groups, and the aptamer is self-assembled on the surface of the carbon fiber electrode through hydrophobic effect, so that the sensing electrode has good stability under physiological conditions and is not easy to interfere with sulfur active molecules in a receptor; meanwhile, the electrode has the advantages of both the carbon fiber electrode and the aptamer, can maintain the flexibility and the specificity recognition capability of the aptamer, and can carry out real-time detection on the neurotransmitter in a living body with high space-time resolution and high specificity; in addition, the sensing electrode is simple in preparation method, the nucleic acid sequence in the nucleic acid aptamer can be replaced according to requirements, and the sensing electrode has a wide application prospect.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Fig. 1 shows a schematic diagram of the preparation of the sensing electrode and the detection of dopamine according to the present invention.
Fig. 2 shows a linear response graph of a sensing electrode to dopamine in example 1 of the present invention, wherein a is an ampere graph for measuring dopamine, and B is a linear relationship graph of a current response value and a concentration.
FIG. 3 is a graph showing selectivity of a sensing electrode to dopamine in example 1 of the present invention, wherein A is an amperometric graph of the electrode to dopamine and interferents, and B is a graph showing a comparison of selectivity between a bare electrode and an aptamer-modified electrode.
Fig. 4 shows an in vivo analysis diagram of a sensing electrode in example 1 of the present invention, wherein a diagram is a diagram of DA release detection by electrode electrical stimulation, and B is a diagram of dopamine response current recorded by the electrode after electrical stimulation.
Fig. 5 shows a flow chart of the preparation of the sensing electrode in example 2 of the present invention.
Fig. 6 shows a linear response graph of a sensing electrode to dopamine in example 2 of the present invention, wherein a is an ampere graph for measuring dopamine, and B is a linear relationship graph of a current response value and a concentration.
Fig. 7 shows a selectivity graph of the sensing electrode to dopamine, wherein the upper graph is an ampere curve graph of the sensing electrode to dopamine and its interferents in example 2, and the lower graph is a selectivity comparison graph of a bare electrode, the sensing electrode in example 1 and the sensing electrode in example 2.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
The DNA sequence of the nucleotide chain in the nucleic acid aptamer is as follows:
the DNA sequence of the nucleotide chain of the aptamer is shown as SEQ ID No. 1:
SEQ ID No.1：GGA CGA CGC CAG TTT GAA GGT TCG TTC GCA GGT GTG GAG TGA CGT CG TCC TTT TTT–Chol
the 3' end of the nucleotide chain of the aptamer is labeled with cholesterol (Chol).
Preparation of a sensing electrode:
soaking the carbon fiber electrode without modification after cleaning in a 1.0M NaOH solution, and performing ampere method treatment for 80-100 s by applying a voltage of +1.5V to perform primary activation; and then, carrying out cyclic voltammetry scanning for multiple circles at a scanning speed of 50mV/s within a voltage range of 0 to +1V until a stable cyclic voltammogram is obtained, and completing the activation of the carbon fiber electrode.
Soaking activated carbon fiber electrode in 0.10M LiClO4And (3) as a supporting electrolyte, circularly scanning for 5-10 circles at a scanning speed of 5mV/s in an ethanol solution containing 5mM n-hexylamine within a potential range of-0.2-1.6V until a stable cyclic voltammogram is obtained, and finishing the modification of the alkyl chain to the glassy carbon fiber electrode. And then repeatedly washing the electrode with ethanol and deionized water for multiple times, then sequentially soaking in the ethanol and the deionized water for 30min to remove substances adsorbed on the surface of the electrode, and finally drying the electrode for later use. When the first circle is scanned, an obvious oxidation peak can be found at a potential of about 1.4V, which shows that amino at the tail end of N-hexylamine loses 1 electron to be changed into an amino free radical, and the amino free radical reacts with carbon on the surface of a carbon fiber electrode to form a C-N bond, so that the N-hexylamine is modified on the surface of the electrode to prepare the carbon fiber electrode modified by the alkyl chain. The contact angle of the surface of the electrode with water is measured to be 85 degrees and is 64 degrees higher than the contact angle of the bare electrode with water, which shows that after n-hexylamine is modified on the electrode, the alkyl chain of the n-hexylamine is vertically and closely arranged on the surface of the electrode, and the electrode is longThe carbon chains face the outside of the electrode, and the hydrophobic degree of the electrode is increased.
The carbon fiber modified with the n-hexane chain on the surface is soaked in PBS buffer solution containing 1 mu M aptamer for 12h at room temperature, and then the electrode is thoroughly washed by PBS and deionized water. Then sealing in 0.5mM cholesterol-polyethylene glycol solution for 1h, washing with deionized water, storing the prepared sensing electrode in PBS solution, and storing in a refrigerator at 4 ℃ for later use. The water contact angle of the surface of the finally prepared sensing electrode is 19 degrees.
Electrochemical sensing performance determination of sensing electrode
The prepared sensing electrode is used as a working electrode, a platinum wire is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, an amperometric method is adopted on an electrochemical workstation, the applied potential is +0.3V, the electrochemical response of Dopamine (DA) with different concentrations is detected in artificial cerebrospinal fluid, and a relation graph between the current response and the dopamine concentration is drawn (figure 2). When different concentrations of dopamine are added into the system, the current response is rapidly and stepwise increased (graph A in figure 2), and in the concentration range of 0.5 mu M to 2 mu M, the current response signal of the sensing electrode is linearly related to the concentration of dopamine (graph B in figure 2).
The results of fig. 2 show that the sensing electrode prepared by the invention has good detection sensitivity under physiological conditions, and the change range of the dopamine concentration in the brain is in the linear response range, so that the electrode can meet the requirement of in vivo electrochemical detection.
Specific selection performance determination of sensing electrode
The specific and selective detection capability of the prepared sensing electrode is further researched, an amperometric method is used in an experiment, the sensing electrode is used as a working electrode, a platinum wire is used as a counter electrode, an Ag/AgCl electrode is used as a reference electrode, the selection voltage is +0.3V, and the current response condition that Dopamine (DA) and common detection interferents, namely dihydroxy phenylacetic acid (DOPAC), Ascorbic Acid (AA), levodopa (L-DOPA) and Norepinephrine (NE), are sequentially and continuously added into artificial cerebrospinal fluid is observed. As shown in a diagram in fig. 3, when the target substance dopamine is added, the current signal is obviously increased, the signal source is the current signal generated by oxidation of the dopamine aptamer capturing dopamine to the electrode surface, and when other interferents are sequentially added to the solution, the current does not change obviously, which indicates that the sensing electrode has good selective sensing capability.
The difference in response of the sensing electrode (AptCFE) to different concentrations of interferents was further compared to the bare Carbon Fiber Electrode (CFE). The current response of 10 μ M dopamine by two electrodes was selected as reference, and the current response values were normalized, the results are shown in B in fig. 3. Compared with a bare Carbon Fiber Electrode (CFE), the response of the sensing electrode to interferents with different concentrations is obviously reduced, particularly the selectivity to the interferents NE and L-DOPA is obviously improved by 4 times and 3 times respectively, and the sensing electrode has good selectivity.
Sensing electrode for detecting dopamine in living body
The in-vivo dopamine sensing capacity of the prepared sensing electrode is explored, the animals are anesthetized by chloral hydrate and fixed by using a stereo positioning instrument before in-vivo detection, the dual-polarization stimulation electrode is implanted into the inner lateral forebrain bundle (MFB), the sensing electrode is implanted into the nucleus accumbens (NAc), and the Ag/AgCl reference electrode and the platinum wire counter electrode are positioned in the dura mater. The stimulation electrodes applied a 3 second pulsed stimulation (frequency 60Hz, current intensity ± 250 μ Α) to trigger dopamine release from the NAc area. Then, the amperometry was used to monitor the change in current response due to the change in dopamine levels during the electrical stimulation at a potential of + 0.3V. After electrical stimulation, the current signal at the sensing electrode rises rapidly, peaking at about 2 μ M in a few seconds, and then falls rapidly to the base level (fig. 4, panel B). The current signal rise is caused by that the electrical stimulation is carried out on the MFB brain area to cause the Dopamine (DA) level in the NAc brain area to rise rapidly (A picture in figure 4), the observed dopamine release amount is consistent with that of the previous research, and the fact that the sensing electrode can be used for sensing the dopamine serving as the in-vivo electrochemical neurotransmitter is proved, and the sensing electrode has good space-time resolution.
Example 2
The aptamer is a DNA tetrahedral nano structure, and the DNA sequence of the nucleotide chain is shown as SEQ ID No.2-SEQ ID No. 5:
SEQ ID No.2：TAT CAC CAG GCA GTT GAC AGT GTA GCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C TT–Chol
SEQ ID No.3：TCA ACT GCC TGG TGA TAA AAC GAC ACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C TT–Chol
SEQ ID No.4：TTC AGA CTT AGG AAT GTG CTT CCC ACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T TT–Chol
SEQ ID No.5：GGA CGA CGC CAG TTT GAA GGT TCG TTC GCA GGT GTG GAG TGA CGT CGT CC TTT TTT ACA TTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAG AGC CGC CAT AGT A
the 3' -ends of the nucleotide chains shown in SEQ ID No.2-SEQ ID No.4 are labeled with cholesterol (Chol).
Preparation of DNA tetrahedral nanostructures: the tetrahedral DNA structure constructed in this example was prepared by mixing four DNA strands (SEQ ID No.2-SEQ ID No.5) in 1 XPBS solution +5mM MgCl2(pH 7.4) was heated to 95 ℃ for 5min and then slowly cooled to room temperature, wherein the final concentration of each DNA added was 1. mu.M. The resulting DNA product was stored at 4 ℃ until use.
Preparation of a sensing electrode:
the process of cleaning and activating the bare carbon fiber electrode was carried out as described in example 1.
And (3) carrying out 5-circle electrochemical cyclic voltammetry scanning on the activated electrode in an ethanol solution containing 5mM of heptamine and 0.1M of lithium perchlorate at a scanning speed of 10mV/s and in a voltage range of-0.2-1.6V to obtain the heptane-based chain modified carbon fiber electrode. And then repeatedly washing the electrode with ethanol and deionized water for multiple times, then sequentially soaking in the ethanol and the deionized water for 30min to remove substances adsorbed on the surface of the electrode, and finally drying the electrode for later use.
The heptane-based chain modified carbon fiber electrode was incubated for 4h in PBS buffer containing previously prepared 1 μ M DNA tetrahedral nanostructures, and the electrode was rinsed with PBS solution and deionized water. Then blocking in 1mM cholesterol-polyethylene glycol solution for 1h to prepare the sensing electrode, as shown in FIG. 5.
Electrochemical sensing performance determination of sensing electrode
The sensing electrode prepared in example 2 was used as a working electrode, a platinum wire was used as a counter electrode, an Ag/AgCl electrode was used as a reference electrode, and an amperometric method was used at an electrochemical workstation to apply a potential of +0.3V, so that the electrochemical response of dopamine at a different concentration was detected in artificial cerebrospinal fluid, and a graph was drawn showing the relationship between the current response and the dopamine concentration (fig. 6). When different concentrations of dopamine were added to the system, the current response rose rapidly and in steps (graph a in fig. 6), and the electrode current response signal and the dopamine concentration were linearly related in the concentration range of 1 μ M to 10 μ M (graph B in fig. 6). The result shows that the electrode has good detection sensitivity under physiological conditions and can meet the requirement of in vivo electrochemical detection.
Specific selection performance determination of sensing electrode
Specific selectivity of bare carbon fiber electrode (bare CFE), single-stranded aptamer electrode (ssDNA/CFE) prepared in example 1 and sensing electrode with DNA tetrahedron structure with 1 (1-Chol/CFE), 2 (2-Chol/CFE) or 3 (3-Chol/CFE) cholesterol number is compared and studied, platinum wire is used as a counter electrode, Ag/AgCl electrode is used as a reference electrode, the selection voltage is +0.3V, and Dopamine (DA) and common detection interferent dihydroxy phenylacetic acid (DOPAC), Ascorbic Acid (AA), epinephrine (E) and Norepinephrine (NE) current response conditions are observed to be sequentially and continuously added into artificial cerebrospinal fluid.
The upper graph in fig. 7 shows that when the sensing electrode with the DNA tetrahedron structure prepared in example 2 is used as a working electrode, a current signal is significantly increased after a target substance Dopamine (DA) is added, a signal source is a current signal generated by oxidation after dopamine is captured by a dopamine aptamer to the surface of the electrode, and the current does not change significantly after other interferents are sequentially added to a solution, which indicates that the sensing electrode has good selective sensing capability.
In the lower graph of fig. 7, it is shown that unmodified bare carbon fiber electrodes have obvious responses to interferents such as DOPAC, AA, E (epinephrine), NE and the like, while the response of sensing electrodes to the interferents is remarkably reduced in the invention, which proves that the selectivity of the carbon fiber electrode is improved by introducing the aptamer on the surface of the electrode. And compared with the single-stranded aptamer sensing electrode prepared in example 1, the response of DNA tetrahedral sensing electrodes with different cholesterol numbers to interferents in example 2 is reduced to different degrees, wherein the response of DNA tetrahedral sensing electrodes with 3 cholesterol numbers to interferents is minimum. The universality of the aptamer modified electrode strategy of the invention is proved, and the introduction of DNA tetrahedron can further improve the electrode selectivity.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
SEQUENCE LISTING
<110> university of Beijing teachers
<120> sensing electrode suitable for in-situ detection of living body and preparation method and application thereof
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Claims (10)
1. A sensing electrode adapted for in vivo testing, comprising:
the surface of the carbon fiber electrode is modified with a first hydrophobic group;
an aptamer modified with a second hydrophobic group;
the aptamer modified with the second hydrophobic group is self-assembled on the surface of the carbon fiber electrode modified with the first hydrophobic group through hydrophobic interaction.
2. The sensing electrode suitable for in-situ detection of a living body according to claim 1, wherein the contact angle of the carbon fiber electrode with the surface modified with the first hydrophobic group and water is 65-120 °;
preferably, the first hydrophobic group is an alkyl chain.
3. The sensing electrode suitable for in vivo detection as claimed in claim 1, wherein the second hydrophobic group is selected from cholesterol, tocopherol or phospholipid polyethylene glycol derivatives; preferably, the number of modified second hydrophobic groups on the single aptamer is 1 to 3.
4. The sensing electrode suitable for in vivo detection as claimed in claim 2, wherein the alkyl chain is modified on the surface of the bare carbon fiber electrode by the following way:
placing the activated bare carbon fiber electrode in an ethanol solution containing alkylamine, and performing cyclic voltammetry scanning for 5-10 circles within a voltage range of-0.2-1.6V at a scanning speed of 5 mV/s; preferably, lithium perchlorate is used as a supporting electrolyte in an ethanol solution of alkylamine.
5. The sensing electrode suitable for in vivo detection as claimed in claim 4, wherein the alkylamine is a primary amine compound; preferably, the alkylamine is selected from n-hexylamine, heptylamine or nonylamine.
6. The sensing electrode suitable for in vivo detection as claimed in claim 4, wherein the activation of the bare carbon fiber electrode comprises the steps of: soaking the cleaned bare carbon fiber electrode in a 1.0M NaOH solution, and performing ampere method treatment for 80-100 s by applying a voltage of +1.5V to perform primary activation; then, a plurality of cycles of cyclic voltammograms are scanned at a scanning speed of 50mV/s in a voltage range of 0 to +1V until a stable cyclic voltammogram is obtained.
7. A method for preparing a sensing electrode suitable for in vivo detection as defined in any one of claims 1 to 6, comprising the steps of:
soaking the carbon fiber electrode with the surface modified with the first hydrophobic group in a buffer solution containing a nucleic acid aptamer, and cleaning the electrode by using the buffer solution and deionized water after 8-16 h;
and sealing the cleaned electrode in 0.5-2mM solution of amphipathic molecules containing second hydrophobic groups for 0.5-2h, and cleaning with deionized water to obtain the sensing electrode.
8. The method according to claim 7, wherein the concentration of the aptamer in the aptamer-containing buffer is 500nM to 1. mu.M.
9. A method of detecting a neurotransmitter in a living subject, comprising:
measuring the neurotransmitter concentration by measuring the current response signal between the working electrode and the counter electrode using the sensing electrode of any one of claims 1 to 6 as the working electrode, the platinum wire as the counter electrode and the Ag/AgCl electrode as the reference electrode.
10. The method according to claim 9, wherein the specific method for detecting the neurotransmitter concentration is cyclic voltammetry and amperometry.
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