CN109596685B - Electrochemical sensor for detecting ATP and preparation method thereof - Google Patents
Electrochemical sensor for detecting ATP and preparation method thereof Download PDFInfo
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- 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
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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
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Abstract
The invention relates to the technical field of biosensors, in particular to a biosensor for detecting ATP by amplifying ATP to approximate Apt-1 and Apt-2 and a branched HCR (human chorionic gonadotropin) signal, wherein a CP-SH layer, an AP layer, an HP1-HP2-HP3-HP4 layer and [ Ru (NH) are sequentially modified on an electrode3)6]3+And (3) a layer. The preparation method comprises the following steps: pretreating the electrode; modifying the CP-SH layer on the surface of the electrode; modifying an AP layer on the surface of an electrode, modifying an HP1-HP2-HP3-HP4 layer on the surface of the electrode, and modifying [ Ru (NH)3)6]3+The layer is decorated to the electrode surface. The high specificity detection of the target ATP is realized by utilizing the specificity recognition of the aptamer; the ATP is used for drawing the two aptamers close, so that the cyclic utilization of the target is realized, and the effect of signal amplification in the first step is achieved. And the branched HCR is utilized to play a role in continuously enhancing signals.
Description
Technical Field
The invention relates to the technical field of electrochemical sensors, in particular to an electrochemical sensor for detecting ATP based on adjacent induced strand displacement and branched HCR, and also relates to a preparation method thereof.
Background
Adenosine Triphosphate (ATP), the most important energy source in cells, plays an important role in intracellular metabolic, synthetic and biochemical processes. Notably, ATP is indicative of a number of human diseases. Typically, abnormal ATP concentrations are strongly associated with diseases such as parkinson's disease, hypoglycemia, hypoxia, ischemia, cardiovascular disease, etc. Therefore, it is urgent and important to construct an effective assay method capable of detecting trace amounts of ATP.
At present, commonly used ATP detection methods comprise mass spectrometry, high performance liquid chromatography and the like, and the methods have the problems of expensive instruments, long analysis period, complex sample pretreatment, high detection cost and the like, and are difficult to adapt to the requirements of convenience, rapidness, sensitivity and the like of ATP detection. Therefore, it is urgently needed to establish a method for detecting trace ATP rapidly, accurately, sensitively and highly specifically.
Disclosure of Invention
In order to solve the problems of low specificity and sensitivity and high cost of the ATP detection method in the prior art, the invention provides the ATP detection electrochemical sensor based on the adjacent induction strand displacement and branch HCR, which has high specificity and sensitivity, low cost and high detection speed. Meanwhile, a preparation method of the electrochemical sensor for detecting ATP based on adjacent induced strand displacement and branched HCR is also provided.
An electrochemical sensor for detecting ATP is characterized in that a CP-SH layer, an AP layer, an HP1-HP2-HP3-HP4 layer and [ Ru (NH) are sequentially modified on an electrode3)6]3+A layer;
the AP layer is obtained by the reaction of homogeneous AP solution, and the raw materials comprise Apt-1, Apt-2, AP-TP hybrid double chains, FP and a target object to be detected;
the CP-SH is a sulfydryl modified at the 5' end of CPHP 1;
the base sequence is as follows:
the Apt-1 sequence is shown in SEQ No. 1;
the Apt-2 sequence is shown in SEQ No. 2;
the sequence of TP is shown in SEQ No. 3;
the AP sequence is shown as SEQ No. 4;
the FP sequence is shown in SEQ No. 5;
the HP1 sequence is shown in SEQ No. 6;
the HP2 sequence is shown in SEQ No. 7;
the HP3 sequence is shown in SEQ No. 8;
the HP4 sequence is shown in SEQ No. 9;
the CP-SH sequence is shown in SEQ No. 10.
In the electrochemical sensor, the mole ratio of HP1, HP2, HP3 and HP4 in an HP1-HP2-HP3-HP4 layer is 1: 1: 1: 1.
the preparation method of the electrochemical sensor comprises the following steps:
(1) pretreating the electrode;
(2) modifying the CP-SH layer on the surface of the electrode;
(3) decorating the AP layer on the surface of the electrode;
(4) modifying an HP1-HP2-HP3-HP4 layer on the surface of the electrode;
(5) will [ Ru (NH)3)6]3+The layer is decorated on the surface of the electrode;
(6) the electrochemical sensor detects ATP.
The operation steps of modifying the CP-SH layer on the surface of the electrode in the step (2) are as follows:
mu.L of 1. mu.M CP-SH was added dropwise to the pretreated electrode surface, incubated at 37 ℃ for 2h, and washed.
The step (3) of modifying the AP layer on the surface of the electrode comprises the following operation steps:
s1 adding 2 mul of 1 XPBS buffer solution, 1 mul Apt-1, 1 mul Apt-2, 1 mul AP-TP hybrid double chains, 1 mul FP and the target object to be detected and 8 mul of sterilized water into a centrifuge tube, shaking for 30S, and putting into a thermostat at 37 ℃ for incubation for 2 h;
s2, dropwise adding the incubated mixed solution to the electrode modified with the CP-SH layer, continuously placing the electrode in a thermostat at 37 ℃ for incubation for 2h, and cleaning.
The operation steps of modifying the HP1-HP2-HP3-HP4 layer on the surface of the electrode in the step (4) are as follows:
adding 2 mu L of 1 XPBS buffer solution, 1 mu M HP1, 1 mu M HP2, 1 mu M HP3 and 1 mu M HP4 into a centrifuge tube, shaking for 30s, dripping onto the modified AP-CP-SH layer electrode, continuously incubating the electrode in a thermostat at 37 ℃ for 2h, and washing.
The step (5) is to react [ Ru (NH)3)6]3+The operation steps of layer modification to the electrode surface are as follows:
mu.L of 50. mu.M [ Ru (NH) ]3)6]3+Dripping onto the electrode after the reaction in step (4), incubating the electrode in a 37 deg.C incubator for 10min, and washing.
The step (1) of pretreating the electrode is to polish the electrode in alumina slurry with the particle size of 0.3 μm and 0.05 μm until the electrode is a mirror surface, and wash the mirror surface with PBS and secondary water.
The electrode in the step (1) is a gold electrode.
The detection condition of the step (6): and taking Ag/AgCl as a reference electrode and a Pt electrode as a counter electrode, setting the potential to be 0-0.5V, the pulse width to be 0.05V and the scanning rate to be 0.06s, reading the change of an electric signal by adopting a differential pulse voltammetry, and detecting the target object to be detected.
Wherein: the PBS buffer was prepared by conventional methods: na (Na)2HPO4(10mM)，NaH2PO4(10mM)，NaCl(140mM)，KCl(1mM)，MgCl2(1mM)，CaCl2(1mM), the pH of the final solution was 7.4. In addition, the prepared PBS buffer solution and ultrapure water are required to be subjected to high-temperature sterilization treatment. The specific method is that PBS and ultrapure water are respectively placed in different conical flasks, and then the sealing is carried out by using tinfoil paper and newspaper. Sterilizing in autoclave at 120 deg.C for 20 min.
The detection mode of the invention is electrochemical detection, and a traditional three-electrode system is utilized. Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, and a modified gold electrode is used as a working electrode. Before detection, CP-SH is first fixed to the electrode surface by Au-S bonds. The homogeneous AP solution after reaction is modified on the surface of an electrode, and then the AP is incubated for 2h at 37 ℃ to open CP-SH. Then, the solution of HP1-HP2-HP3-HP4 is added dropwise and incubated at 37 ℃ for 2h to form branched HCR. Adding [ Ru (NH)3)6]3+After that, incubation was continued for 10 min. The signal peak was then detected with a three electrode working system. Setting the potential to be 0-0.5V, the pulse width to be 0.05V and the scanning rate to be 0.06s, reading the change of an electric signal by adopting a differential pulse voltammetry, and detecting the target object to be detected.
The invention has used 10 pieces of DNA chains altogether, its sequence is respectively:
Apt-1:5’-ACC TGG GGG AGT ATA TAA GCA CCA CAT CTC AT-3’
Apt-2:5’-CCT TAT GCT GCT TAT TTG CGG AGG AAG GT-3’
TP:5’-CAG TAT GAG ATG TGG TAG CCA GTA TGA GAT GTG GTA GCA TAA GG-3’
AP:5’-AAT AGC TAC CAC ATC TCA TAC TG-3’
FP:5’-GCT ACC ACA TCT CAT ACT GGC TAC CAC ATC TCA TAC TG-3’
HP1:5’-CAG TAT GAG ATG TGG TAG GCT TAG TTA ACG
-3’
HP2:5’-CTA CCA CAT CTC ATA CTG 
TCC ATC-3’
HP3:5’-TTA ACG TCC ATC AGT TTC 
-3’
HP4:5’-GAA ACT GAT GGA CGT TAA 
-3’
CP-SH:5’-SH-TTTTT CAG TAT GAG ATG TGG TAG GCT TAG TTA ACG
-3’
the single underlined parts of Apt-1 and Apt-2 are aptamers cleaved by ATP, and in the presence of ATP, the single underlined parts can be pulled close to Apt-1 and Apt-2, so that the two double underlined sequences are adjacent to each other and jointly enter the next reaction.
The double-underlined and single-underlined portions of the TP are identical sequences and can simultaneously hybridize to the single-underlined portion of the AP to form a double strand. This part requires hybridization before it can be added to the reaction system. After the ATP is pulled close to Apt-1 and Apt-2, the pulled ends can hybridize to the italic portion of the TP, replacing the AP hybridized to the single underlined portion of the TP. FP then hybridizes to the single and double underlined portion of the TP, replacing both the AP and Apt-1-Apt-2-ATP portions of the double underlined portion of the TP.
The single underlined sequence of AP opens the single underlined sequence of HP1, the double underlined sequence of HP1 opens the double underlined portion of HP2, the single underlined portion of HP2 opens and continues to open HP1, forming a first step HCR (hybrid chain reaction). The underline portions of both HP1 and HP2 are connected by HCR to open the double underline portion of HP3, the open double underline sequence of HP3 opens the double underline portion of HP4, and the single underline portion of HP4 continues to open HP3, forming a second HCR, i.e., a branched HCR portion.
The 5' end of the CP-SH is modified with sulfydryl (-SH) which can form a stable Au-S bond with the gold electrode so as to be fixed on the surface of the electrode; it has the same sequence and functions as HP1, and SH is modified to immobilize the resulting branched HCR moieties on the electrode surface. Adding trichlorohexaammine ruthenium [ Ru (NH)3)6]3+The probe can be electrostatically adsorbed on a DNA chain, and an obviously enhanced electric signal can be obtained by an electrochemical differential pulse voltammetry, namely the aim of quantitatively detecting a target object is fulfilled by detecting the signal.
The reactions that occur in homogeneous phases are mainly: in the presence of the target ATP, Apt-1 and Apt-2 are pulled by ATP, and the AP hybridized with TP single underline in the TP-AP double chain is jointly replaced. After addition of FP, FP can continue to replace the AP and Apt-1-Apt-2-ATP portions that hybridize double underlined to TP. Apt-1-Apt-2-ATP continues to replace TP-AP, which is the first loop amplification. The released two portions of AP are able to open the CP-SH that has been modified on the electrode. After addition of HP1, HP2, HP3, HP4, AP-HP1-HP2-HP3-HP4 branched HCR aggregates were formed, which is the second step of signal amplification. Adding [ Ru (NH)3)6]3+It is then capable of electrostatic adsorption on the DNA strand, resulting in a significantly enhanced electrical signal. In the homogeneous reaction, the reaction conditions were all 37 ℃ and the reaction time was 2 h.
The ATP detection is realized on the electrode, and the signal is increased in a one-step cycle and one-step amplification mode, so that the high-sensitivity ATP detection is realized, and a lower detection lower limit is obtained.
The invention has the beneficial effects that:
1. detection limit is low
The high specificity detection of the target ATP is realized by utilizing the specificity recognition of the aptamer; the ATP is used for drawing the two aptamers close, so that the cyclic utilization of the target is realized, and the effect of signal amplification in the first step is achieved; by utilizing the branched HCR reaction, the second-step amplification of the signal is realized, the high-sensitivity detection of a target object is realized, and the detection sensitivity is improved; the detection limit was 1 pM.
2. The detection speed is high
The main process of the detection principle is realized on the electrode, so that the reaction speed is improved, the complexity of operation is reduced, and the rapid, simple and sensitive detection of the target object is realized; the sensor has mild reaction conditions and high reaction speed.
3. The gold electrode is used in the industrial production, so that the electrode is simple, convenient, miniaturized, easy to carry and capable of being used for multiple times; the preparation method is simple, stable in performance and good in electrode repeatability, and is suitable for the detection of trace ATP and the practical application of biosensor industrialization; the process for manufacturing the electrode has low cost and is suitable for the requirement of low price in industrialization.
Drawings
FIG. 1 is a schematic diagram of the experiment;
FIG. 2 is a diagram showing the results of CP-SH concentration optimization detection in example 1;
FIG. 3 is a graph showing the results of the optimized assay of HP1 concentration in example 2;
FIG. 4 is a graph showing the operation of the sensor for detecting ATP according to example 3.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
A preparation method of the electrochemical biosensor comprises the following steps:
a. polishing the gold electrode in 0.3 and 0.05 μm alumina slurry until the gold electrode is mirror-finished, and repeatedly washing with PBS and secondary water;
b. mu.L (0. mu.M, 0.5. mu.M, 1. mu.M, 1.5. mu.M, 2. mu.M) of CP-SH was added dropwise to the electrode surface, incubated at room temperature for 2h, and washed.
The modification process of the electrode is described in the paragraph, and the main steps in the homogeneous reaction are as follows:
a. adding 1 XPBS buffer solution, 1 mu M Apt-1, 1 mu M Apt-2, 1 mu M AP-TP hybrid double chains, 2 mu L of each of 1 mu M FP and the target object to be detected and 8 mu L of sterilized water into a centrifuge tube, oscillating for 30s, and putting into a thermostat at 37 ℃ for incubation for 2 h;
b. and (b) dropwise adding the solution (10 mu L) obtained after the reaction in the step (a) onto the electrode which is modified with CP-SH in advance. Then continuously placing the electrode in a constant temperature box at 37 ℃ for 2 h;
c. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
d. adding 2 mu L of 1 XPBS buffer solution, 1 mu M HP1, 1 mu M HP2, 1 mu M HP3 and 1 mu M HP4 into a centrifuge tube, shaking for 30s, dripping onto the electrode reacted in the step c, and continuously putting the electrode into a thermostat at 37 ℃ to incubate for 2 h;
e. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
f. mu.L of 50. mu.M [ Ru (NH) ]3)6]3+Dripping the solution on the electrode after the reaction in the step e, and then continuously placing the electrode in a thermostat at 37 ℃ for 10 min;
g. the electrodes were washed 3 times with a magnetic stirrer in PBS solution for 10min each time.
And (2) detecting a signal by using a Differential Pulse Voltammetry (DPV) by using Ag/AgCl as a reference electrode and a Pt electrode as a counter electrode, wherein the base solution for the DPV detection is PBS (10mM, and the pH is 7.4), the potential is set to be 0-0.5V, the scanning rate is 0.06s, and the pulse width is 0.05V.
As a result, as shown in FIG. 2, it can be seen that the detected current signal increases as the CP-SH concentration increases in the interval of 0-1. mu.M, and when the concentration exceeds 1. mu.M, the current tends to stabilize, so that the optimum concentration of CP-SH is 1. mu.M.
Example 2
A preparation method of the electrochemical biosensor comprises the following steps:
a. polishing the gold electrode in 0.3 and 0.05 μm alumina slurry until the gold electrode is mirror-finished, and repeatedly washing with PBS and secondary water;
b. 10 μ L of 1 μ M CP-SH was added dropwise to the electrode surface, incubated at room temperature for 2h, and washed.
The modification process of the electrode is described in the paragraph, and the main steps in the homogeneous reaction are as follows:
a. adding 1 XPBS buffer solution, 1 mu M Apt-1, 1 mu M Apt-2, 1 mu M AP-TP hybrid double chains, 2 mu L of each of 1 mu M FP and the target object to be detected and 8 mu L of sterilized water into a centrifuge tube, oscillating for 30s, and putting into a thermostat at 37 ℃ for incubation for 2 h;
b. and (b) dropwise adding the solution (10 mu L) obtained after the reaction in the step (a) onto the electrode which is modified with CP-SH in advance. Then continuously placing the electrode in a constant temperature box at 37 ℃ for 2 h;
c. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
d. adding 1 XPBS buffer solution, (0 mu M, 0.5 mu M, 1 mu M, 1.5 mu M, 2 mu M) HP1, 1 mu M HP2, 1 mu M HP3 and 2 mu L of 1 mu M HP4 into a centrifuge tube, shaking for 30s, dripping the solution onto the electrode of the reaction in the step c, and continuously placing the electrode in an incubator at 37 ℃ for incubation for 2 h;
e. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
f. mu.L of 50. mu.M [ Ru (NH) ]3)6]3+Dripping the solution on the electrode after the reaction in the step e, and then continuously placing the electrode in a thermostat at 37 ℃ for 10 min;
g. the electrodes were washed 3 times with a magnetic stirrer in PBS solution for 10min each time.
And (2) detecting a signal by using a Differential Pulse Voltammetry (DPV) by using Ag/AgCl as a reference electrode and a Pt electrode as a counter electrode, wherein the base solution for the DPV detection is PBS (10mM, and the pH is 7.4), the potential is set to be 0-0.5V, the scanning rate is 0.06s, and the pulse width is 0.05V.
As a result, as shown in FIG. 3, it can be seen that the detected current signal increases as the concentration of HP1 increases in the interval of 0-1 μ M, and when the concentration exceeds 1 μ M, the current tends to stabilize, so that the optimum concentration of HP1 is 1 μ M. In addition, since HP1, HP2, HP3 and HP4 are able to produce branched HCR reactions under the action of AP, the concentration ratio thereof should be 1: 1: 1: 1. Namely: the optimum concentrations of HP1, HP2, HP3 and HP4 were all 1. mu.M.
Example 3
A preparation method of the electrochemical biosensor comprises the following steps:
a. polishing the gold electrode in 0.3 and 0.05 μm alumina slurry until the gold electrode is mirror-finished, and repeatedly washing with PBS and secondary water;
b. 10 μ L of 1 μ M CP-SH was added dropwise to the electrode surface, incubated at room temperature for 2h, and washed.
The modification process of the electrode is described in the paragraph, and the main steps in the homogeneous reaction are as follows:
a. adding 2 mu L of 1 XPBS buffer solution, 1 mu M Apt-1, 1 mu M Apt-2, 1 mu M AP-TP hybrid double chains, 1 mu M FP, a target substance to be detected (0pM, 1pM, 10pM, 100pM, 1nM, 10nM, 1 mu M, 5 mu M) and 8 mu L of sterilized water into a centrifuge tube, shaking for 30s, and putting the centrifuge tube into a thermostat at 37 ℃ for incubation for 2 h;
b. and (b) dropwise adding the solution (10 mu L) obtained after the reaction in the step (a) onto the electrode which is modified with CP-SH in advance. Then continuously placing the electrode in a constant temperature box at 37 ℃ for 2 h;
c. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
d. adding 2 mu L of 1 XPBS buffer solution, 1 mu M HP1, 1 mu M HP2, 1 mu M HP3 and 1 mu M HP4 into a centrifuge tube, shaking for 30s, dripping onto the electrode reacted in the step c, and continuously putting the electrode into a thermostat at 37 ℃ to incubate for 2 h;
e. washing the electrode in PBS solution with magnetic stirrer for 10min each time for 3 times;
f. mu.L of 50. mu.M [ Ru (NH) ]3)6]3+Dripping the solution on the electrode after the reaction in the step e, and then continuously placing the electrode in a thermostat at 37 ℃ for 10 min;
g. the electrodes were washed 3 times with a magnetic stirrer in PBS solution for 10min each time.
And (2) detecting a signal by using a Differential Pulse Voltammetry (DPV) by using Ag/AgCl as a reference electrode and a Pt electrode as a counter electrode, wherein the base solution for the DPV detection is PBS (10mM, and the pH is 7.4), the potential is set to be 0-0.5V, the scanning rate is 0.06s, and the pulse width is 0.05V.
The results are shown in FIG. 4. As can be seen from FIG. 4A, the detected current signal increases with increasing concentration of the target in the interval 1pM to 1. mu.M, and FIG. 4B shows that the logarithm of the ATP concentration is proportional to the magnitude of the current peak, fitting a curve: i ═ 0.83+0.50lg (C/pM) (correlation coefficient 0.95771, where C represents the ATP concentration), while we continued to test towards lower concentrations on the basis of a concentration of 1pM, it was tested that the logarithm of the ATP concentration and the magnitude of the current peak just no longer fit the law of the fitted curve, i.e. the lowest value of the current peak in the graph, when the concentration is below 1 pM. Therefore, the lower detection limit of this method is 1 pM.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
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Claims (10)
1. An electrochemical sensor for detecting ATP is characterized in that a CP-SH layer, an AP layer, an HP1-HP2-HP3-HP4 layer and [ Ru (NH) are sequentially modified on an electrode3)6]3+A layer;
the AP layer is obtained by the reaction of homogeneous AP solution, and the raw materials comprise Apt-1, Apt-2, AP-TP hybrid double chains, FP and a target object to be detected;
the CP-SH is a sulfydryl modified at the 5' end of the CP;
the Apt-1 sequence is shown in SEQ No. 1;
the Apt-2 sequence is shown in SEQ No. 2;
the sequence of TP is shown in SEQ No. 3;
the AP sequence is shown as SEQ No. 4;
the FP sequence is shown in SEQ No. 5;
the HP1 sequence is shown in SEQ No. 6;
the HP2 sequence is shown in SEQ No. 7;
the HP3 sequence is shown in SEQ No. 8;
the HP4 sequence is shown in SEQ No. 9;
the CP-SH sequence is shown in SEQ No. 10.
2. The electrochemical sensor according to claim 1, wherein the mole ratio of HP1, HP2, HP3 and HP4 in the HP1-HP2-HP3-HP4 layer is 1: 1: 1: 1.
3. a method of manufacturing an electrochemical sensor according to claim 1 or 2, comprising the steps of:
(1) pretreating the electrode;
(2) modifying the CP-SH layer on the surface of the electrode;
(3) decorating the AP layer on the surface of the electrode;
(4) modifying an HP1-HP2-HP3-HP4 layer on the surface of the electrode;
(5) will [ Ru (NH)3)6]3+The layer is decorated on the surface of the electrode;
(6) the electrochemical sensor detects ATP.
4. The method according to claim 3, wherein the step (2) of modifying the CP-SH layer on the surface of the electrode comprises the following steps:
mu.L of 1. mu.M CP-SH was added dropwise to the pretreated electrode surface, incubated at 37 ℃ for 2h, and washed.
5. The method according to claim 3, wherein the step (3) of applying the AP layer to the surface of the electrode comprises the following steps:
s1 adding 2 mul of 1 XPBS buffer solution, 1 mul Apt-1, 1 mul Apt-2, 1 mul AP-TP hybrid double chains, 1 mul FP and the target object to be detected and 8 mul of sterilized water into a centrifuge tube, shaking for 30S, and putting into a thermostat at 37 ℃ for incubation for 2 h;
s2, dropwise adding the incubated mixed solution to the electrode modified with the CP-SH layer, continuously placing the electrode in a thermostat at 37 ℃ for incubation for 2h, and cleaning.
6. The method according to claim 3, wherein the step (4) of modifying the HP1-HP2-HP3-HP4 layer on the surface of the electrode comprises the following steps:
adding 2 mu L of 1 XPBS buffer solution, 1 mu M HP1, 1 mu M HP2, 1 mu M HP3 and 1 mu M HP4 into a centrifuge tube, shaking for 30s, dripping onto the modified AP-CP-SH layer electrode, continuously incubating the electrode in a thermostat at 37 ℃ for 2h, and washing.
7. The method according to claim 3, wherein the step (5) is carried out by reacting [ Ru (NH)3)6]3+The operation steps of layer modification to the electrode surface are as follows:
mu.L of 50. mu.M [ Ru (NH) ]3)6]3+Dripping onto the electrode after the reaction in step (4), incubating the electrode in a 37 deg.C incubator for 10min, and washing.
8. The preparation method according to claim 3, wherein the pretreatment operation of the electrode in the step (1) is to polish the electrode in 0.3 μm and 0.05 μm alumina slurry until the electrode is mirror-finished, and to wash the electrode with PBS and secondary water.
9. The method according to claim 3, wherein the electrode of step (1) is a gold electrode.
10. The method according to claim 3, wherein the detection conditions of step (6): and taking Ag/AgCl as a reference electrode and a Pt electrode as a counter electrode, setting the potential to be 0-0.5V, the pulse width to be 0.05V and the scanning rate to be 0.06s, reading the change of an electric signal by adopting a differential pulse voltammetry, and detecting the target object to be detected.
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