CN111721819B - Electrochemical aptamer sensor for simultaneously detecting multiple disease markers - Google Patents

Electrochemical aptamer sensor for simultaneously detecting multiple disease markers Download PDF

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CN111721819B
CN111721819B CN202010537179.XA CN202010537179A CN111721819B CN 111721819 B CN111721819 B CN 111721819B CN 202010537179 A CN202010537179 A CN 202010537179A CN 111721819 B CN111721819 B CN 111721819B
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邱彬
赵华楠
罗子伊
傅志宏
夏伟耀
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Chuang Wei Lai Pingtan Technology Co ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention discloses an electrochemical aptamer sensor for simultaneously detecting multiple disease markers, which comprises the following steps: selecting materials and instruments: the DNA sequence is synthesized by the company of bioengineering GmbH, the name comprises CEA-Apt1 (A1), the sequence is 5'-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATGAAGCATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), the sequence is 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the invention solves the problems that only one disease marker can be independently detected at present, only a plurality of sensors can be utilized when a plurality of diseases are required to be detected, the sensitivity is low, the anti-interference capability is poor, and the responsiveness is poor.

Description

Electrochemical aptamer sensor for simultaneously detecting multiple disease markers
Technical Field
The invention relates to the technical field of medical detection, in particular to an electrochemical aptamer sensor for simultaneously detecting multiple disease markers.
Background
Disease markers play an increasingly important role in cancer risk assessment and screening, improving prognosis, and assessing response to biological therapy. Early and sensitive detection of disease markers can greatly improve the efficiency of treatment of many diseases. Carcinoembryonic antigen (CEA) is a highly glycosylated glycoprotein, highly expressed in a variety of tumors, and is a broad spectrum of disease markers for cancer diagnosis. Platelet-derived growth factor (PDGF), a protein of growth factor found in human platelets, is becoming increasingly important because of its role in regulating cell growth and division. PDGF dimers, consisting of two different types of disulfide-linked polypeptide chains (designated a and B), occur in three subtypes: PDGF-BB, PDGF-AB and PDGF-AA. Among these subtypes, PDGF-BB is directly involved in the cellular transformation process and tumor growth and progression. It is expressed at undetectable or low levels in normal cells, but is found overexpressed in human malignancies.
As a potential protein marker, sensitive and rapid detection of PDGF-BB is particularly important in the early diagnosis, treatment and prognosis of cancer. At present, only one disease marker can be detected independently, a plurality of sensors can be utilized when a plurality of diseases are detected, the sensitivity is poor, the anti-interference capability is poor, and the responsiveness is poor.
Disclosure of Invention
The invention aims to provide an electrochemical aptamer sensor for simultaneously detecting multiple disease markers, and solves the problems that only one disease marker can be independently detected at present, only multiple sensors can be utilized when multiple diseases need to be detected, and the sensitivity is poor, the anti-interference capability is poor and the responsiveness is poor.
In order to achieve the purpose, the invention provides the following technical scheme: an electrochemical aptamer sensor for simultaneous detection of multiple disease markers comprising the steps of:
step 1: selecting materials and instruments: the DNA sequence is synthesized by the biological engineering of biology GmbH, the name of which comprises CEA-Apt1 (A1), 5'-SH-TATCCAGCTTATTCAATT-3' and CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATATACCC-3' and PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGCACCATGATCCTG-3' and PDGF-BB Apt2 (B2), and 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), sugar chain antigen 125 (CA 125), mucoidProtein (MUC 1), platelet-derived growth factor (PDGF-BB), prostate Specific Antigen (PSA), alpha-fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises electrolyte solution and DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a table acidity meter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision pipetting gun;
step 2: and (3) synthesis of PEI-Au: rapidly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au;
and step 3: preparing a metal ion probe: with PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg EDC and 0.25g NHS into 100 mu L of 10 mu M A2 solution, mixing uniformly, and reacting for 1h at 37 ℃;100 μ L PEI-Au was added to the above system, shaken at 37 ℃ at 300rpm for 12h, centrifuged at 8000rpm for 20min to remove unattached A2, and redispersed in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, after shaking reaction at 37 ℃ and 300rpm for 12h and reaction at 8000rpm for 20min, centrifuging and washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use;
and 4, step 4: preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using alumina powder with the diameter of 0.3 mu m and alumina powder with the diameter of 0.05 mu m respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the gold electrode is dried by nitrogen, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, blowing dry with nitrogen, and dripping 10.0 μ L of target (CEA or CEA) with different concentrationsPDGF-BB), rinsing the electrode with ultrapure water, and drying the electrode with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use;
and 5: detection of single disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are 0.0V-0.5V and-0.5-0.2V respectively;
step 6: detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, wherein the DPV scanning range of simultaneous detection is-0.6V.
Preferably, in step 1, the reagent and the solvent are both analytically pure and can be used without further purification.
Preferably, in step 1, the experimental water is ultrapure water (resistivity 18.2 M.OMEGA.. Multidot.cm) purified by Millipore Milli-Q system.
Preferably, in step 1, the electrolyte solution (Tris-HCl buffer, pH = 7.4) is 0.1mol/L Tris,0.5mol/L NaCl,0.1mol/L KCl,0.1mol/L MgCl 2 。
Preferably, in step 1, the DNA buffer (1X NEBuffer 2): 50mmol/L NaCl,10mmol/L Tris-HCl,10mmol/L MgCl 2 1mmol/L DTT, pH 7.9.
Preferably, in the step 1, the model of the digital display intelligent temperature control magnetic stirrer is SZCL-4, the manufacturer is Zhengzhou Kehua Instruments and equipment Limited company, the model of the transmission electron microscope is HT7700, the manufacturer is TEM, HITACHI, japan and the model of the desktop acidometer is PB-10, the manufacturer is German Saedolis Instruments and equipment Limited company, the model of the electrochemical workstation is CHI-660A, the manufacturer is CH Instruments, USA, milli-Q, the model of the ultrapure water system is Academic, the manufacturer is Michibo China Limited company, the model of the precision pipetting gun is Eppendorf, and the manufacturer is Aibend China Limited company.
Preferably, when the target substance to be added dropwise in step 4 is CEA, the reaction is carried out at 37 ℃ for 90min.
Preferably, in step 4, the preparation method can be used for constructing a sensor of a single target object PDGF-BB or CEA, and can also be used for simultaneously detecting multiple target objects CEA and PDGF-BB.
Preferably, in step 5, the detection of CEA in the range of 0.05ng/mL to 20ng/mL shows a good linear relationship, and the linear equation is as follows: I.C. A pa (μA)＝0.3520+0.0854C(μM)，R 2 =0.990, where C is the CEA concentration, with a detection limit of 16pg/mL (S/N = 3).
Preferably, in step 6, 6.0mmol/L Cu is used 2+ And 30.0mmol/L Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ 。
Compared with the prior art, the invention has the beneficial effects that: the electrochemical aptamer sensor for simultaneously detecting multiple disease markers has good conductivity of gold balls, can enhance conductivity, accelerates transfer of electrons on the surface of an electrode, is simple in operation, high in sensitivity, good in responsiveness and strong in anti-interference capability, and can realize the advantages of detection of a single target and simultaneous detection of two targets.
Detailed Description
The present invention will now be described in more detail by way of examples, which are given by way of illustration only and are not intended to limit the scope of the present invention in any way.
The invention provides a technical scheme that: an electrochemical aptamer sensor for simultaneous detection of multiple disease markers comprising the steps of:
step 1: selecting materials and instruments: the DNA sequence is synthesized by the biological engineering of biology GmbH, the name of which comprises CEA-Apt1 (A1), 5'-SH-TATCCAGCTTATTCAATT-3' and CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATATACCC-3' and PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGCACCATGATCCTG-3' and PDGF-BB Apt2 (B2), and 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), carbohydrate chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate-specific antigen (PSA), alpha-fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises electrolyte solution and DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a desk acidimeter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision liquid-transferring gun;
step 2: and (3) synthesis of PEI-Au: quickly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au;
and step 3: preparing a metal ion probe: with PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg of EDC and 0.25g of NHS into 100 mu L of 10 mu M A2 solution, uniformly mixing, and reacting for 1h at 37 ℃; adding 100 μ L PEI-Au to the above system, shaking at 37 deg.C and 300rpm for reaction 12h, and centrifuging at 8000rpm for 20min to remove non-attached PEI-AuA2, redispersion in 200.0. Mu.L of Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, after shaking reaction at 37 ℃ and 300rpm for 12h and reaction at 8000rpm for 20min, centrifuging and washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use;
and 4, step 4: preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using 0.3 mu m and 0.05 mu m of alumina powder respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dripping 10.0 mu L of target substances (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying the electrode with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use;
and 5: detection of individual disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are 0.0V-0.5V and-0.5-0.2V respectively;
and 6: detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain metal ion probes for simultaneous detection, and DPV scanning for simultaneous detectionThe range is-0.6V.
The first embodiment is as follows:
selecting materials and instruments: the DNA sequence is synthesized by the company of bioengineering GmbH, the name comprises CEA-Apt1 (A1), the sequence is 5'-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATGAAGCATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), the sequence is 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), sugar chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate Specific Antigen (PSA), alpha Fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises an electrolyte solution and a DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a desk acidimeter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision liquid-transferring gun; and (3) synthesis of PEI-Au: rapidly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au; preparation of a metal ion probe: with PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg EDC and 0.25g NHS into 100 mu L of 10 mu M A2 solution, mixing uniformly, and reacting for 1h at 37 ℃; adding 100 μ L PEI-Au to the above system, shaking at 37 deg.C and 300rpm for reaction 12h, centrifuging at 8000rpm for 20min to remove non-attached A2, and redispersing in 200.0 μ L Cu (NO) 3 ) 2 In the solution, the reaction was carried out at 37 ℃ for 12h, and the supernatant was removed by centrifugation at 8000rpm for 20min and redispersed in a 1xB2 buffer solution to prepare PEI-Au-A2-Cu 2+ The same method was used to prepare PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, after shaking reaction at 37 ℃ and 300rpm for 12h and reaction at 8000rpm for 20min, centrifuging and washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use; preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using alumina powder with the diameter of 0.3 mu m and alumina powder with the diameter of 0.05 mu m respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, drying with nitrogen, dripping 10.0 μ L of target (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use; detection of single disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are 0.0V-0.5V and-0.5-0.2V respectively; detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, wherein the DPV scanning range of simultaneous detection is-0.6V.
Example two:
in the first embodiment, the following steps are added:
in step 1, the reagents and solvents were all analytical grade, and were used without further purification, and the experimental water was ultrapure water (resistivity 18.2 M.OMEGA.. Multidot.cm) purified by Millipore Milli-Q system, electrolyte solution (Tr)is-HCl buffer, pH = 7.4) 0.1mol/L Tris,0.5mol/L NaCl,0.1mol/L KCl,0.1mol/L MgCl 2 DNA buffer (1X NEBuffer 2): 50mmol/L NaCl,10mmol/L Tris-HCl,10mmol/L MgCl 2 1mmol/L DTT, pH 7.9, the model of a digital display intelligent temperature control magnetic stirrer is SZCL-4, the manufacturer is Zhengzhou Kehua instrument and equipment Limited company, the model of a transmission electron microscope is HT7700, the manufacturer is TEM, HITACHI, the model of Japan and a desktop acidimeter is PB-10, the manufacturer is Germany Saedolis instrument and limited company, the model of an electrochemical workstation is CHI-660A, the manufacturer is CH Instruments, USA, milli-Q and an ultrapure water system is academy, the manufacturer is Millibo China Limited company, the model of a precision pipetting gun is Eppendorf, and the manufacturer is Aibend China Limited company can avoid searching materials which are not prepared in the process of operation.
Selecting materials and instruments: the DNA sequence is synthesized by the biological engineering of biology GmbH, the name of which comprises CEA-Apt1 (A1), 5'-SH-TATCCAGCTTATTCAATT-3' and CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATATACCC-3' and PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGCACCATGATCCTG-3' and PDGF-BB Apt2 (B2), and 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), sugar chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate Specific Antigen (PSA), alpha Fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises electrolyte solution and DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a table acidometer, an electrochemical workstation and Milli-Q ultra-pureWater systems, precision pipette guns; and (3) synthesis of PEI-Au: quickly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au; preparing a metal ion probe: by using PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg EDC and 0.25g NHS into 100 mu L of 10 mu M A2 solution, mixing uniformly, and reacting for 1h at 37 ℃; adding 100 μ L PEI-Au to the above system, shaking at 37 deg.C and 300rpm for reaction 12h, centrifuging at 8000rpm for 20min to remove non-attached A2, and redispersing in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, after shaking reaction at 37 ℃ and 300rpm for 12h and reaction at 8000rpm for 20min, centrifuging and washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use; preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using 0.3 mu m and 0.05 mu m of alumina powder respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying with nitrogen, dripping 10.0 μ L of 1mmol/L MCH, and reacting at 37 deg.C in dark for 40min; rinsing the electrode with ultrapure water, drying with nitrogen, dripping 10.0 μ L of target (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use; detection of individual disease markers: DPV was selected as the detection method in the experiment, and measured using a three-electrode working system (working electrode was Au electrode; reference electrode was Ag/AgCl electrode; counter electrode was Pt electrode), DPV test was performed in 2.0mL of 0.1mol/L Tris-HCl buffer (pH = 7.4), CEA and PDGF-BBThe DPV scanning ranges are respectively 0.0V-0.5V and-0.5-0.2V; detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, wherein the DPV scanning range of simultaneous detection is-0.6V.
Example three:
in the second embodiment, the following steps are added:
in step 4, when the dropwise added target is CEA, the reaction is carried out for 90min at 37 ℃, and the preparation method can be used for constructing a sensor of a single target PDGF-BB or CEA, and can also be used for simultaneously detecting multiple targets of CEA and PDGF-BB, so that the operation can obtain the best effect.
Selecting materials and instruments: the DNA sequence is synthesized by the company of bioengineering GmbH, the name comprises CEA-Apt1 (A1), the sequence is 5'-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATGAAGCATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), the sequence is 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), sugar chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate Specific Antigen (PSA), alpha Fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises electrolyte solution and DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a desk acidimeter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision liquid-transferring gun;and (3) synthesis of PEI-Au: rapidly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au; preparing a metal ion probe: by using PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg of EDC and 0.25g of NHS into 100 mu L of 10 mu M A2 solution, uniformly mixing, and reacting for 1h at 37 ℃;100 μ L PEI-Au was added to the above system, shaken at 37 ℃ at 300rpm for 12h, centrifuged at 8000rpm for 20min to remove unattached A2, and redispersed in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, shaking at 37 ℃ and 300rpm for reaction for 12h, and centrifuging at 8000rpm for 20min, washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use; preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using 0.3 mu m and 0.05 mu m of alumina powder respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying with nitrogen, dripping 10.0 μ L of 1mmol/L MCH, and reacting at 37 deg.C in dark for 40min; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dripping 10.0 mu L of target substances (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying the electrode with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use; detection of single disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are respectively 0.0V0.5V and-0.5-0.2V; detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to the formula 1:1 to obtain the metal ion probe for simultaneous detection, wherein the DPV scanning range of simultaneous detection is-0.6V.
Example four:
in example three, the following procedure was added:
in step 5, in the range of 0.05 ng/mL-20 ng/mL, the detection of CEA presents a good linear relation, and the linear equation is as follows: i is pa (μA)＝0.3520+0.0854C(μM)，R 2 =0.990, where C is the CEA concentration, and the detection limit is 16pg/mL (S/N = 3), ensuring the accuracy of the detection.
Selecting materials and instruments: the DNA sequence is synthesized by the biological engineering of biology GmbH, the name of which comprises CEA-Apt1 (A1), 5'-SH-TATCCAGCTTATTCAATT-3' and CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATATACCC-3' and PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGCACCATGATCCTG-3' and PDGF-BB Apt2 (B2), and 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), carbohydrate chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate-specific antigen (PSA), alpha-fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises an electrolyte solution and a DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a desk acidimeter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision liquid-transferring gun; PEI-Au combinationsThe composition is as follows: rapidly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au; preparing a metal ion probe: by using PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg EDC and 0.25g NHS into 100 mu L of 10 mu M A2 solution, mixing uniformly, and reacting for 1h at 37 ℃;100 μ L PEI-Au was added to the above system, shaken at 37 ℃ at 300rpm for 12h, centrifuged at 8000rpm for 20min to remove unattached A2, and redispersed in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method was used to prepare PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, shaking at 37 ℃ and 300rpm for reaction for 12h, and centrifuging at 8000rpm for 20min, washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use; preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using 0.3 mu m and 0.05 mu m of alumina powder respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, drying with nitrogen, dripping 10.0 μ L of target (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use; detection of single disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are 0.0V-0.5V and-0V respectively5-0.2V; detection of multiple disease markers: in contrast to the detection of individual disease markers, cu was used separately 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, and the DPV scanning range of the simultaneous detection is-0.6V.
Example five:
in the fourth example, the following steps were added:
in step 6, 6.0mmol/L Cu was used, respectively 2+ And 30.0mmol/L Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ To obtain the best effect.
Selecting materials and instruments: the DNA sequence is synthesized by the company of bioengineering GmbH, the name comprises CEA-Apt1 (A1), the sequence is 5'-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATGAAGCATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), the sequence is 5 '-COOH-CAGGCTACGGCACGTAGCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl) 4 ·4H 2 O), tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), and potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), magnesium chloride (MgCl) 2 ) Potassium ferricyanide (K) 3 Fe(CN) 6 ) Potassium ferrocyanide (K) 4 Fe(CN) 6 ·3H 2 O), carcinoembryonic antigen (CEA), sugar chain antigen 125 (CA 125), mucin (MUC 1), platelet-derived growth factor (PDGF-BB), prostate Specific Antigen (PSA), alpha Fetoprotein (AFP), polyethyleneimine (PEI), 6-mercapto-1-hexanol (MCH), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC); the solution comprises electrolyte solution and DNA buffer solution; the main instruments comprise a digital display intelligent temperature control magnetic stirrer, a transmission electron microscope, a desk acidimeter, an electrochemical workstation, a Milli-Q ultrapure water system and a precision liquid-transferring gun; and (3) synthesis of PEI-Au: 1.0mL of 4% PEI solution was quickly added to 10.0mL of 1.2mmol/L chloroauric acid solution, with magnetic stirringHeating to 80 ℃, keeping heating for 20min, and cooling to room temperature to obtain PEI-Au; preparation of a metal ion probe: by using PEI-Au-A2-Cu 2+ The preparation is as follows: firstly, adding 1.0mg of EDC and 0.25g of NHS into 100 mu L of 10 mu M A2 solution, uniformly mixing, and reacting for 1h at 37 ℃; adding 100 μ L PEI-Au to the above system, shaking at 37 deg.C and 300rpm for reaction 12h, centrifuging at 8000rpm for 20min to remove non-attached A2, and redispersing in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, shaking at 37 ℃ and 300rpm for reaction for 12h, and centrifuging at 8000rpm for 20min, washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use; preparation of electrochemical aptamer sensor: firstly, polishing the surface of a gold electrode (with the diameter of 3.0 mm) by using 0.3 mu m and 0.05 mu m of alumina powder respectively, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 (A1 or B1) is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed overnight at room temperature; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dripping 10.0 mu L of target substances (CEA or PDGF-BB) with different concentrations, rinsing the electrode with ultrapure water, and drying the electrode with nitrogen; finally, 10.0 mu L of metal ion probe solution (PEI-Au-CEA-A2-Cu) is dripped 2+ Or PEI-Au-B2-Pb 2+ ) Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use; detection of individual disease markers: DPV is selected as a detection method in the experiment, a three-electrode working system (the working electrode is an Au electrode; the reference electrode is an Ag/AgCl electrode; and the counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0mL of 0.1mol/L Tris-HCl buffer solution (pH = 7.4), and the DPV scanning ranges of CEA and PDGF-BB are 0.0V-0.5V and-0.5-0.2V respectively; detection of multiple disease markers: unlike single disease marker detection, isUse of Cu 2+ And Pb 2+ Synthesis of two Metal probes Au-A2-Cu 2+ And Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, and the DPV scanning range of the simultaneous detection is-0.6V.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (7)

1. A method for simultaneously detecting multiple disease markers using an electrochemical aptamer sensor, comprising: the method comprises the following steps:
step 1: selecting materials and instruments: the name of the DNA sequence comprises that the DNA sequence A1 is CEA-Apt1, the sequence is 5'-SH-TATCCAGCTTATTCAATT-3', the DNA sequence A2 is CEA-Apt2, the sequence is 5 '-COOH-AGGGTGAAGGGATATACCC-3', the DNA sequence B1 is PDGF-BBApt1, the sequence is 5 '-SH-CAGGCTACGGCACGTAGCATCACCATGATCCTG-3', the DNA sequence B2 is PDGF-BB Apt2, and the sequence is 5 '-COOH-CAGGCTAGCACGTAGCACCATGATCCTG-3';
and 2, step: and (3) synthesis of PEI-Au: rapidly adding 1.0mL of 4-percent PEI solution into 10.0mL of 1.2mmol/L chloroauric acid solution, heating to 80 ℃ under magnetic stirring, keeping continuously heating for 20min, and cooling to room temperature to obtain PEI-Au;
and step 3: preparing a metal ion probe: PEI-Au-A2-Cu 2+ The preparation of (1): firstly, adding 1.0mg of EDC and 0.25g of NHS into 100 mu L of 10 mu M A2 solution, uniformly mixing, and reacting for 1h at 37 ℃;100 μ L PEI-Au was added to the above system, shaken at 37 ℃ at 300rpm for 12h, centrifuged at 8000rpm for 20min to remove unattached A2, and redispersed in 200.0 μ L Cu (NO) 3 ) 2 Reacting at 37 ℃ for 12h, centrifuging at 8000rpm for 20min to remove supernatant, re-dispersing in 1xB2 buffer solution to prepare the PEI-Au-A2-Cu 2+ The same method is used for preparing PEI-Au-B2-Pb 2+ Except that it was dispersed in 200.0. Mu.L of Pb (NO) 3 ) 2 In the solution, shaking at 37 ℃ and 300rpm for reaction for 12h, and centrifuging at 8000rpm for 20min, washing for several times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing at 4 ℃ for later use;
and 4, step 4: preparation of electrochemical aptamer sensor: firstly, respectively polishing the surface of a gold electrode by using 0.3 mu m and 0.05 mu m of alumina powder, washing the surface by using ultrapure water, and carrying out ultrasonic treatment for 15s in an ultrapure water solution; at 0.5mol/L H 2 SO 4 CV scanning is carried out in the solution, the surface of the gold electrode is cleaned, 10 mu L of 1 mu M Apt1 is dripped on the surface of the gold electrode immediately after the nitrogen is dried, and the gold electrode is placed at room temperature overnight; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dropwise adding 10.0 mu L of 1mmol/L MCH, and reacting at 37 ℃ in a dark place for 40min; rinsing the electrode with ultrapure water, drying the electrode with nitrogen, dripping 10.0 mu L of target CEA or PDGF-BB with different concentrations, rinsing the electrode with ultrapure water, and drying the electrode with nitrogen; finally, 10.0 mu L of metal ion probe solution PEI-Au-A2-Cu is dripped 2+ Or PEI-Au-B2-Pb 2+ Reacting at 37 ℃ for 120min, washing the electrode with 0.1mol/L Tris-HCl, completing the preparation of the sensor, and storing at 4 ℃ for later use;
and 5: detection of multiple disease markers: using Cu respectively 2+ And Pb 2+ Synthesis of two kinds of metal probes PEI-Au-A2-Cu 2+ And PEI-Au-B2-Pb 2+ According to 1:1 to obtain the metal ion probe for simultaneous detection, and the DPV scanning range of the simultaneous detection is-0.6V.
2. The method of claim 1, wherein the electrochemical aptamer sensor is used for simultaneously detecting multiple disease markers, and the method comprises the following steps: in step 1, the electrolyte solution is a Tris-HCl buffer solution, pH =7.4, 0.1mol/L Tris,0.5mol/L NaCl,0.1mol/L KCl,0.1mol/L MgCl 2 。
3. The method of claim 1, wherein the electrochemical aptamer sensor is used for simultaneously detecting multiple disease markers, and the method comprises the following steps: in step 1, DNA buffer 1X NEBuffer2:50mmol/L NaCl,10mmol/L Tris-HCl,10mmol/L MgCl 2 1mmol/LDTT, pH 7.9.
4. The method of claim 1 for simultaneously detecting multiple disease markers using an electrochemical aptamer sensor, wherein the electrochemical aptamer sensor comprises: in step 4, when the target substance to be added dropwise is CEA, the reaction is carried out at 37 ℃ for 90min.
5. The method of claim 1, wherein the electrochemical aptamer sensor is used for simultaneously detecting multiple disease markers, and the method comprises the following steps: in step 4, the preparation method can be used for constructing a sensor of a single target object PDGF-BB or CEA, and can also be used for simultaneously detecting multiple target objects CEA and PDGF-BB.
6. The method of claim 1, wherein the electrochemical aptamer sensor is used for simultaneously detecting multiple disease markers, and the method comprises the following steps: in the step 5, the detection of CEA shows a good linear relation in the range of 0.05 ng/mL-20 ng/mL, and the linear equation is as follows: i is pa (μA)＝0.3520+0.0854C(μM)，R 2 =0.990, where C is CEA concentration and detection limit is 16pg/mL (S/N = 3).
7. The method of claim 1, wherein the electrochemical aptamer sensor is used for simultaneously detecting multiple disease markers, and the method comprises the following steps: in step 5, 6.0mmol/L Cu was used 2+ And 30.0mmol/L Pb 2+ Synthesis of two kinds of metal probes PEI-Au-A2-Cu 2+ And PEI-Au-B2-Pb 2+ 。
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