CN111721819A - 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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CN111721819A
CN111721819A CN202010537179.XA CN202010537179A CN111721819A CN 111721819 A CN111721819 A CN 111721819A CN 202010537179 A CN202010537179 A CN 202010537179A CN 111721819 A CN111721819 A CN 111721819A
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cea
pdgf
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CN111721819B (en
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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 sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-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 disease marker 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, multiple sensors can be utilized when multiple diseases need to be 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 sequences are allSynthesized by biological engineering GmbH, the name of which comprises CEA-Apt1 (A1), the sequence is 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), and the sequence is 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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;
step 2: and (3) synthesis of PEI-Au: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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: by using PEI-Au-A2-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/LH2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 1 mmol/LMCH, and reacting for 40 min at 37 ℃ in a dark place; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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 (a working electrode is an Au electrode; a reference electrode is an Ag/AgCl electrode; a counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL0.1 mol/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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for 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 18.2M Ω. cm) purified by a Millipore Milli-Q system.
Preferably, in step 1, the electrolyte solution (Tris-HCl buffer, pH = 7.4) is 0.1mol/L of LTris, 0.5 mol/L of NaCl, 0.1mol/L of KCl, 0.1mol/L of MgCl2。
Preferably, in step 1, the DNA buffer (1X NEBuffer 2): 50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L MgCl21mmol/L DTT, pH 7.9.
Preferably, when the target substance to be added dropwise in step 4 is CEA, the reaction is carried out at 37 ℃ for 90 min.
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 the step 5, the detection of CEA exhibits a good linear relationship within a range of 0.05 ng/mL to 20 ng/mL, and the linear equation is as follows: i ispa（μA）= 0.3520 + 0.0854C（μM），R2= 0.990, where C is CEA concentration and detection limit is 16 pg/mL (S/N = 3).
Preferably, in step 6, 6.0 mmol/L Cu is used respectively2+And 30.0 mmol/L Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+。
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 sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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;
step 2: and (3) synthesis of PEI-Au: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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: by using PEI-Au-A2-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/LH2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 1 mmol/LMCH, and reacting for 40 min at 37 ℃ in a dark place; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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 (a working electrode is an Au electrode; a reference electrode is an Ag/AgCl electrode; a counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL0.1 mol/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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for simultaneous detection is-0.6V.
The first embodiment is as follows:
selecting materials and instruments: the DNA sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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: at 10.0 mL of 1.2 mmol/LQuickly adding 1.0 mL of 4% PEI solution into the 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-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/L H2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 40 min; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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 was selected as the detection method in the experiment, and was 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 2.0 mL of 0.1mol/L Tris-HCl buffer (pH = 7.4), CThe DPV scanning ranges of EA 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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for 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 used without further purification, the experimental water was ultrapure water purified by Millipore Milli-Q system (resistivity 18.2 M.OMEGA.cm), electrolyte solution (Tris-HCl buffer, pH = 7.4) 0.1mol/L Tris, 0.5 mol/L NaCl, 0.1mol/L KCl, 0.1mol/L MgCl2DNA buffer (1X NEBuffer 2): 50 mmol/L NaCl, 10 mmol/L Tris-HCl, 10 mmol/L MgCl21mmol/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 sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main agent comprises Uric Acid (UA) and N-hydroxylSulfosuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution at 4 ℃ for later use; preparation of electrochemical aptamer sensor: first, the surface of a gold electrode (diameter: 3.0 mm) was polished with 0.3 μm and 0.05 μm alumina powders, respectively, and washed with ultrapure water, in which ultrapure water was addedCarrying out ultrasonic treatment for 15 s in the solution; at 0.5 mol/L H2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 40 min; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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; the electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL 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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for 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 90 min 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 has the best effect.
Selecting materials and instruments: the DNA sequences are synthesized by the company of bioengineering GmbH, and the names include CEA-Apt1 (A1), 5 ' -SH-TATCCAGCTTATTCAATT-3 ', CEA-Apt2 (A2), 5 ' -COOH-AGGGGGTGAAGGGATACCC-3 ', PDGF-BBApt1 (B1), and 5 ' -SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3 ', PDGF-BB Apt2 (B2), the sequence is 5 ' -COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3 '; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution at 4 ℃ for later use; electrochemical aptamer deliveryPreparation of the Sensors: 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 15 s in an ultrapure water solution; at 0.5 mol/L H2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 40 min; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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; the electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL 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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for simultaneous detection is-0.6V.
Example four:
in the third embodiment, the following steps are added:
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 ispa（μA）= 0.3520 + 0.0854C（μM），R2= 0.990, where C is the CEA concentration and the detection limit is 16 pg/mL (S/N = 3), ensuring the accuracy of the detection.
Selecting materials and instruments: the DNA sequences are synthesized by Biotechnology engineering Ltd, nameComprises CEA-Apt1 (A1), the sequence is 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), the sequence is 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), the sequence is 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2), and the sequence is 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/L H2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 40 min; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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; the electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL 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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for simultaneous detection is-0.6V.
Example five:
in the fourth example, the following steps were added:
in step 6, 6.0 mmol/L Cu was used2+And 30.0 mmol/L Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+To obtain the best effect.
Selecting materials and instruments: the DNA sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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-Cu2+The preparation is as follows: firstly, adding 1.0 mg EDC and 0.25 g NHS into 100 μ L of 10 μ M A2 solution, mixing uniformly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used for the preparation of PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/L H2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 40 min; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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; the electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL 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 separately2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for 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 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 (9)

1. An electrochemical aptamer sensor for simultaneously detecting multiple disease markers, comprising: the method comprises the following steps:
step 1: selecting materials and instruments: the DNA sequences are synthesized by the company of bioengineering GmbH, and the names of the DNA sequences comprise CEA-Apt1 (A1), 5 '-SH-TATCCAGCTTATTCAATT-3', CEA-Apt2 (A2), 5 '-COOH-AGGGGGTGAAGGGATACCC-3', PDGF-BBApt1 (B1), 5 '-SH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3', PDGF-BB Apt2 (B2) and 5 '-COOH-CAGGCTACGGCACGTAGAGCATCACCATGATCCTG-3'; the main reagents include Uric Acid (UA), N-hydroxysuccinimide (NHS), chloroauric acid (HAuCl)4·4H2O), Tris (hydroxymethyl) aminomethane (Tris), potassium chloride (KCl), sodium chloride (NaCl), potassium ferrocyanide (K)4Fe(CN)6·3H2O), magnesium chloride (MgCl)2) Potassium ferricyanide (K)3Fe(CN)6) Potassium ferrocyanide (K)4Fe(CN)6·3H2O), 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;
step 2: and (3) synthesis of PEI-Au: quickly adding 1.0 mL of 4% PEI solution into 10.0 mL of 1.2 mmol/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: by using PEI-Au-A2-Cu2+The preparation is as follows: first, 100. mu.L of 10. mu. M A2 solution was added with 1.0 mg EDC and 0.25 g NHS,mixing evenly, and reacting for 1 h at 37 ℃; adding 100 μ L PEI-Au into the above system, shaking at 37 deg.C and 300 rpm for 12 h, centrifuging at 8000 rpm for 20min to remove the unattached A2, and redispersing in 200.0 μ L Cu (NO)3）2Reacting in the solution at 37 ℃ for 12 h, centrifuging at 8000 rpm for 20min to remove supernatant, and redispersing in 1xB2 buffer solution to obtain the final PEI-Au-A2-Cu2+The same method was used to prepare PEI-Au-B2-Pb2+Except that it was dispersed in 200.0. mu.L of Pb (NO)3）2In the solution, shaking the solution at 37 ℃ and 300 rpm for 12 h, centrifuging the solution at 8000 rpm for 20min, washing the solution for a plurality of times, dispersing the obtained metal probe in a 1xB2 buffer solution, and storing the solution 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 15 s in an ultrapure water solution; at 0.5 mol/LH2SO4CV 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 immediately dropped on the surface of the gold electrode after being 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 1 mmol/LMCH, and reacting for 40 min at 37 ℃ in a dark place; 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 dripped2+Or PEI-Au-B2-Pb2+) Reacting at 37 ℃ for 120 min, 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 (a working electrode is an Au electrode; a reference electrode is an Ag/AgCl electrode; a counter electrode is a Pt electrode) is adopted for measurement, the DPV test is carried out in 2.0 mL0.1 mol/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 single disease marker detection, C was used separatelyu2+And Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+According to 1: 1 to obtain the metal ion probe for simultaneous detection, wherein the scanning range of the DPV for simultaneous detection is-0.6V.
2. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: in step 1, the reagent and the solvent are analytically pure and can be used without further purification.
3. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: the experimental water was ultrapure water (resistivity 18.2 M.OMEGA.. multidot.cm) purified by a Millipore Milli-Q system in step 1.
4. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: in step 1, the electrolyte solution (Tris-HCl buffer, pH = 7.4) is 0.1mol/L Tris, 0.5 mol/L NaCl, 0.1mol/L KCl, 0.1mol/L MgCl2。
5. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: in step 1, DNA buffer (1X NEBuffer 2): 50 mmol/L NaCl, 10 mmol/L LTris-HCl, 10 mmol/L MgCl21mmol/L DTT, pH 7.9.
6. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: in step 4, when the target substance to be added dropwise is CEA, the reaction is carried out at 37 ℃ for 90 min.
7. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: 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.
8. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: 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 ispa（μA）= 0.3520 + 0.0854C（μM），R2= 0.990, where C is CEA concentration and detection limit is 16 pg/mL (S/N = 3).
9. The electrochemical aptamer sensor for simultaneous detection of multiple disease markers according to claim 1, wherein: in step 6, 6.0 mmol/L Cu was used2+And 30.0 mmol/L Pb2+Synthesis of two Metal probes Au-A2-Cu2+And Au-B2-Pb2+。
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