CN116008370B - Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof - Google Patents
Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof Download PDFInfo
- Publication number
- CN116008370B CN116008370B CN202211615834.4A CN202211615834A CN116008370B CN 116008370 B CN116008370 B CN 116008370B CN 202211615834 A CN202211615834 A CN 202211615834A CN 116008370 B CN116008370 B CN 116008370B
- Authority
- CN
- China
- Prior art keywords
- peg
- pedot
- aunps
- pollution
- electrode
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 68
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 35
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims abstract description 31
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims abstract description 31
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 30
- 238000000835 electrochemical detection Methods 0.000 claims abstract description 4
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 claims description 70
- IMQLKJBTEOYOSI-UHFFFAOYSA-N Phytic acid Natural products OP(O)(=O)OC1C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C(OP(O)(O)=O)C1OP(O)(O)=O IMQLKJBTEOYOSI-UHFFFAOYSA-N 0.000 claims description 70
- 229940068041 phytic acid Drugs 0.000 claims description 70
- 235000002949 phytic acid Nutrition 0.000 claims description 70
- 239000000467 phytic acid Substances 0.000 claims description 70
- 230000003373 anti-fouling effect Effects 0.000 claims description 27
- 238000001514 detection method Methods 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 13
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 10
- 238000011534 incubation Methods 0.000 claims description 10
- GKWLILHTTGWKLQ-UHFFFAOYSA-N 2,3-dihydrothieno[3,4-b][1,4]dioxine Chemical compound O1CCOC2=CSC=C21 GKWLILHTTGWKLQ-UHFFFAOYSA-N 0.000 claims description 9
- 239000010931 gold Substances 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- 229910052737 gold Inorganic materials 0.000 claims description 7
- 239000003431 cross linking reagent Substances 0.000 claims description 6
- 238000000151 deposition Methods 0.000 claims description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 6
- VUFNRPJNRFOTGK-UHFFFAOYSA-M sodium;1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 VUFNRPJNRFOTGK-UHFFFAOYSA-M 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- PCWLNNZTBJTZRN-AVGNSLFASA-N Pro-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 PCWLNNZTBJTZRN-AVGNSLFASA-N 0.000 claims description 3
- CICQXRWZNVXFCU-SRVKXCTJSA-N Ser-His-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O CICQXRWZNVXFCU-SRVKXCTJSA-N 0.000 claims description 3
- FPCGZYMRFFIYIH-CIUDSAMLSA-N Ser-Lys-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O FPCGZYMRFFIYIH-CIUDSAMLSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000003745 diagnosis Methods 0.000 claims description 3
- 108010062381 leucyl-threonyl-valyl-seryl-prolyl-tryptophyl-tyrosine Proteins 0.000 claims description 3
- 239000002105 nanoparticle Substances 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 108010077112 prolyl-proline Proteins 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 238000011010 flushing procedure Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- 239000004323 potassium nitrate Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000001179 sorption measurement Methods 0.000 abstract description 16
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 108090000623 proteins and genes Proteins 0.000 abstract description 15
- 239000000463 material Substances 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 3
- 239000000863 peptide conjugate Substances 0.000 abstract 3
- 210000001124 body fluid Anatomy 0.000 abstract 1
- 239000010839 body fluid Substances 0.000 abstract 1
- 238000011896 sensitive detection Methods 0.000 abstract 1
- 229920001609 Poly(3,4-ethylenedioxythiophene) Polymers 0.000 description 59
- 239000002202 Polyethylene glycol Substances 0.000 description 57
- 229920001223 polyethylene glycol Polymers 0.000 description 57
- 210000002966 serum Anatomy 0.000 description 12
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 7
- 238000011109 contamination Methods 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 6
- 238000000329 molecular dynamics simulation Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 4
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 4
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 4
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 4
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 239000002131 composite material Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 229960001701 chloroform Drugs 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 230000036571 hydration Effects 0.000 description 3
- 238000006703 hydration reaction Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000011056 performance test Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000004088 simulation Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- MYBITMLLBJNNSS-UHFFFAOYSA-N 1-(cyclohexanecarbonyloxy)-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound OS(=O)(=O)C1CC(=O)N(OC(=O)C2CCCCC2)C1=O MYBITMLLBJNNSS-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 238000000026 X-ray photoelectron spectrum Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000002519 antifouling agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001940 conductive polymer Polymers 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005315 distribution function Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 239000002659 electrodeposit Substances 0.000 description 1
- 238000004070 electrodeposition Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 229940098197 human immunoglobulin g Drugs 0.000 description 1
- 229940094342 human immunoglobulin m Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000003746 surface roughness Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明属于电化学检测技术领域，公开了一种基于PEG‑多肽偶联物的抗污染电化学免疫传感器及其制备方法与应用。本发明创新性的将PEG‑多肽偶联物作为抗污染材料，集成优化两类重要生物分子的功能，以协同增强抗污染效果。本发明制备的生物传感器可高效抵抗复杂体液环境中蛋白质等生物分子的非特异性吸附，实现在复杂真实样本中对HER2的灵敏检测。经实验验证，所述PEG‑多肽偶联物的确比单一的PEG或多肽具有更为突出的抗污染效果和较为广阔的实际应用前景。
The present invention belongs to the field of electrochemical detection technology, and discloses an anti-pollution electrochemical immunosensor based on PEG-peptide conjugates, and a preparation method and application thereof. The present invention innovatively uses PEG-peptide conjugates as anti-pollution materials, and integrates and optimizes the functions of two important biological molecules to synergistically enhance the anti-pollution effect. The biosensor prepared by the present invention can effectively resist the nonspecific adsorption of biological molecules such as proteins in complex body fluid environments, and realize sensitive detection of HER2 in complex real samples. It has been experimentally verified that the PEG-peptide conjugate does have a more prominent anti-pollution effect and a broader practical application prospect than a single PEG or polypeptide.
Description
Technical Field
The invention belongs to the technical field of electrochemical detection, and particularly relates to an anti-pollution electrochemical immunosensor based on a PEG-polypeptide conjugate, a preparation method thereof and application thereof in quantitative detection of human epidermal growth factor receptor-2 (HER 2).
Background
Early and accurate diagnosis of cancer has been an important topic in disease treatment, playing a vital role in the life and social development of people. Over the past few years, more and more methods have been used for cancer detection, and in particular some advanced biosensors have attracted considerable attention from researchers. However, under normal operating conditions of the biosensor, biological contamination caused by non-specific protein adsorption is a non-negligible problem, which not only affects the detection response of the biosensor to the target analyte, but also generates false positive or negative signals, causes sensitivity and accuracy degradation, and severely causes malfunction of the biosensor. The problem greatly influences the reliability and stability of the detection of the sensor in a real sample (such as human blood and serum), so that the actual detection of the biosensor in a complex medium is limited, and the application prospect is not wide. Therefore, the development of a sensing interface that is effective against biological attachment and reduces the adsorption of non-specific proteins is a major issue in the construction of an anti-fouling electrochemical immunosensor.
The anti-pollution electrochemical immunosensor is a novel sensor developed by combining an anti-pollution material with an electrochemical biological sensing technology, not only maintains the advantages of high sensitivity, good specificity, simple operation, low cost and the like of the electrochemical sensor, but also endows the anti-biomolecule nonspecific adsorption performance due to the introduction of the anti-pollution material.
The non-specific adsorption of the protein on the sensing interface is mainly caused by two large acting forces of hydrophobic action and charge attraction, so that the construction of the pollution-resistant interface with good electric neutrality and hydrophilicity is easy to reduce the electrostatic attraction between the sensing interface and pollutants, and a hydration layer coating interface is formed, thereby effectively preventing the adhesion of the non-specific protein. Zwitterionic materials such as zwitterionic polymers, zwitterionic peptides, polyethylene glycol (PEG) and the like have good hydrophilicity and satisfactory electric neutrality, and are ideal interface anti-pollution materials. They are used to construct anti-fouling biosensors and are successfully applied to the detection of biomarkers in complex media. However, an anti-fouling biosensor based on any one of the anti-fouling materials is not completely resistant to the interference of contaminants. Therefore, it remains a challenge and has significant implications to explore the development of biosensors with excellent anti-fouling properties.
Disclosure of Invention
In view of this, the present invention has devised a novel anti-fouling PEG-polypeptide conjugate and constructed a practical electrochemical biosensor.
In addition, the combination of polyethylene glycol and polypeptide or protein can effectively improve the stability and water solubility, and the polyethylene glycol-polypeptide composite material is proved to be an effective anti-pollution material better than pure peptide in human serum. Therefore, the design of the composite anti-pollution material is an effective way for improving the interface anti-pollution performance.
In order to achieve the aim, the invention discloses an anti-pollution electrochemical immunosensor based on a PEG-polypeptide conjugate, which specifically adopts the following technical scheme:
An anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate uses a glassy carbon electrode as a substrate, and sequentially electrodeposits phytic acid doped 3, 4-ethylenedioxythiophene and gold nanoparticles, and connects PEG through gold-ammonia bond, and then the polypeptide is coupled by a cross-linking agent; and, in addition, the method comprises the steps of,
The sequence of the polypeptide is Pep: cys Pro Pro Pro Pro Lys Ser GIu SER LYS SER GIu Ser His Leu Thr Val SerPro Trp Tyr;
And, the structural formula of PEG is:
it is important to note that biological contamination caused by non-specific protein adsorption and the like is a serious problem facing electrochemical immunosensors, which greatly affects the reliability and stability of sensor detection in real biological samples (such as human blood and serum), and limits the practical application of the biosensor in complex biological media. The anti-pollution sensor constructed in the invention effectively solves the problem that the biosensor is adsorbed by nonspecific proteins in practical application.
In particular, the design of the composite anti-pollution material is an effective way for improving the interface anti-pollution performance, and the combination of polyethylene glycol and polypeptide or protein can improve the stability and the water solubility, and can effectively solve the adhesion of biomolecules in an actual sample at the interface. According to the invention, by utilizing the strong affinity of amino modified PEG to a gold interface and coupling PEG with polypeptide to synthesize a novel self-assembled molecular structure such as a PEG-polypeptide conjugate, the integrated optimization of functions of two important biomolecules can be realized, and the anti-pollution capability of a single polypeptide is enhanced.
A second object of the present invention is to provide a method for preparing an anti-fouling electrochemical immunosensor based on a PEG-polypeptide conjugate as described above.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a preparation method of an anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate comprises the following steps:
I. pretreatment of a glassy carbon electrode: firstly, polishing an interface by using alumina powder, and then cleaning the interface by adopting water, absolute ethyl alcohol and water in turn under an ultrasonic condition so as to pretreat a glassy carbon electrode;
preparation of I, auNPs/PEDOT (PA) modified electrode: soaking the glassy carbon electrode pretreated in the step I in a mixed solution containing 3, 4-ethylenedioxythiophene EDOT and phytic acid PA, and preparing a PEDOT (PA) modified electrode by adopting a constant potential method at room temperature; then soaking the prepared PEDOT (PA) modified electrode in chloroauric acid HAuCl 4 solution containing potassium nitrate KNO 3, and preparing the AuNPs/PEDOT (PA) modified electrode by adopting a potentiostatic method at room temperature;
Assembling I, I and electrochemical immunosensor: and (3) connecting the four-arm amino PEG to an AuNPs/PEDOT (PA) modified electrode by using a gold-ammonia bond, and then connecting the polypeptide to the PEG/AuNPs/PEDOT (PA) modified electrode by using a cross-linking agent Sulfo-SMCC, so as to finally prepare the anti-pollution electrochemical immunosensor based on the PEG-polypeptide conjugate.
Further, the specific method in the step II is as follows:
Immersing the pretreated glassy carbon electrode in a solution containing 0.005-0.02M of 3, 4-ethylenedioxythiophene EDOT and 0.005-0.02M of phytic acid PA, and depositing for 20-100 s at room temperature by adopting a constant potential of 1.1V to obtain a PEDOT (PA) modified electrode; the PEDOT (PA) modified electrode is soaked in 0.5mM HAuCl 4 solution containing 0.5mM KNO 3, and deposited for 30-60 s at-0.5V, so that the AuNPs/PEDOT (PA) modified electrode is prepared.
Further, the specific method in the step III is as follows:
Incubating the AuNPs/PEDOT (PA) modified electrode in PEG solution with concentration of 1.0-4.0mg.mL -1 for two hours, and connecting PEG to the AuNPs/PEDOT (PA) modified interface through gold-ammonia bond to prepare the PEG/AuNPs/PEDOT (PA) modified electrode; and then activating the prepared PEG/AuNPs/PEDOT (PA) electrode for 1 hour through a cross-linking agent Sulfo-SMCC, and incubating the activated PEG/AuNPs/PEDOT (PA) electrode in a polypeptide solution with the concentration of 0.1-0.5 mg.mL -1 for 3 hours to obtain the anti-pollution electrochemical immunosensor Pep/PEG/AuNPs/PEDOT (PA)/GCE based on the PEG-polypeptide conjugate.
It is worth to say that the invention combines the anti-pollution polypeptide and the anti-pollution PEG for the first time, and designs a novel composite anti-pollution material of the PEG-polypeptide conjugate. The anti-pollution polypeptide is neutral in electricity and strong in hydrophilicity, the tetra-amino PEG and the gold interface have strong affinity, a layer of stable and compact film can be formed on the interface, and the effect of well covering the sensing interface to prevent other biomolecules from being adsorbed on the interface is achieved. The PEG-polypeptide conjugates exhibit better anti-fouling properties compared to the polypeptide and PEG alone.
A third object of the present invention is to provide the use of an anti-fouling electrochemical immunosensor based on a PEG-polypeptide conjugate as described above for in vitro diagnosis/detection of complex biological media.
Further, the sensor is applied to detection of human epidermal growth factor receptor-2 (HER 2).
Still further, the use includes for quantitative detection of human epidermal growth factor receptor-2 (HER 2); the specific operation steps of the quantitative detection are as follows:
placing the prepared anti-pollution electrochemical immunosensor based on the PEG-polypeptide conjugate in target HER2 solutions with different concentrations, incubating at constant temperature, then flushing an electrode interface by using PBS buffer to wash off the uncaptured target HER2, and carrying out electrochemical detection on the obtained electrode;
and recording current signal changes of the sensing interface before and after incubation in the target solution in a voltage range of-0.2-0.6V by using a differential pulse voltammetry method, so as to realize detection of the target HER 2.
Wherein the temperature of constant temperature incubation is room temperature (23-28 ℃), and the incubation time is 15-90min, so as to ensure sufficient binding with the target HER2 and sufficient specific recognition of the target HER 2.
The linear detection range of the HER2 detection was 1.0 pg/mL -1-1.0ug·mL-1, and the detection limit was 0.443 pg/mL -1 (S/N).
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention discloses the use of PEG-polypeptide conjugates as anti-pollution materials in sensors for the first time.
(2) The anti-pollution electrochemical immunosensor of the PEG-polypeptide conjugate disclosed by the invention integrates the functions of two important biomolecules, synergistically enhances the anti-pollution effect, has more excellent anti-pollution effect than a single biomolecule, and has more excellent application potential.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a construction diagram of the anti-pollution electrochemical immunosensor.
FIG. 2 is a graph of PEG concentration optimization.
FIG. 3 is a graph of optimization of polypeptide concentration.
Fig. 4 is a graph of HER2 incubation time optimization.
Fig. 5 is a scanning electron microscope image of different modified electrodes.
FIG. 6 is a theoretical calculation chart (A) and Zeta potential test chart (B) of the charged condition of the polypeptide.
Fig. 7 is a graph of contact angles for different modified electrodes.
Fig. 8 is a graph of contact angle of oil in water for different modified electrodes.
FIG. 9 is a molecular dynamics simulation of different modified electrodes.
Fig. 10 is a graph of DPV response curves for different working electrodes.
FIG. 11 is a full scan X-ray photoelectron spectrum of a different modified electrode.
FIG. 12 is a graph of anti-contamination tests of different modified electrodes against non-specific adsorption in different concentrations of human blood.
FIG. 13 is a graph comparing the anti-fouling performance of different modified electrodes in different concentrations of human blood.
FIG. 14 is an anti-contamination test pattern of different modified electrodes against non-specific adsorption in human serum at different concentrations.
FIG. 15 is a graph comparing the anti-fouling performance of different modified electrodes in human serum at different concentrations.
FIG. 16 is an anti-contamination test pattern of different modified electrodes against non-specific adsorption in different concentrations of animal serum.
FIG. 17 is a graph comparing the anti-fouling performance of different modified electrodes in animal serum at different concentrations.
FIG. 18 is a graph of long-term anti-fouling performance test of different modified electrodes in 20% human serum.
FIG. 19 is a confocal laser photograph of a different modified electrode incubated in 0.2mg/mL fluorescent protein for 2 hours.
Fig. 20 is a graph (a) and a linear curve (B) of DPV response of an anti-fouling electrochemical immunosensor to HER2 at different concentrations.
FIG. 21 is a bar graph comparing the analytical performance of the present method and ELISA.
FIG. 22 is a graph showing the stability of an anti-fouling electrochemical immunosensor in a continuous CV method test.
FIG. 23 is a graph of storage stability test of an anti-fouling electrochemical immunosensor in PBS (10 mM, pH 7.4).
FIG. 24 is a bar graph of the current response of an anti-fouling electrochemical immunosensor to 1.0 μg/mL HER2, 100 μg/mL IgM, CEA, AFP, CA, igG, and Mix.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Examples of certain embodiments of the invention are given below and are not intended to limit the scope of the invention.
The device comprises: the interface was characterized by scanning electron microscopy (Hitachi Co., japan) using a Hitachi S-4800 scanning electron microscope. ESCALAB 250Xi spectrometer (Thermo FISHER SCIENTIFIC, U.K.) was used to perform X-ray photoelectron spectroscopy (XPS) on the interface. The wettability of the electrode interface was characterized by JC2000 contact angle instrument (Shanghai instruments limited). Electrochemical testing was done on a CHI 760E electrochemical workstation (Shanghai Chen Hua instruments Co., ltd.) using a conventional three electrode system: a platinum wire electrode is used as a counter electrode, a saturated calomel electrode is used as a reference electrode and a bare or modified glassy carbon electrode is used as a working electrode.
Reagent: fluorescein isothiocyanate labeled BSA (FITC-BSA), potassium chloride (KCL), phytic Acid (PA), carcinoembryonic antigen (CEA) and Alpha Fetoprotein (AFP) were all purchased from Hadamard reagent Co., ltd. Sodium 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl ester (Sulfo-SMCC) was purchased from Sigma Aldrich (U.S.). Trichloromethane (CHCl 3) is from the company Tianjin Fuyu fine chemical Co., ltd. Chloroauric acid (HAuCl 4) was offered by chinese national medicine, chemical company limited. Human immunoglobulin G (IgG), human immunoglobulin M (IgM), carbohydrate antigen 125 (CA 125) and HER2 are purchased from beijing solebao technologies limited. 3, 4-Ethylenedioxythiophene (EDOT) was ordered by ala Ding Shiji limited (Shanghai, china). HER2 enzyme-linked immunosorbent assay (ELISA) kit was purchased from chinese enzyme-linked immunosorbent assay (ELISA) limited. Blood samples from healthy adult blood donors were provided by the Qingdao eighth people hospital (China). Fetal Bovine Serum (FBS) was purchased from the middle-aged biotechnology company of beijing euphoriaceae. All chemical reagents used in this experiment were analytical pure as required, and all water used in this experiment was secondary pure water prepared from Milli-Q (18 mΩ/cm) purification system for all aqueous solutions.
The antipollution polypeptide sequence used was Pep: cys Pro Pro Pro Pro Lys Ser GIu SER LYS SER GIu Ser His Leu ThrVal SerPro Trp Tyr, synthesized by polypeptide Co., ltd (China); the structural formula of the 4-arm PEG-NH 2 (2000 Da) used is:
Purchased from Shanghai punk Biotechnology Inc. (China);
Example 1
The construction process of the anti-pollution electrochemical immunosensor is shown in fig. 1:
(1) Electrode pretreatment: before carrying out interface modification, polishing an interface by using alumina powder, and then respectively cleaning the interface by using water, absolute ethyl alcohol and water for 2min under an ultrasonic condition;
(2) Preparation of AuNPs/PEDOT (PA) modified electrode: immersing the pretreated glassy carbon electrode in a mixed solution containing 0.02M EDOT and 0.02M PA, uniformly dispersing the glassy carbon electrode by ultrasonic treatment, and performing electrodeposition by adopting constant potential of 1.1V at room temperature to synthesize a PEDOT (PA) modified electrode, wherein the potential is 1.1V, and the time is 20s; then, immersing the PEDOT (PA) modified electrode in a 0.5M HAuCl 4 solution containing 0.5M KNO 3, and depositing gold nano particles (AuNPs) for 30s at-0.5V to prepare the AuNPs/PEDOT (PA) modified electrode;
(3) Preparation of the sensor: the AuNPs/PEDOT (PA) modified electrode is soaked in PEG solution with the concentration of 2.5 mg.mL -1, the PEG/AuNPs/PEDOT (PA) is prepared by connecting through a gold amine bond for 2 hours, the PEG/AuNPs/PEDOT (PA) is activated for 1 hour through a cross-linking agent Sulfo-SMCC, and finally the polypeptide solution with the concentration of 0.3 mg.mL -1 is incubated on the activated PEG/AuNPs/PEDOT (PA) electrode, and the PEG-polypeptide conjugate is obtained through thiol-amino reaction for 3 hours, namely the electrochemical immunosensor.
Example 2-N
To obtain more excellent sensing performance, we have optimized several important experimental conditions.
First is the optimization of PEG concentration: PEG solutions of different concentrations were prepared, with the current signal increasing with increasing PEG concentration (1.0 mg. ML -1 increasing to 4.0 mg. ML -1). When the concentration is 2.5 mg.mL -1, the current signal is no longer changed with the increase of the concentration, as shown in FIG. 2;
In addition, the concentration of the peptide was optimized for optimal reaction signal. With increasing peptide concentration (0.1 mg.ml -1 to 0.5 mg.ml -1), electrode anti-pollution performance is gradually enhanced, then polypeptide concentration is continuously increased, and anti-pollution performance is weakened, so that 0.3 mg.ml -1 is selected as the optimal modified concentration of polypeptide, at the moment, current signals are in a dynamic balance state and are not changed with the increase of concentration, as shown in fig. 3;
And, the incubation time of the target object is optimized, and the biosensor is incubated with HER2 of 1 mg.mL -1 for different times. Theoretically, as the incubation time increases from 15min to 90min, the current signal will change accordingly. As can be seen from fig. 4, the current signal tends to be dynamically balanced when the incubation time is 60 min. At this time, the biosensor can maximally bind to HER2, thereby improving the detection performance of the biosensor.
In addition, in order to further verify the excellent performance of the anti-pollution electrochemical immunosensor prepared by the invention, the inventor also performs the following experiment:
experimental example 1
Characterization of PEDOT (PA), auNPs/PEDOT (PA) and PEG/AuNPs/PEDOT (PA) modified electrodes
In order to more intuitively demonstrate the interface modification process, the morphology of different modified electrodes was characterized by using a scanning electron microscope (fig. 5). As shown in fig. 5A, the formed PEDOT (PA) film exhibits an irregular three-dimensional network structure. After deposition of AuNPs, the spherical AuNPs were uniformly and densely covered on the PEDOT (PA) film as shown in fig. 5B. Next, PEG was immobilized by a self-assembly process of Au-NH 2 bonds. As shown in FIG. 5C, the AuNPs/PEDOT (PA) surface was uniformly covered with PEG, and the surface roughness was lower than that of the AuNPs/PEDOT (PA) modified electrode.
Experimental example 2
Characterization of polypeptides and PEG-polypeptide conjugates:
As shown in FIG. 6, the electrical properties of the designed polypeptides were studied by calculation with a polypeptide property simulator (panel A) and zeta potential measurement (panel B). The net charge of the designed peptide at ph=7 was almost 0, and the isoelectric point (pI) was calculated to be 7.09 (panel a). Meanwhile, the zeta potential of the designed peptide was very close to 0.0mV (Panel B), consistent with the theoretical calculation result. This indicates that the polypeptide is close to a neutral state.
In addition, the hydrophilicity of the designed polypeptides was tested by testing the contact angle of water in air and the contact angle of oil in water. As shown in FIG. 7, the static contact angles of the different modified electrodes are bare GCE(61.58°±1.93°)、AuNPs/PEDOT(PA)/GCE(42.92°±3.75°)、PEG/AuNPs/PEDOT(PA)/GCE(27.82°±1.63°)、Pep/PEG/AuNPs/PEDOT(PA)/GCE(15.83°±2.34°).
From the experimental data, it can be seen that: with the immobilization of the designed peptide fragment, the contact angle was further reduced from (27.82 ° ± 1.63 °) to (15.83 ° ± 2.34 °). The contact angle of the Pep/PEG modified interface is the smallest (15.83+/-2.34 degrees), so that the hydrophilicity of the PEG-polypeptide conjugate modified sensing interface is greatly enhanced.
Subsequently, the contact angle of the different modified electrodes in water was measured using chloroform as the oil phase medium, as shown in fig. 8. And as opposed to the contact angle result of water, the Pep/PEG/AuNPs/PEDOT (PA) modified electrode shows obvious repulsive action on chloroform, and the oil contact angle is 128.32 degrees. The contact angle test results show that the Pep/PEG modified electrode has good hydrophilicity and oleophobicity, and experimental data are all in line with the expectations of us.
The strong hydration layer formed on the surface can reduce dirt and reduce the adsorption of nonspecific proteins, thereby improving the sensing performance. Depending on the nature of the PEG and polypeptide, it is believed that the hydrophilicity will be better after the two are combined. To demonstrate this, we performed Molecular Dynamics (MD) simulations using OPLSS-AA force fields and MKTOP to parameterize all atoms, such as bond parameters, angle parameters, dihedral angles, etc.
The hydrophilicity of the different molecules was studied using Molecular Dynamics (MD) simulation (fig. 9). 20 molecules A (Peptide) (FIG. 9A), 20 molecules B (Pep/PEG) (FIG. 9B) and 14069 water molecules were randomly inserted into the simulation box (8.000X8.000X8.000X 3) respectively. From the dynamic equilibrium diagram after the molecular dynamics simulation process, it can be seen that under the same condition, more water molecules are combined at the interface modified by the PEG-polypeptide conjugate, so that a larger hydration layer is formed on the sensing interface, and the anti-pollution performance of the interface is improved.
Furthermore, according to the radial distribution function of water (RDFs) (fig. 9D), the number of water molecules bound to the PEG-polypeptide conjugate increases at (r=0.2 nm), on the other hand also manifests itself as the formation of hydrogen bonds between molecules (fig. 9C). The above results are also in line with our expectations.
Experimental example 3
Characterization of anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugates
(1) Electrochemical characterization of the immunosensor process:
Evaluation of HER2 biosensor by DPV measurement the construction process (fig. 10) shows that the peak current signal of the PEDOT (PA) modified electrode (curve b) is significantly increased compared to the bare GCE (curve a) as seen in fig. 10. This is because PEDOT (PA) exhibits good conductivity on the one hand, and because the three-dimensional network structure of the conductive polymer provides a larger specific surface area, which can further promote electron transfer. After deposition of AuNPs, the peak current signal increases further (curve c) because AuNPs have good conductivity. The peak current at the Pep/PEG/AuNPs/PEDOT (PA) modified interface (curve d, curve e) decreased to different extents, respectively, due to the poor conductivity of Pep/PEG. The specific recognition of HER2 at the Pep/PEG modified interface (curve e) results in a further decrease of the current signal, because the insulation and steric effects of the HER2 protein hinder electron transfer.
(2) Immune sensor sensing procedure XPS characterization:
The assembly interface was XPS characterized to further monitor the immunosensor manufacturing process, as shown in fig. 11. The PEDOT (PA) electrode is mainly composed of carbon (C), oxygen (O), sulfur (S) and phosphorus (P) elements. After deposition of AuNPs, a distinct gold element (Au) peak appears. After the Pep/PEG was modified to the interface, a new nitrogen (N) peak appeared, indicating that Pep/PEG was successfully immobilized on the electrode surface.
Experimental example 4
Anti-pollution performance test one:
Bare GCE, auNPs/PEDOT (PA)/GCE, PEG/AuNPs/PEDOT (PA)/GCE, pep/AuNPs/PEDOT (PA)/GCE and Pep/PEG/AuNPs/PEDOT (PA)/GCE were immersed in complex biological medium solutions of different concentrations, and the change of current signal due to adsorption of biomolecules was recorded with A being GCE, B being AuNPs/PEDOT (PA)/GCE, C being PEG/AuNPs/PEDOT (PA)/GCE, D being Pep/AuNPs/PEDOT (PA)/GCE, and E being Pep/PEG/AuNPs/PEDOT (PA)/GCE). And the change of the current signal is represented by a signal suppression rate = (Δi/I 0)×100％,ΔI＝I0-I,I0 and I represent the DPV peak current values before and after the electrode soaking, respectively).
As shown in FIG. 12, the anti-fouling ability of the Pep/PEG/AuNPs/PEDOT (PA)/GCE modified electrode was significantly better than the other interfaces, and this result was related to the sufficient coverage of the interface with the PEG-polypeptide conjugate to give it excellent anti-fouling ability.
As shown in FIG. 13, the signal inhibition rate of Pep/PEG/AuNPs/PEDOT (PA)/GCE after being soaked in 100% human blood is 15.59%, and the anti-fouling performance of the anti-fouling agent is superior to other modified interfaces, which benefits from the synergistic anti-fouling effect of PEG and polypeptide.
Similar results were obtained for non-specific adsorption assays performed in human serum (FIGS. 14, 15) and fetal bovine serum (FIGS. 16, 17), further indicating good anti-fouling ability of the PEG-polypeptide conjugate modified electrode surfaces.
In addition, to ensure the accuracy of the anti-contamination experiments, pep/AuNPs/PEDOT (PA)/GCE and Pep/PEG/AuNPs/PEDOT (PA)/GCE were immersed in high concentration HER2 (1 ug mL -1) before the experiments were performed, preventing the specific recognition from being affected by HER2 in human serum and human blood.
The long-term anti-fouling performance of the different modified electrodes is shown in FIG. 18 to reflect the practical application capabilities of the developed biosensor. The biosensor still has good anti-pollution performance after being soaked in 20% human serum for 24 hours, and is far superior to other modified interfaces. The excellent anti-pollution performance can ensure that the biosensor can keep good sensing performance in a complex environment, the detection limit is improved, and higher sensitivity is obtained.
Experimental example 5
Anti-pollution performance test II:
Naked GCE, auNPs/PEDOT (PA)/GCE, pep/AuNPs/PEDOT (PA)/GCE, PEG/AuNPs/PEDOT (PA)/GCE and Pep/PEG/AuNPs/PEDOT (PA)/GCE were incubated in PBS buffer (10 mM, pH 7.4) containing -1 FITC-BSA for 2h and then fluorescent protein adsorption at the different interfaces was recorded using a fluorescence microscope as shown in FIG. 19.
As can be seen from fig. 19, the adsorption amount of protein at the interface is reflected by the fluorescence signal intensity, the fluorescence intensity of the bare GCE surface (fig. 19A) is strongest, and next to the AuNPs/PEDOT-PA modified interface (fig. 19B), a small amount of green fluorescence is observed on PEG/AuNPs/PEDOT (PA)/GCE (fig. 19C) and Pep/AuNPs/PEDOT (PA)/GCE (fig. 19D); and little green fluorescence was observed at the electrode interface modified by Pep/PEG/AuNPs/PEDOT (PA)/GCE (fig. 19E). Experimental results show that the interface co-modified by Pep/PEG has more effective protein adsorption resistance than other interfaces.
Furthermore, as is clear from the above experimental examples 4 to 5, pep/PEG/AuNPs/PEDOT (PA)/GCE has superior and stable contamination resistance against a single protein even for a truly complex biological sample.
Experimental example 6
Quantitative detection of human epidermal growth factor receptor-2 (HER 2):
Under optimal experimental conditions, the sensing performance of the electrochemical biosensor under investigation was measured using DPV. As shown in fig. 20A, the peak DPV current gradually decreases with the change in HER2 concentration from 1 pg-mL -1 to 1 ug-mL -1 due to steric hindrance caused by specific recognition and insulation of the target molecule. There is a good linear relationship between the logarithm of HER2 concentration and the DPV current change (Δi), and the results are shown in fig. 20B below. The detection limit of the biosensor is calculated to be 0.443 pg.mL -1 (S/N=3), and the biosensor has good sensing performance.
To measure the potential value of HER2 biosensors in practical applications, we collected actual serum samples from 5 breast cancer patients and analyzed using our biosensors and commercial ELISA methods. As shown in fig. 21, the experimental results obtained by the two methods are consistent, and the variation range of the relative standard deviation (1.95% -9.09%) is satisfactory. These results indicate that the biosensor constructed based on the PEG-polypeptide conjugate has good capability of resisting non-specific protein adsorption and has great practical application potential.
Experimental example 7
Stability of anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate
The stability of the biosensor (Pep/PEG/AuNPs/PEDOT (PA)/GCE) was measured by continuous CV scanning in the formulated PBS solution. As shown in fig. 22, even after 100 cycles of CV scan, the peak signal and potential of the CV curve did not change significantly. Meanwhile, the current signal of the sensor was detected at intervals within fifteen days, and the signal retention rate was still greater than 90% (fig. 23).
Experimental example 8
Selectivity of anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate
In actual detection, there are some interfering proteins that coexist with the target (HER 2), such as IgM, igG, AFP, CA and CEA. The specificity of HER2 biosensor was measured by DPV and the response signals of the biosensor to the target and interferents were recorded as shown in fig. 24. Even though the target concentration is one percent of the interfering concentration, the constructed biosensor still has a superior signal response to HER2, indicating that the biosensor has good selectivity.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. An anti-pollution electrochemical immunosensor based on a PEG-polypeptide conjugate is characterized in that the sensor takes a glassy carbon electrode as a substrate, and 3, 4-ethylenedioxythiophene doped with phytic acid, gold nanoparticles and the PEG-polypeptide conjugate are sequentially modified; and, in addition, the method comprises the steps of,
The polypeptide sequence of the PEG-polypeptide conjugate is Pep: cys Pro Pro Pro Pro Lys Ser GIu SER LYS SER GIu Ser His Leu ThrVal SerPro Trp Tyr;
And, the structural formula of PEG is:
the preparation method of the anti-pollution electrochemical immunosensor based on the PEG-polypeptide conjugate comprises the following steps:
I. pretreatment of a glassy carbon electrode: firstly, polishing an interface by using alumina powder, and then cleaning the interface by adopting water, absolute ethyl alcohol and water in turn under an ultrasonic condition so as to pretreat a glassy carbon electrode;
Preparation of I, auNPs/PEDOT-PA modified electrode: soaking the glassy carbon electrode pretreated in the step I in a mixed solution containing 3, 4-ethylenedioxythiophene EDOT and phytic acid PA, and preparing a PEDOT-PA modified electrode by adopting a constant potential method at room temperature; then soaking the prepared PEDOT-PA modified electrode in a chloroauric acid HAuCl 4 solution containing potassium nitrate KNO 3, and preparing the AuNPs/PEDOT-PA modified electrode by adopting a potentiostatic method at room temperature;
Assembling I, I and electrochemical immunosensor: and (3) connecting the four-arm amino PEG to the AuNPs/PEDOT-PA modified electrode by using a gold-ammonia bond, and then connecting the polypeptide to the PEG/AuNPs/PEDOT-PA modified electrode by using a cross-linking agent Sulfo-SMCC, so as to finally prepare the anti-pollution electrochemical immunosensor based on the PEG-polypeptide conjugate.
2. The anti-fouling electrochemical immunosensor based on a PEG-polypeptide conjugate of claim 1, wherein the specific method of step II is:
Immersing the pretreated glassy carbon electrode in a mixed solution containing 0.005-0.02M of 3, 4-ethylenedioxythiophene EDOT and 0.005-0.02M of phytic acid PA, and depositing for 20-100 s at room temperature by adopting a constant potential of 1.1V to obtain a PEDOT-PA modified electrode; the PEDOT-PA modified electrode is soaked in 0.5mM HAuCl 4 solution containing 0.5mM KNO 3, and deposited for 30-60 s at-0.5V, so that the AuNPs/PEDOT-PA modified electrode is prepared.
3. The anti-fouling electrochemical immunosensor based on a PEG-polypeptide conjugate of claim 1, wherein the specific method of step III is:
Incubating the AuNPs/PEDOT-PA modified electrode in PEG solution with concentration of 1.0-4.0mg.mL -1 for two hours, and connecting PEG to the AuNPs/PEDOT-PA modified interface through gold-ammonia bond to prepare the PEG/AuNPs/PEDOT-PA modified electrode; and then activating the prepared PEG/AuNPs/PEDOT-PA modified electrode for 1 hour through a cross-linking agent Sulfo-SMCC, and incubating the electrode in a polypeptide solution with the concentration of 0.1-0.5 mg.mL -1 for 3 hours to obtain the anti-pollution electrochemical immunosensor Pep/PEG/AuNPs/PEDOT-PA/GCE based on the PEG-polypeptide conjugate.
4. Use of an anti-fouling electrochemical immunosensor based on a PEG-polypeptide conjugate as defined in claim 1 for in vitro diagnosis/detection of complex biological media.
5. The use according to claim 4, wherein the anti-fouling electrochemical immunosensor is for human epidermal growth factor receptor-2 detection.
6. The use according to claim 5, wherein the anti-pollution electrochemical immunosensor is for quantitative detection of human epidermal growth factor receptor-2; the specific operation steps of the quantitative detection are as follows:
placing the prepared anti-pollution electrochemical immunosensor based on the PEG-polypeptide conjugate in target human epidermal growth factor receptor-2 solutions with different concentrations, incubating at constant temperature, then flushing an electrode interface by using PBS buffer solution to wash out the non-captured target human epidermal growth factor receptor-2, and carrying out electrochemical detection on the obtained electrode;
and recording the current signal change of the sensing interface before and after incubation in the target solution in a voltage range of-0.2-0.6V by using a differential pulse voltammetry, so as to realize detection of the target human epidermal growth factor receptor-2.
7. The use according to claim 6, wherein the incubation temperature is 23-28 ℃ and the incubation time is 15-90min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211615834.4A CN116008370B (en) | 2022-12-15 | 2022-12-15 | Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211615834.4A CN116008370B (en) | 2022-12-15 | 2022-12-15 | Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116008370A CN116008370A (en) | 2023-04-25 |
| CN116008370B true CN116008370B (en) | 2024-11-15 |
Family
ID=86030907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211615834.4A Active CN116008370B (en) | 2022-12-15 | 2022-12-15 | Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116008370B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117969837B (en) * | 2023-10-07 | 2024-09-20 | 青岛科技大学 | Preparation method of anti-pollution electrochemical sensor based on polypeptide nano-particles |
| CN118032900A (en) * | 2024-02-28 | 2024-05-14 | 青岛科技大学 | An anti-pollution electrochemical biosensor with thorny vine-like micro-nano scale and its preparation method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114216943A (en) * | 2021-11-26 | 2022-03-22 | 青岛科技大学 | Anti-pollution electrochemical immunosensor based on DNA-polypeptide conjugate and preparation method and application thereof |
| CN114324526A (en) * | 2021-12-27 | 2022-04-12 | 青岛农业大学 | A biosensor for detecting prostate specific antigen in human serum and its preparation method and application |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170045505A1 (en) * | 2014-04-16 | 2017-02-16 | Salve Regina University | Electrochemical detection systems and methods using modified coated multi-labeled magnetic beads with polymer brushes |
| CN112505121B (en) * | 2020-12-11 | 2022-09-13 | 江苏知至生物科技有限公司 | Anti-interference photoelectrochemical disease marker sensor and preparation method thereof |
| CN112964765B (en) * | 2021-02-05 | 2022-12-27 | 重庆医科大学 | Electrochemical immunosensor for detecting CEA and preparation and application thereof |
| CN114924074B (en) * | 2022-05-22 | 2024-07-19 | 重庆医科大学 | Electrochemical immunosensor for detecting breast cancer HER2 and detection method thereof |
| CN115403672A (en) * | 2022-08-29 | 2022-11-29 | 上海大学 | Biotinylated anti-lysozyme antibody and construction method thereof |
-
2022
- 2022-12-15 CN CN202211615834.4A patent/CN116008370B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114216943A (en) * | 2021-11-26 | 2022-03-22 | 青岛科技大学 | Anti-pollution electrochemical immunosensor based on DNA-polypeptide conjugate and preparation method and application thereof |
| CN114324526A (en) * | 2021-12-27 | 2022-04-12 | 青岛农业大学 | A biosensor for detecting prostate specific antigen in human serum and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116008370A (en) | 2023-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114216943B (en) | An anti-pollution electrochemical immune sensor and its preparation method and application | |
| CN116008370B (en) | Anti-pollution electrochemical immunosensor based on PEG-polypeptide conjugate, and preparation method and application thereof | |
| Knichel et al. | Utilization of a self-assembled peptide monolayer for an impedimetric immunosensor | |
| Wang et al. | Application of impedance spectroscopy for monitoring colloid Au-enhanced antibody immobilization and antibody–antigen reactions | |
| Shi et al. | A novel label-free amperometric immunosensor for carcinoembryonic antigen based on redox membrane | |
| Wang et al. | Antifouling and ultrasensitive biosensing interface based on self-assembled peptide and aptamer on macroporous gold for electrochemical detection of immunoglobulin E in serum | |
| Han et al. | Highly conducting gold nanoparticles–graphene nanohybrid films for ultrasensitive detection of carcinoembryonic antigen | |
| Huang et al. | Selective and sensitive glycoprotein detection via a biomimetic electrochemical sensor based on surface molecular imprinting and boronate-modified reduced graphene oxide | |
| Wang et al. | Ultrasensitive detection of carcinoembryonic antigen by a simple label-free immunosensor | |
| Zhu et al. | Amperometric immunosensor for simultaneous detection of three analytes in one interface using dual functionalized graphene sheets integrated with redox-probes as tracer matrixes | |
| CN111693571B (en) | Method for detecting GPC3 based on light addressing potential sensor | |
| Zhu et al. | Simultaneous detection of four biomarkers with one sensing surface based on redox probe tagging strategy | |
| CN104833718B (en) | Preparation method and application of a pH release type immunosensor | |
| Karaboğa et al. | A novel silanization agent based single used biosensing system: Detection of C-reactive protein as a potential Alzheimer’s disease blood biomarker | |
| CN111505077A (en) | A method for detecting GPC3 based on RGO-Hemin/Au NPs nanocomposites | |
| Ling et al. | Study on immunosensor based on gold nanoparticles/chitosan and MnO2 nanoparticles composite membrane/Prussian blue modified gold electrode | |
| Rezaei et al. | Stainless steel modified with an aminosilane layer and gold nanoparticles as a novel disposable substrate for impedimetric immunosensors | |
| Xie et al. | An electrochemical aptasensor for Mycobacterium tuberculosis ESAT-6 antigen detection using bimetallic organic framework | |
| CN115201302A (en) | Electrochemical sensor, preparation method and application thereof | |
| Wang et al. | Antibody-functionalized magnetic nanoparticles for electrochemical immunoassay of α-1-fetoprotein in human serum | |
| Li et al. | Label-free detection of glypican-3 using reduced graphene oxide/polyetherimide/gold nanoparticles enhanced aptamer specific sensing interface on light-addressable potentiometric sensor | |
| Wang et al. | Immunosensors Based on Layer‐by‐Layer Self‐Assembled Au Colloidal Electrode for the Electrochemical Detection of Antigen | |
| Shi et al. | Amplification of antigen–antibody interactions via back-filling of HRP on the layer-by-layer self-assembling of thionine and gold nanoparticles films on Titania nanoparticles/gold nanoparticles-coated Au electrode | |
| CN113155930A (en) | Electrochemical immunosensing method for detecting leukemia stem cell tumor marker CD123 by using multiple signal amplification technology | |
| CN111004836A (en) | Ratiometric electrochemical aptamer sensor with bidirectional amplification and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 266061 No. 99 Songling Road, Qingdao City, Shandong Province Applicant after: QINGDAO University OF SCIENCE AND TECHNOLOGY Address before: 266042 Zhengzhou Road, Shibei District, Qingdao, Shandong 53 Applicant before: QINGDAO University OF SCIENCE AND TECHNOLOGY |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |