CN117147654A - Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof - Google Patents
Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof Download PDFInfo
- Publication number
- CN117147654A CN117147654A CN202311121531.1A CN202311121531A CN117147654A CN 117147654 A CN117147654 A CN 117147654A CN 202311121531 A CN202311121531 A CN 202311121531A CN 117147654 A CN117147654 A CN 117147654A
- Authority
- CN
- China
- Prior art keywords
- bsa
- mxene
- pei
- solution
- 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.)
- Pending
Links
- 229920002873 Polyethylenimine Polymers 0.000 title claims abstract description 95
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000000463 material Substances 0.000 title claims abstract description 23
- 239000012888 bovine serum Substances 0.000 title claims abstract description 18
- 108091008102 DNA aptamers Proteins 0.000 claims abstract description 25
- 239000002086 nanomaterial Substances 0.000 claims abstract description 14
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 55
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 37
- 229940098773 bovine serum albumin Drugs 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 14
- 229920001661 Chitosan Polymers 0.000 claims description 12
- KRHYYFGTRYWZRS-UHFFFAOYSA-N Fluorane Chemical compound F KRHYYFGTRYWZRS-UHFFFAOYSA-N 0.000 claims description 12
- 238000002791 soaking Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 230000003213 activating effect Effects 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 125000003277 amino group Chemical group 0.000 claims description 4
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical group O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- 238000000835 electrochemical detection Methods 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 15
- 230000008859 change Effects 0.000 description 13
- 238000001903 differential pulse voltammetry Methods 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 238000004132 cross linking Methods 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 9
- 238000000576 coating method Methods 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 8
- 239000011248 coating agent Substances 0.000 description 7
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 6
- 229910010293 ceramic material Inorganic materials 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 108091023037 Aptamer Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 4
- 238000004833 X-ray photoelectron spectroscopy Methods 0.000 description 4
- 230000003373 anti-fouling effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000027756 respiratory electron transport chain Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000000439 tumor marker Substances 0.000 description 3
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005530 etching Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000013112 stability test Methods 0.000 description 2
- 241000235648 Pichia Species 0.000 description 1
- 238000000026 X-ray photoelectron spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 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
- 238000004090 dissolution Methods 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000009881 electrostatic interaction Effects 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
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 238000002546 full scan Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000000733 zeta-potential measurement Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Urology & Nephrology (AREA)
- Oncology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Hospice & Palliative Care (AREA)
- Microbiology (AREA)
- Nanotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a biosensor based on bovine serum albumin-polyethyleneimine materials and a preparation method thereof, which relate to the technical field of electrochemical detection and comprise a BSA solution, a PE I solution, a nano material MXene and a DNA aptamer, wherein the BSA solution and the PE I solution are covalently crosslinked to a glassy carbon electrode modified with the conductive nano material MXene, and then the DNA aptamer is covalently connected to prepare the sensor.
Description
Technical Field
The invention relates to the technical field of electrochemical detection, in particular to a biosensor based on bovine serum albumin-polyethyleneimine material and preparation thereof.
Background
The electrochemical sensing technology has great potential in the aspect of disease biomarker detection due to the advantages of quick response, low cost, simple preparation, sensitive detection and the like, but in practical use, pollution problems in biological fluid have great threat to detection accuracy, stability and sensitivity, so that great attention has been paid to searching anti-pollution materials with excellent performance to construct an effective anti-pollution sensing interface in recent years, and the commonly used anti-pollution materials which are found at present are polyethylene glycol (PEG) and derivatives thereof, zwitterionic polymers, anti-pollution polypeptides and the like, but the materials still have the problems of poor stability, complex synthesis and the like.
In recent years, the anti-pollution performance of protein-based materials has been paid attention to, wherein, natural Bovine Serum Albumin (BSA) has wide application in biochemical experiments due to the advantages of low cost, high abundance, good biocompatibility and the like, for example, the natural BSA protein can be used as a blocking agent to be anchored on the surface of the materials, and can also be used as a passivating agent of a sensing interface to block active sites, thereby preventing the adsorption of nonspecific proteins to a certain extent.
In the prior art, BSA protein physically adsorbed on an interface is poor in stability, and can seriously block charge transfer on the surface of an electrode, so that the anti-pollution performance and sensitivity of the interface are limited, the BSA protein modified interface presents stronger electronegativity, and the interface has certain hydrophobicity, so that the anti-pollution performance of the interface is limited, and the preparation and the use of an actual biosensor are not facilitated.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a biosensor based on bovine serum albumin-polyethyleneimine material and a preparation method thereof, and solves the problems of limited anti-pollution performance and interface electron transfer inhibition of single protein.
Technical proposal
In order to achieve the above purpose, the invention is realized by the following technical scheme: the biosensor based on bovine serum albumin-polyethyleneimine material comprises a BSA solution, a PEI solution, a nanomaterial MXene and a DNA aptamer, wherein the BSA solution and the PEI solution are covalently crosslinked on a glassy carbon electrode modified with a conductive nanomaterial MXene, and then the sensor is prepared by covalently connecting the DNA aptamer, and the preparation method of the sensor comprises the following steps:
sp1: electrode pretreatment, namely uniformly mixing the prepared MXene with a CS solution, then dripping the mixture on a pretreated bare glassy carbon electrode, and naturally drying the mixture at room temperature to obtain an MXene-CS modified electrode;
sp2: preparing a BSA-PEI/MXene-CS modified electrode, activating the MXene-CS modified electrode by glutaraldehyde, and then soaking the activated MXene-CS modified electrode in a mixed solution of BSA and PEI to obtain the BSA-PEI/MXene-CS modified electrode;
sp3: and (3) forming and preparing the sensor, wherein the BSA-PEI/MXene-CS modified electrode is soaked in the DNA aptamer solution activated by the EDC/NHS solution, and the sensor is obtained.
Preferably, in the electrode pretreatment process, 8mgTi is used as a catalyst 3 AlC 2 Slowly adding the powder into 32mL hydrofluoric acid solution with volume of 40% and mass fraction, continuously stirring at high speed at room temperature for 48 hr, repeatedly washing the material with secondary water and absolute ethanol, centrifuging until the supernatant reaches pH not less than 6, and vacuum drying the precipitate at 60deg.C for 12 hr to obtain Ti 3 C 2 T x MXene powder is evenly mixed with CS solution with the mass fraction of 0.2 percent according to the volume ratio of 1:2 after being dissolved by secondary water ultrasonic, and then 20 mu L of the mixture is dripped on a pretreated bare glassy carbon electrode, and the mixture is naturally dried at room temperature.
Preferably, in the preparation of the BSA-PEI/MXene-CS modified electrode, 5% glutaraldehyde is used for activating an amino group on the MXene-CS modified electrode for 50min, then the BSA-PEI/MXene-CS modified electrode is soaked in a mixed solution of BSA and PEI, the reaction is carried out for 16h at room temperature, after the reaction is finished, secondary water is used for washing, and the BSA-PEI/MXene-CS modified electrode which is not covalently crosslinked is removed, thus obtaining the BSA-PEI/MXene-CS modified electrode.
Preferably, in the preparation of the sensor, EDC/NHS solution is firstly used for activating carboxyl groups on the DNA aptamer for 0.5h, then a BSA-PEI/MXene-CS modified electrode is soaked in the DNA aptamer solution with the concentration of not less than 1 mu M, the reaction is carried out for 2h at room temperature, and after the reaction is finished, secondary water is used for washing to remove the unfixed DNA aptamer, thus obtaining the sensor.
Preferably, in the mixed solution of BSA and PEI used in the preparation of the BSA-PEI/MXene-CS modified electrode, the concentration of BSA is 1mg/mL, and the mass percentage of PEI is 3wt%.
Preferably, the BSA solution and the PEI solution are a bovine serum albumin solution and a polyethylenimine solution, respectively, and the CS solution is a chitosan solution.
Advantageous effects
The invention provides a biosensor based on bovine serum albumin-polyethyleneimine material and preparation thereof. The beneficial effects are as follows:
the invention adopts covalent crosslinking of bovine serum albumin-polyethyleneimine to a glassy carbon electrode modified with MXene, and then covalent connection of a DNA aptamer, thus a stable anti-pollution electrochemical biosensor is constructed, excellent conductivity of MXene and excellent performance of a bovine serum albumin-polyethyleneimine crosslinking coating are integrated, the sensor prepared by the invention can effectively resist unnecessary biological pollution, and realizes specific, simple and accurate detection of a cancer marker CA125 in a real sample, and experiments prove that the modification interface of the bovine serum albumin-polyethyleneimine crosslinking coating has more excellent anti-pollution performance and higher sensitivity than that of a single bovine serum albumin modification interface.
Drawings
FIG. 1 is a schematic diagram showing the construction process of the biosensor and the detection process of the target CA 125;
FIG. 2 is a scanning electron microscope image of a different modified electrode provided in an embodiment of the present invention;
FIG. 3 is a full-scan X-ray photoelectron spectrum of different modified electrodes provided by an embodiment of the present invention;
FIG. 4 is a graph of zeta potential data of BSA, PEI and BSA-PEI provided by an embodiment of the present invention;
FIG. 5 is a graph of water contact angles for different modified interfaces provided by embodiments of the present invention;
FIG. 6 is a graph showing DPV response curves for different working electrodes provided by an embodiment of the present invention;
FIG. 7 is a graph showing DPV test of different modified electrodes in human serum with different concentrations according to an embodiment of the present invention;
FIG. 8 is a comparative bar graph of anti-fouling performance of different modified electrodes provided in the examples of the present invention in human serum of different concentrations;
FIG. 9 is a confocal image of laser after incubation of different modified interfaces provided in the examples of the present invention in 0.2mg/mL fluorescent protein for 2h and co-culture with cells for 24 h;
FIG. 10 is a graph (a) and a linear curve (b) of the DPV response of the sensor to CA125 at various concentrations according to an embodiment of the present invention;
FIG. 11 is a diagram showing a specific test of a biosensor according to an embodiment of the present invention;
FIG. 12 is a chart showing a stability test of a biosensor according to an embodiment of the present invention;
FIG. 13 is a reproduction test chart of a biosensor according to an embodiment of the present invention;
FIG. 14 is a bar graph showing analytical performance of the detection method and ELISA detection method according to the embodiment of the present invention;
FIG. 15 is a graph of PEI concentration optimization provided by an example of the present invention (signal change of modified electrode immersed in 20% human serum for 30 min);
FIG. 16 is a graph of BSA concentration optimization provided by an example of the present invention (signal change of modified electrode immersed in 20% human serum for 30 min);
FIG. 17 is a graph of optimized cross-linking time for BSA-PEI coatings provided by examples of the present invention (modified electrode signal change in 20% human serum for 30 min);
FIG. 18 is a graph of optimization of DNA aptamer concentration provided in an embodiment of the invention;
fig. 19 is an optimized view of the incubation time of the target object according to the embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. 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.
Detailed description of the preferred embodiments
As shown in fig. 1 to 19, the biosensor based on bovine serum albumin-polyethyleneimine material and the preparation thereof comprise a BSA solution, a PEI solution, a nanomaterial MXene and a DNA aptamer, wherein the BSA solution and the PEI solution are covalently crosslinked to a glassy carbon electrode modified with a conductive nanomaterial MXene, and then the DNA aptamer is covalently connected to prepare the sensor, and the preparation method of the sensor comprises the following steps:
sp1: before the electrode is subjected to interface modification, polishing the interface by using alumina powder with the particle size of 0.3 mu m and 0.05 mu m respectively, and then ultrasonically cleaning water, absolute ethyl alcohol and water respectively for 1-2min;
sp2: preparation of BSA-PEI/MXene-CS modified electrode 8mgTi 3 AlC 2 Slowly adding the powder into 32mL hydrofluoric acid solution with volume of 40% and mass fraction, continuously stirring at high speed at room temperature for 48 hr, repeatedly washing the material with secondary water and absolute ethanol, centrifuging until the supernatant reaches pH not less than 6, and vacuum drying the precipitate at 60deg.C for 12 hr to obtain Ti 3 C 2 T x Uniformly mixing MXene powder with CS solution with the mass fraction of 0.2% according to the volume ratio of 1:2 after ultrasonic dissolution by secondary water, then dripping 20 mu L of the mixture on a pretreated bare glassy carbon electrode, naturally drying at room temperature, activating amino groups on an MXene-CS modified electrode by 5% glutaraldehyde for 50min, soaking the modified electrode in a mixed solution of BSA and PEI, reacting for 16h at room temperature, washing by secondary water after the reaction is finished, and removing non-covalent cross-linked BSA-PEI to obtain the BSA-PEI/MXene-CS modified electrode;
sp3: the method comprises the steps of (1) preparing a sensor by molding, activating carboxyl on a DNA aptamer with EDC/NHS solution for 0.5h, soaking a BSA-PEI/MXene-CS modified electrode in the DNA aptamer solution with the concentration of not less than 1 mu M, reacting for 2h at room temperature, and washing with secondary water after the reaction is finished to remove the unfixed DNA aptamer, thus obtaining the anti-pollution sensor.
In the preparation process of the sensor, an instrument adopted is a CHI650E electrochemical workstation in sequence for electrochemical testing and characterization, a traditional three-electrode system is adopted, a glassy carbon electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode and a platinum wire is used as a counter electrode, a Hitachi S-4800 scanning electron microscope is used for characterizing the surface morphology of different modified interfaces, an ESCALAB250Xi spectrometer is used for performing X-ray photoelectron spectroscopy on the different modified interfaces, a JC2000D1 water contact angle measuring instrument is used for characterizing the surface wettability of the different modified interfaces, zeta potential measurement is performed on a ZETASIZERNano-ZS, and non-specific adsorption testing of fluorescent marker proteins and cells is performed on a TCSSP5 confocal laser microscope.
The adopted reagent and material and the source thereof are 98 percent of mass fraction and 200 meshes of Ti 3 AlC 2 Powders were purchased from gosman technologies, inc, 40wt% hydrofluoric acid was purchased from hadamard reagent, inc, branched polyethylenimine PEI, LS measured for average Mw-25,000 da, gpc measured for average Mn-10,000 da was purchased from sigma, chitosan (CS), glutaraldehyde (GA), N-hydroxysuccinimide (NHS) were all from michelin biochemistry, inc, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was obtained from pichia pharmaceutical technologies, bovine Serum Albumin (BSA), fluorescein isothiocyanate labeled BSA (FITC-BSA) was purchased from hadamard reagent, inc, saccharide antigen 125 (CA 125), CA125 aptamer and Fluorescein Diacetate (FDA), all reagents were analytical grade, all experiments were used with ultrapure water, and water purification systems were provided by bedford li-Q systems, massachusetts, usa.
The stable anti-pollution electrochemical biosensor is constructed by covalently crosslinking bovine serum albumin-polyethyleneimine to a glassy carbon electrode modified with MXene and then covalently connecting a DNA aptamer. The sensor prepared by the invention can effectively resist unnecessary biological pollution, realizes the specificity, simplicity, sensitivity and accuracy detection of a cancer marker CA125 in a real sample, and has more excellent pollution resistance and higher sensitivity compared with a single bovine serum albumin modified interface through experimental verification.
Use of the prepared biosensor for detecting cancer marker CA125, wherein the biosensor is soaked in solutions containing targets with different concentrations, incubated at constant temperature for not less than 1.5h, then washed with PBS buffer solution to remove nonspecifically captured CA125, and the electrode is soaked in [ Fe (CN) containing 0.1MKCl 6 ] 3-/4- In the solution of (2), the differential pulse voltammetry is utilized, the voltage range is-0.2V-0.6V, the electric signal change before and after the target object is identified is recorded, the specific detection of the target object CA125 is realized, the linear range of the target object CA125 is detected to be 0.01-1000U/mL, and the detection limit is 2.6mU/mL.
Second embodiment
As shown in FIGS. 1-19, experiments were performed on the characterization of BSA-PEI/MXene-CS modified electrodes, and the following are specifically shown:
scanning electron microscope characterization of different modified interface surface topography, in particular as can be seen from the scanning electron microscope image in FIG. 2A, ti 3 AlC 2 The porous ceramic material has a typical compact bulk structure, gaps among layers are not left, the ceramic material is shown in fig. 2B, an Al layer is removed after hydrofluoric acid etching, the MXene has an accordion-shaped layered structure with obvious gaps, the interesting structure enables the MXene to have a larger specific surface area and excellent conductivity, the ceramic material is shown in fig. 2C, after the ceramic material is further mixed with chitosan, the MXene is stably attached to an interface due to film forming effect of the chitosan and electrostatic interaction between CS and the MXene, the surface of the ceramic material is not provided with an obvious bulk structure after BSA-PEI is connected in fig. 2D, a cross-linked coating is successfully modified on the interface, and a BSA-PEI/MXene-CS modified electrode is successfully prepared.
XPS characterization of the different modified interfaces, in particular further analysis of the chemical composition of the different modified interfaces by X-ray photoelectron spectroscopy (XPS), as shown in FIG. 3A, ti 3 AlC 2 Modification of interfacial memoryTypical peaks of F1S appear at C1S, O1S, ti2p and Al2p, typical peaks of Al2p disappear at MXene modified interfaces, typical peaks of Al2p appear due to successful etching of Al layers by hydrofluoric acid, and the appearance of typical peaks of N1S originate from amino groups on chitosan, which proves successful modification of MXene-CS, and typical characteristic peaks of S2S and S2p appear after further modification of BSA-PEI crosslinked coating, and N1S typical peaks are obviously enhanced due to disulfide bonds in BSA and nitrogen-rich characteristics of BSA and PEI, respectively, in FIG. 3D.
Detailed description of the preferred embodiments
As shown in FIGS. 1-19, the characterization of BSA-PEI was tested, as shown in FIG. 4, by zeta potential test it was seen that BSA had a very large electronegativity (about-18.9 mV), whereas due to the cationic nature of PEI (about +30.3 mV), the mixed solution of PEI and BSA was close to electroneutrality (about-0.59 mV), indicating that the BSA-PEI modified interface had the potential to resist non-specific protein adsorption, and then the hydrophilicity of the BSA-PEI modified interface was evaluated by water contact angle test, as shown in FIG. 5, by MXene hydrophilicity, the water contact angle was reduced to 46.85 ℃compared to the bare electrode (64.68 ℃) followed by a significant decrease in interface hydrophilicity after covalent cross-linking with a single bovine serum albumin, the water contact angle was significantly increased to 72.72℃and after cross-linking with BSA-PEI, the modified interface water contact angle was significantly reduced to 19.69℃indicating that the interface had excellent hydrophilicity and great potential to resist non-specific protein adsorption, and the BSA-PEI modified interface was subjected to FIGS. 2, 4, 6, 8) (5B) and salt strength (1 aCl, caCl) at different pH values 2 、MgCl 2 、Na 2 SO 4 ) After 3h of soaking in the solution (FIG. 5C), the water contact angle is not changed obviously, the hydrophilicity is still good, and the modified interface has good acid resistance, alkali resistance and salt resistance.
Detailed description of the preferred embodiments
Electrochemical characterization of anti-fouling electrochemical biosensors based on BSA-PEI crosslinked conductive nanomaterials, as shown in FIGS. 1-19, the sensor construction process was characterized by electrochemical DPV testing in the presence of a negative electrical probe [ Fe (CN) 6 ] 3-/4- Is carried out in the iron standard solution of (2) as shown in FIG. 6, after incubation with MXene-CS, since MXene has a large sizeThe specific surface area and the excellent conductivity are obviously increased, after the BSA-PEI coating is crosslinked, the electron transfer on the surface of the electrode is blocked to a certain extent due to the insulating property of protein and polymer, the electric signal is reduced, and it is worth noting that compared with the interface of physically adsorbing BSA and only crosslinking BSA, the BSA-PEI crosslinked coating can better maintain the electron transfer to the bottom electrode due to the property of being close to the electric neutrality of the interface modified by the crosslinking with the conductive nanomaterial, has relatively high electric signal, and can further block the electron transfer on the surface of the electrode after the DNA aptamer and the recognition target are sequentially connected, the electric signal is sequentially reduced, and the change of the DPV signal indicates the successful construction of the sensor and the successful detection of the target.
Detailed description of the preferred embodiments
As shown in fig. 1 to 19, an anti-contamination assay was performed on the prepared biosensor as follows:
soaking different modified interfaces in human serum with different dilution concentrations for 30min, respectively, and adding 0.1MKCl [ Fe (CN) 6 ] 3-/4- The DPV test was performed in the solution of (2) and the change in the electrical signal before and after soaking was recorded, as shown in FIG. 7, the BSA-PEI/MXene-CS modified interface was able to maintain most of the original signal to the greatest extent in human serum at different concentrations compared to the naked, MXene-CS and BSA/MXene-CS modified interfaces, and more importantly, as shown in FIG. 8, after soaking in 100% human serum, the signal inhibition ratio (%) = (I 0 -I)/I 0 Wherein I 0 And I represents the electrical signal values before and after soaking respectively) is only 10.24%, which indicates that the BSA-PEI modified interface can effectively resist nonspecific adsorption and has more excellent anti-pollution performance than a single BSA coating.
The bare, MXene-CS, BSA/MXene-CS and BSA-PEI/MXene-CS modified interfaces were incubated in 0.2mg/mLFITC-BSA solution for 2H in the dark, and the adsorption of the non-specific proteins by the different modified interfaces was evaluated by TCS-SP5 confocal laser microscopy, as seen in the presence of significant green fluorescence on the bare (FIG. 9A), MXene-CS (FIG. 9B) and BSA/MXene-CS modified interfaces (FIG. 9C), while the BSA-PEI/MXene-CS modified interfaces (FIG. 9D) were almost non-fluorescent, and in the same way, co-cultured with hela cells for 24H, and after further staining with 50. Mu.g/mLFDA, similar results were obtained by cell imaging, with the bare (FIG. 9E), mne-CS (FIG. 9F) and BSA/MXene-CS modified interfaces (FIG. 9G) all showing that there was significant cell adsorption, whereas the non-specific adsorption of the modified interfaces on the-PEI/MXene-CS modified interfaces was almost non-excellent.
Description of the preferred embodiments
As shown in FIGS. 1-19, the analysis performance of an anti-pollution electrochemical biosensor based on BSA-PEI crosslinked conductive nanomaterial was tested by immersing the biosensor in a solution containing targets at different concentrations, incubating at constant temperature for not less than 1.5 hours, washing with PBS buffer solution to remove non-specifically captured CA125, and placing the above electrode in a solution containing 0.1MKCl [ Fe (CN) 6 ] 3-/4- In (a) and recording the change of the electric signal before and after identifying the target object by differential pulse voltammetry (voltage range-0.2V-0.6V), as shown in FIG. 10A (a-g represents the CA125 concentration of the target object as 0,0.01,0.1,1, 10, 100, 1000U/mL, respectively), the electric signal gradually decreases with the increase of the CA125 concentration, as shown in FIG. 10B, within the target object detection range of 0.01-1000U/mL, the signal change value (DeltaI=I 0 -I, wherein I 0 And I is the DPV current value before and after identifying the target object and the logarithm (LgC) of the concentration of the target object, wherein the linear equation is delta I (mu A) =4.28 LgC (U/mL) +12.36, and the correlation coefficient R 2 0.999 and a minimum detection Limit (LOD) of 2.6mU/mL.
Detailed description of the preferred embodiments
As shown in FIGS. 1-19, the selectivity test of an anti-fouling electrochemical biosensor based on BSA-PEI crosslinked conductive nanomaterial, the sensor was immersed in a solution containing 10U/mLCA125, 1. Mu.g/mLIgG, CEA, HSA, NSE and their mixed solutions, respectively, for 1.5 hours, and then the electrode was placed in a solution containing 0.1MKCl [ Fe (CN) 6 ] 3-/4- As shown in FIG. 11, even in the case where the concentration of the interfering protein is far greater than that of the target protein, only negligible changes in the electrical signal before and after the identification of each protein were generated by differential pulse voltammetry recordingThe response of the electrical signal indicates that the sensor of the invention has good selectivity.
Description of the preferred embodiments
As shown in fig. 1-19, stability test of anti-pollution electrochemical biosensor based on BSA-PEI crosslinked conductive nanomaterial: the sensor was placed in a solution containing 0.1MKCl [ Fe (CN) 6 ] 3-/4- In the solution of (2), a continuous CV scan of 100 turns was performed, and as shown in FIG. 12A, the electric signal was not significantly changed, and the stability was good.
Detailed description of the preferred embodiments
As shown in FIGS. 1-19, reproducibility test of anti-contamination electrochemical biosensor based on BSA-PEI crosslinked conductive nanomaterial, 7 sensors prepared under the same conditions were immersed in a solution containing 10U/mLCA125, incubated at constant temperature for not less than 1.5h, rinsed with PBS buffer solution to remove non-specifically captured CA125, and the above electrodes were placed in a solution containing 0.1MKCl [ Fe (CN) 6 ] 3-/4- The change of the electric signal before and after the identification of the target was recorded by differential pulse voltammetry (voltage range-0.2V-0.6V), and the result was shown in fig. 13, and the relative standard deviation was 3.82%, indicating that the sensor prepared by the present invention had good reproducibility.
Detailed description of the preferred embodiments
As shown in figures 1-19, the analysis performance of the provided detection method is compared with that of an ELISA detection method, the sensor prepared by the invention is used for detecting CA125 in an actual human serum sample, and the test result is compared with that of the ELISA method, as shown in figure 14, in the detection of five human serum samples, the sensor prepared by the invention has basically consistent detection results with the ELISA method, and the difference rate is less than 10%, so that the sensor prepared by the invention has great practical application potential.
Detailed description eleven of the invention
As shown in FIGS. 1-19, to obtain optimal anti-fouling and sensing properties, parameters (such as PEI concentration, BSA-PEI crosslinking time, DNA aptamer concentration and target incubation time) during sensor construction were optimized, and BSA-PEI was used for repairElectrical signal change of the decorative interface before and after soaking in 20% human serum for 30min (Δi=i 0 -I，I 0 And I represents the DPV electrical signal values before and after soaking in 20% human serum), respectively), the anti-contamination performance of the sensor is optimized, the smaller the electrical signal change, the better the anti-contamination performance of the modified interface is, as shown in fig. 15, when the PEI concentration increases by 3wt% from 0.5wt%, the electrical signal change decreases, and the concentration increases again, the electrical signal change increases, so 3wt% is selected as the optimal PEI concentration, and similarly, as shown in fig. 16, 1mg/mL is selected as the optimal BSA concentration; as shown in fig. 17, 16h was selected as the optimal BSA-PEI crosslinking time, and then, in order to obtain the optimal sensing performance, the concentration of the DNA aptamer and the target incubation time were optimized, and as shown in fig. 18, when the concentration of the aptamer reached 1 μm, the decrease value of the electric signal no longer increased with the increase in the concentration of the aptamer, and the interface binding amount reached saturation, so 1 μm was selected as the optimal aptamer concentration, and similarly, 90min was selected as the optimal target incubation time.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises an element.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. The biosensor based on bovine serum albumin-polyethyleneimine material comprises BSA solution, PEI solution, nanomaterial MXene and DNA aptamer, and is characterized in that: the BSA solution and the PEI solution are covalently crosslinked on a glassy carbon electrode modified with a conductive nanomaterial MXene, and then are covalently connected with a DNA aptamer to prepare a sensor, and the preparation method of the sensor is as follows:
sp1: electrode pretreatment, namely uniformly mixing the prepared MXene with a CS solution, then dripping the mixture on a pretreated bare glassy carbon electrode, and naturally drying the mixture at room temperature to obtain an MXene-CS modified electrode;
sp2: preparing a BSA-PEI/MXene-CS modified electrode, activating the MXene-CS modified electrode by glutaraldehyde, and then soaking the activated MXene-CS modified electrode in a mixed solution of BSA and PEI to obtain the BSA-PEI/MXene-CS modified electrode;
sp3: and (3) forming and preparing the sensor, wherein the BSA-PEI/MXene-CS modified electrode is soaked in the DNA aptamer solution activated by the EDC/NHS solution, and the sensor is obtained.
2. The bovine serum albumin-polyethylenimine material-based biosensor and preparation according to claim 1, wherein: in the electrode pretreatment process, 8mgTi is used for 3 AlC 2 Slowly adding the powder into 32mL hydrofluoric acid solution with volume of 40% and mass fraction, continuously stirring at high speed at room temperature for 48 hr, repeatedly washing the material with secondary water and absolute ethanol, centrifuging until the supernatant reaches pH not less than 6, and vacuum drying the precipitate at 60deg.C for 12 hr to obtain Ti 3 C 2 T x MXene powder is evenly mixed with CS solution with the mass fraction of 0.2 percent according to the volume ratio of 1:2 after being dissolved by secondary water ultrasonic, and then 20 mu L of the mixture is dripped on a pretreated bare glassy carbon electrode, and the mixture is naturally dried at room temperature.
3. The bovine serum albumin-polyethylenimine material-based biosensor and preparation according to claim 1, wherein: in the preparation of the BSA-PEI/MXene-CS modified electrode, 5% glutaraldehyde is used for activating an amino group on the MXene-CS modified electrode for 50min, the BSA-PEI/MXene-CS modified electrode is soaked in a mixed solution of BSA and PEI, the reaction is carried out for 16h at room temperature, after the reaction is finished, secondary water is used for washing, and the BSA-PEI/MXene-CS modified electrode which is not covalently crosslinked is removed, thus obtaining the BSA-PEI/MXene-CS modified electrode.
4. The bovine serum albumin-polyethylenimine material-based biosensor and preparation according to claim 1, wherein: in the preparation of the sensor, EDC/NHS solution is firstly used for activating carboxyl on DNA aptamer for 0.5h, then BSA-PEI/MXene-CS modified electrode is soaked in DNA aptamer solution with concentration not less than 1 mu M, reaction is carried out for 2h at room temperature, after the reaction is finished, secondary water is used for washing, and unfixed DNA aptamer is removed, thus obtaining the sensor.
5. A biosensor based on bovine serum albumin-polyethylenimine material and preparation according to claim 3, characterized in that: in the mixed solution of BSA and PEI used in the preparation of the BSA-PEI/MXene-CS modified electrode, the concentration of BSA is 1mg/mL, and the mass percentage of PEI is 3wt%.
6. The bovine serum albumin-polyethylenimine material-based biosensor and preparation according to claim 1, wherein: the BSA solution and the PEI solution are respectively a bovine serum albumin solution and a polyethyleneimine solution, and the CS solution is a chitosan solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311121531.1A CN117147654A (en) | 2023-08-31 | 2023-08-31 | Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311121531.1A CN117147654A (en) | 2023-08-31 | 2023-08-31 | Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117147654A true CN117147654A (en) | 2023-12-01 |
Family
ID=88909515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311121531.1A Pending CN117147654A (en) | 2023-08-31 | 2023-08-31 | Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117147654A (en) |
-
2023
- 2023-08-31 CN CN202311121531.1A patent/CN117147654A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lin et al. | Antifouling strategies in advanced electrochemical sensors and biosensors | |
| Pei et al. | Amplification of antigen–antibody interactions based on biotin labeled protein–streptavidin network complex using impedance spectroscopy | |
| Finklea et al. | Formation of an organized monolayer by solution adsorption of octadecyltrichlorosilane on gold: electrochemical properties and structural characterization | |
| Goss et al. | Application of (3-mercaptopropyl) trimethoxysilane as a molecular adhesive in the fabrication of vapor-deposited gold electrodes on glass substrates | |
| 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 | |
| JP2011516651A (en) | Method of assembling two surfaces or one surface and a target molecule | |
| CN112595765B (en) | Preparation method of anti-pollution electrochemical biosensor based on temperature-sensitive western blotting gel | |
| CN112903781B (en) | Molecularly imprinted electrochemical sensor of electro-copper-based MOFs sensitive membrane modified electrode and preparation method and detection method thereof | |
| Liang et al. | Fabrication, characterization, and application of potentiometric immunosensor based on biocompatible and controllable three-dimensional porous chitosan membranes | |
| CN105907844B (en) | Electrochemical DNA biosensor based on three-dimensional graphene-dendritic nanogold and preparation method thereof | |
| Shen et al. | Molecularly imprinted electrochemical sensor for advanced diagnosis of alpha-fetoprotein | |
| Wang et al. | Immunosensors Based on Layer‐by‐Layer Self‐Assembled Au Colloidal Electrode for the Electrochemical Detection of Antigen | |
| CN114216943B (en) | Anti-pollution electrochemical immunosensor and preparation method and application thereof | |
| CN113671002A (en) | Method for detecting bioactive substances based on gold surface modified cucurbit [7] uril | |
| CN117147654A (en) | Biosensor based on bovine serum albumin-polyethyleneimine material and preparation method thereof | |
| Solanki et al. | Self-assembled monolayer based impedimetric platform for food borne mycotoxin detection | |
| CN115791907A (en) | Nano-fold and albumin assembly modified protein pollution resistant electrochemical biosensor and preparation method and application thereof | |
| Tang et al. | Direct and rapid detection of diphtherotoxin via potentiometric immunosensor based on nanoparticles mixture and polyvinyl butyral as matrixes | |
| CN112763553B (en) | Electrochemical detection method for protein based on molecular imprinting technology | |
| Gheorghiu et al. | Electrochemical behavior of oxazoline‐based plasma polymers for biosensing applications | |
| Yeh et al. | One-step electrochemical deposition of antifouling polymers with pyrogallol for biosensing applications | |
| CN114324526B (en) | Biosensor for detecting prostate specific antigen in human serum and preparation method and application thereof | |
| Sun et al. | Electrochemical immunosensors based on different Au modified dendritic mesoporous silica nanoparticles tag for simultaneous detection of eggs and milk proteinaceous binders used in ancient wall paintings | |
| Fu et al. | Reagentless immunosensing assay via electrochemical impedance for hepatitis B surface antigen monitoring based on polypyrrole and gold nanoparticles as matrices | |
| EP3284581B1 (en) | Method to fabricate chemically-stable plasma-etched substrates for direct covalent biomolecule immobilization |
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 |