CN107167608B - A kind of graphene tumor marker sensor and preparation method thereof based on multifunctional nano grade protein films - Google Patents
A kind of graphene tumor marker sensor and preparation method thereof based on multifunctional nano grade protein films Download PDFInfo
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- CN107167608B CN107167608B CN201710219519.2A CN201710219519A CN107167608B CN 107167608 B CN107167608 B CN 107167608B CN 201710219519 A CN201710219519 A CN 201710219519A CN 107167608 B CN107167608 B CN 107167608B
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- 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
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- 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
Abstract
The graphene tumor marker sensor and preparation method thereof based on multifunctional nano grade protein films that the present invention relates to a kind of, including the graphene, multifunctional nano grade protein films and reaction chamber being transferred in silicon dioxide substrates.Preparation method includes: that (1) graphene is transferred to titanium electrode surface；(2) inactivation BSA protein films are grapheme modified；(3) photoetching and etching；(4) preparation protection insulating layer；(5) coupled antibody.Modification of the present invention is simply easily operated, versatile, can carry out different modifications according to different needs to realize the high sensitivity and specific detection of different target molecule, have a good application prospect.
Description
Technical field
It is the invention belongs to graphene biosensor field, in particular to a kind of based on multifunctional nano grade protein films
Graphene tumor marker sensor and preparation method thereof.
Background technique
With the appearance of the nano materials such as nano wire, carbon nanotube and graphene, life is utilized by electrical detection method
The charge of object itself can more efficiently realize the detection of biomolecule.In bio-sensing interface the selection of sensing material for
Constructing highly sensitive, reasonable price biosensor has critical effect.Two-dimensional graphene material has sp2Hydridization
Carbon atom is arranged in single layer atomic structure in a manner of honeycomb lattice.Excellent machinery, optics, electric property and high-ratio surface
Product, these features make graphene become a kind of ideal bioelectronics sensing material.Biosensor based on graphene exists
There is diversified application in the detection of the biomolecule such as albumen, DNA, nanogold and bacterium.
In order to the electric property for keeping graphene itself excellent, non-covalent method of modifying is normally used for graphene
In the building of biosensor.These non-covalent crosslinking agents are usually adsorbed on the surface of graphene by way of self assembly,
But there are adsorbances to be difficult to control, is readily incorporated the problems such as false positive signal.
In the building of traditional graphene biosensor, the production of device and the modification at interface are usually two independences
Part.And in the production of device, the techniques such as production of photoetching, etching and insulating layer can all introduce table on the surface of graphene
Face pollution, influences the performance of graphene and the modification of bioprobe, causes unfavorable shadow to the building of graphene biosensor
It rings, therefore has great importance using new method to construct biosensor.
Summary of the invention
It is swollen that technical problem to be solved by the invention is to provide a kind of graphenes based on multifunctional nano grade protein films
Tumor markers sensor and preparation method thereof, the sensor modification is simply easily operated, versatile, can be according to difference
Demand carry out different modifications realize different target molecule high sensitivity and specificity detection, there is good application
Prospect.
A kind of graphene tumor marker sensor based on multifunctional nano grade protein films of the invention, the sensing
Device includes the graphene being transferred in silicon dioxide substrates, multifunctional nano grade protein films and reaction chamber；Wherein, multi-functional
Nanoscale protein films non-covalent modification is in graphene surface；The multifunctional nano grade protein films are that inactivation BSA albumen is thin
Film.
It is described inactivation BSA protein films with a thickness of 9nm-15nm.It can be realized by changing the condition of modification to graphite
The different types of doping of alkene and the modification of different-thickness, and can reduce graphene to biology point in the way of this modification
The non-specific adsorption of son, to avoid the appearance of false positive signal.
The deactivation temperature of the inactivation BSA protein films is 60-90 DEG C, deactivation time 1-3min.
Inactivation BSA protein films surface coupled antibody.
The antibody is modified by EDC/NHS in inactivation BSA protein films surface.
A kind of preparation side of graphene tumor marker sensor based on multifunctional nano grade protein films of the invention
Method, comprising:
(1) titanium electrode is made in silicon dioxide substrates, transfers graphene to titanium electrode surface；
(2) BSA solution is added dropwise and carries out heat inactivation processing on the surface of graphene, cleaned, inactivated after the completion of inactivation
The graphene of BSA protein films modification；
(3) it using the channel dimensions of the graphene of lithographic definition inactivation BSA protein films modification, is carried out using plasma
It etches, is dissolved channel surface photoresist with acetone after the completion of etching；
(4) the graphene-channel surface that SU-8 photoresist is spin-coated on inactivation BSA protein films modification is insulated as protection
Layer；
(5) it in inactivation BSA protein films surface coupled antibody, is closed, will be reacted using BSA solution after the completion of coupling
Chamber be fixed on the surface of device to get.
Graphene in the step (1) is single-layer graphene.Titanium thickness of electrode is respectively 10nm and 100nm.
The concentration of BSA solution in the step (2) is 1%-10%.
The time of plasma etching in the step (3) is 4min-8min.
The protective layer of graphene is used as in the step (3) using the BSA protein films of inactivation, the middle inactivation of step (4)
BSA protein films avoid influence of the SU-8 glue to graphene as the protective layer of graphene.
Using EDC and NHS as crosslinking agent coupled antibody in the step (5), EDC concentration is 3mg/ml-6mg/ml,
NHS concentration is 0.6mg/ml-1.2mg/ml, antibody concentration 1mg/ml-3mg/ml.
The concentration of BSA solution in the step (5) is 1%, and off-period is 0.5-2 hours.
The present invention using inactivation BSA protein films not only as the protective layer of graphene device manufacturing process, but also
Non covalent crosslinker as graphene is coupled bioprobe, constructs graphene biosensor.The characteristics of sensor is logical
Multi-functional inactivation BSA film is crossed, can solve the problem of device introduces surface contamination in the production process, improves the property of device
Can, and the highly sensitive and special of target molecule is realized in inactivation BSA film surface coupled antibody using EDC and Sulfo-NHS
The detection of property.
Beneficial effect
The present invention modifies inactivation BSA protein films on the surface of graphene, acts not only as the guarantor of graphene device production
Sheath, while it being coupled bioprobe as the non covalent crosslinker of graphene, it by liquid grid graphene field effect pipe can be real
The detection of existing target molecule；Modification is simply easily operated, versatile, can carry out different repair according to different needs
The detection for adoring to realize the high sensitivity and specificity of different target molecule, has a good application prospect.
Detailed description of the invention
Fig. 1 is the structural diagram of the present invention；Wherein, 1 is silicon substrate, and 2 be silica, and 3 be drain terminal, and 4 be source, 5
For graphene, 6 be inactivation BSA protein films, and 7 and 8 be SU-8 photoresist, and 9 be reaction chamber, and 10 be antibody, and 11 be electrolyte, 12
It is Ag/AgCl reference electrode for target molecule antigen, 13；
Fig. 2 (a)~(g) is preparation technology flow chart of the invention；
Fig. 3 is the increment of source-drain current of the present invention with carcinomebryonic antigen (Carcinoembryonic Antigen, CEA) concentration
Change curve；Fig. 4 is specific detection curve of the invention.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Embodiment 1
As shown in Figure 1, the preparation process of the present embodiment is as follows:
The silica for having 300nm on a silicon substrate, by removing (lift-off) technique in silicon dioxide meter wheat flour
Make titanium electrode respectively as drain terminal and source, single-layer graphene is shifted in gold electrode surfaces, shown in processing step such as Fig. 2 (a).
BSA solution is added dropwise on the surface of graphene, the BSA protein films that 3min graphene surface forms inactivation, work are inactivated at 80 DEG C
Shown in skill step such as Fig. 2 (b).Graphene-channel size is determined by photoetching process and will be other than channel with oxygen plasma etch
Partial etching fall, shown in processing step such as Fig. 2 (c) and Fig. 2 (d).Finally the photoresist of ditch pipe protection is dissolved with acetone, work
Skill such as Fig. 2 (e) is shown, insulating protective layer of the spin coating SU-8 photoresist as electrode, and the SU- of electrode surface is left after exposure development
8 photoresists, shown in processing step such as Fig. 2 (f) and 2 (g).Inactivation BSA protein films are all used as graphene in entire process flow
Protective layer avoid surface contamination.
It is thin in inactivation BSA using EDC and NHS after graphene device based on inactivation BSA protein films modification completes
Film surface is coupled the antibody (Anti-CEA) of CEA, is closed 1 hour after the completion of modification with 1%BSA, in device table after the completion of closing
Reaction chamber is fixed in face, and the phosphate buffer of 1 μM of addition is as electrolyte, using the Ag/AgCl reference electrode and electricity of electrochemistry
Solution liquid cooperatively constitutes grid.By source in graphene field effect pipe and leakage both ends plus fixed voltage Vds and and
Grid source both ends are plus fixed voltage Vgs, by the way that the target molecule CEA antigen of various concentration is added, by measuring source-drain current
Variation realize target molecule detection.
The process of specific detection antigen is as follows:
(1) test buffer of 200ul is added in the reactor chamber, Anti-CEA is modified for the detection of various concentration antigen
Graphene field effect pipe source electrode ground connection, drain electrode add 0.1V voltage, grid add -0.3V voltage, record be added not
The increment of source-drain current under synantigen concentration, as shown in Figure 3.From the figure 3, it may be seen that as electronegative target molecule CEA is by Anti-
CEA captures the increase of quantity, and the conductance of graphene-channel increases, so that the graphene field effect pipe source and drain electricity of Anti-CEA modification
Stream increases.
(2) specific detection, test condition is identical as described in (1), and the serum for sequentially adding 2ul in the reactor chamber is dilute
Release liquid (cotrol), squamous cell carcinoma (Squamous Cell Carcinoma, SCC), cytokeratin 19 fragment
(Cytokerantin-19-fragment, CYFRA21-1) and CEA antigen carries out the electric current between GFET source and drain both ends
Monitoring, the change curve of electric current are as shown in Figure 4 in real time.Stone as can be seen from the results based on inactivation BSA protein films modification
Black alkene field-effect tube is apparently higher than the response of control group to the response of target molecule CEA albumen, thus may determine that the device has
There is good specificity.
Claims (5)
1. a kind of preparation method of the graphene tumor marker sensor based on multifunctional nano grade protein films, comprising:
(1) titanium electrode is made in silicon dioxide substrates, transfers graphene to titanium electrode surface；
(2) BSA solution is added dropwise and carries out heat inactivation processing on the surface of graphene, cleaned after the completion of inactivation, obtain inactivation BSA egg
The graphene of white film modification；
(3) it using the channel dimensions of the graphene of lithographic definition inactivation BSA protein films modification, is carved using plasma
It loses, is dissolved channel surface photoresist with acetone after the completion of etching；
(4) SU-8 photoresist is spin-coated on the graphene-channel surface of inactivation BSA protein films modification as protection insulating layer；
(5) it in inactivation BSA protein films surface coupled antibody, is closed after the completion of coupling using BSA solution, reaction chamber is consolidated
Be scheduled on the surface of device to get.
2. a kind of graphene tumor marker sensor based on multifunctional nano grade protein films according to claim 1
Preparation method, it is characterised in that: graphene in the step (1) is single-layer graphene.
3. a kind of graphene tumor marker sensor based on multifunctional nano grade protein films according to claim 1
Preparation method, it is characterised in that: the concentration of the BSA solution in the step (2) be 1%-10%.
4. a kind of graphene tumor marker sensor based on multifunctional nano grade protein films according to claim 1
Preparation method, it is characterised in that: the time of the plasma etching in the step (3) be 4min-8min.
5. a kind of graphene tumor marker sensor based on multifunctional nano grade protein films according to claim 1
Preparation method, it is characterised in that: using EDC and NHS as crosslinking agent coupled antibody in the step (5), EDC concentration is
3mg/ml-6mg/ml, NHS concentration are 0.6mg/ml-1.2mg/ml, antibody concentration 1mg/ml-3mg/ml.
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| CN108956743B (en) * | 2018-07-24 | 2021-01-08 | 哈尔滨工程大学 | Detection method of field effect transistor biosensor enhanced by AuNPs |
| CN111307912B (en) * | 2020-03-05 | 2022-08-09 | 苏州微湃医疗科技有限公司 | Field-effect tube biosensor and preparation method thereof |
| CN111938633A (en) * | 2020-08-10 | 2020-11-17 | 中国科学院上海微系统与信息技术研究所 | Preparation method and structure of brain electrode capable of being flexibly disassembled from scalp based on brain function partition |
| US11810953B2 (en) * | 2020-11-05 | 2023-11-07 | Graphenea Semiconductor SLU | Sensor having graphene transistors |
| CN115032246A (en) * | 2022-06-09 | 2022-09-09 | 大连理工大学 | Field-effect tube biosensor based on laser-induced graphene |
| CN115389586A (en) * | 2022-08-30 | 2022-11-25 | 松山湖材料实验室 | Graphene biosensor and preparation method thereof |
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