CN105044072B - One kind detects method of protein based on graphene sensor - Google Patents
One kind detects method of protein based on graphene sensor Download PDFInfo
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- CN105044072B CN105044072B CN201510509569.5A CN201510509569A CN105044072B CN 105044072 B CN105044072 B CN 105044072B CN 201510509569 A CN201510509569 A CN 201510509569A CN 105044072 B CN105044072 B CN 105044072B
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Abstract
Method of protein is detected based on graphene sensor the invention discloses one kind, including it is as follows：(1) graphene sensor is made：Face is provided with graphene film on a substrate, and the graphene film two ends are provided with electrode；(2) pretreatment of detected sample：Fluorescent dye is added in sample to be detected, for marking the protein in sample, fluorescent dye protein complex is obtained；(3) in sample protein content detection：Detected sample by pretreatment is added drop-wise to graphene film surface, fluorescence is quenched under ultraviolet light, graphene film resistance is changed, and resistance change is directly proportional to fluorescent dye protein complex content in sample, and the content of protein in sample is detected accordingly.Method of the present invention can be high that accurately protein is detected with highly sensitive, and compared with traditional fluorescence excitation probe method, method is simple, and measuring speed is fast.
Description
Technical field
The present invention relates to the technical field of biochemical analysis detection, it is related specifically to a kind of based on graphene sensor
Detect method of protein.
Background technology
Protein is the important matrix composition of all cells and tissue in organism, is the important undertaker of vital movement,
With nutrition, enzyme, virus, immune, heredity etc. suffer from it is close associate, therefore, carrying out analysis to the content of protein has ten
Divide important meaning, be essential in medical diagnosis on disease, biomedical research and trophic analysis evaluation.At present, it is conventional
Method for detecting protein content includes amino acid analysis method, Kjeldahl's method, absorption photometry, colorimetric method, uv-spectrophotometric
Method etc. is measured.XRF is method for detecting protein content developed in recent years, high with sensitivity, linearly
The characteristics of scope is wide.Mainly used using the method for fluorescence spectrometry protein content after fluorescent dye combined with protein,
There are energy transfer characteristics in spectrum, causes Fluorescence Increasing or decrease, i.e. sensitized fluorescence and fluorescent quenching phenomenon.And in certain egg
In white matter concentration range, fluorescence signal enhancing or decrease are directly proportional to the concentration of protein, it is possible thereby to measure protein
Content.But fluorescent dye combined with protein caused by fluorescence signal change density it is small, measure albumen just with it
Matter content is difficult.Therefore, it is in the prior art to utilize surfactants to expand the signal density that detected more.Fluorescence
Dye molecule easily passes through hydrophobic interaction formation dimer or polymer, polymer in the solution containing surfactant
Formation cause fluorescence intensity to reduce, then add protein solution, cause polymer depolymerization and increase fluorescence intensity, one
Determine fluorescence signal intensity in the range of protein concentration to be directly proportional to the concentration of protein.But it is due to that the reaction mechanism mechanism of reaction is complicated, no
Easily infer reaction mechanism, limit application of this method in meterological.
In recent years, with the appearance of graphene, graphene and its graphene oxide are in life science, particularly albumen
Application in the detection research of matter increases increasingly.Graphene is the Two-dimensional Carbon atomic crystal of monoatomic thickness, with big ratio table
A series of peculiar properties such as area, excellent biological chemical performance, cause the great interest of numerous scientific research personnel due to graphite
Alkene surface functional group is less, is difficult to be combined with biomolecule, therefore, often by graphene oxidation generation graphene oxide, at it
Surface produces the functional groups such as more carboxyl, substantially increases its application, be successfully realized to nucleic acid, protein with
And the detection of other biological small molecule.
Method of protein is detected using graphene oxide, such as patent 201410832817.5 is proposed《One kind is based on
The detection method of graphene oxide chip DNA unwindase》, mainly comprise the following steps：(1) preparing has graphene oxide point
The slide of battle array；(2) double chain DNA sequence is connected to the slide with graphene oxide dot matrix；(3) detection of DNA helicase：Plus
Enter the DNA helicase of varying number under different action times, after PBS is rinsed, be scanned and analyze, it is strong by FAM fluorescence
The change of degree, is detected.The invention fixes the double-stranded DNA of FAM (Fluoresceincarboxylic acid) marks on graphene oxide surface, when
When DNA is changed into single-stranded from double-strand, FAM and the distance on graphene oxide surface change, and graphene oxide passes through fluorescence
The degree that FAM fluorescence can be quenched in resonant energy transmission (FRET) is different, based on this feature, passes through the change of FAM fluorescence intensities
DNA helicase activity is detected.The invention can detect to DNA helicase in high sensitivity, and under cost, but
It is due to graphene oxide easy self aggregation under complex environment, can greatly has influence on the precision of detection；Simultaneously because facing
Bed sample composition is complicated, and the protein and other biological molecule contained can be adsorbed in nano-material surface to some extent, to inspection
Specificity is surveyed to impact.Therefore, this method is very difficult to apply in the example of clinical practice.So, to how preferably to play
With the characteristic using graphene, detection and analysis for protein provide more convenient and efficient method, in addition it is also necessary to further
Research and discussion.
The content of the invention
It is a kind of based on graphene sensor detection method of protein it is an object of the invention to propose, with high sensitivity
And precision, and method is simple, measuring speed is fast.
Therefore, the present invention uses following technical scheme：
One kind detects method of protein based on graphene sensor, comprises the following steps：
(1) graphene sensor is made：Face is provided with graphene film on a substrate, and the graphene film two ends are provided with
Electrode；
(2) pretreatment of detected sample：Fluorescent dye is added in sample to be detected, for marking the egg in sample
White matter, obtains fluorescent dye-protein complex；
(3) in sample protein content detection：Detected sample by pretreatment is added drop-wise to graphene film table
Face, descends fluorescence to be quenched for a period of time in ultraviolet light, and graphene film resistance changes, resistance change and sample
The content of middle fluorescent dye-protein complex is directly proportional, and the content of protein in sample is detected accordingly.
It is preferred that, the substrate is PET, PC, PEN, PP, PS or PMMA.
It is preferred that, the graphene film is single-layer graphene film.
It is preferred that, the material of the electrode is Au, Ag, Cu or Ti.
It is preferred that, the fluorescent dye is fluorescein isothiocynate (FITC), Fluoresceincarboxylic acid (FAM) or phycoerythrin
(PE)。
It is preferred that, the ultraviolet light time is 1min-10min.
The present invention uses above technical scheme, and fluorescent dye-protein complex is obtained with fluorochrome label protein,
Under ultraviolet light, fluorescent dye can produce π-π and stack in graphenic surface causes electro transfer to be adsorbed onto graphenic surface,
Fluorescent quenching is produced, while also causing electro transfer to be adsorbed onto on graphenic surface because π-π are stacked, makes the load of graphene film
Sub- concentration increase is flowed, resistivity reduces, and graphene film resistance reduction, graphene film resistance change contaminates with fluorescence in sample
Material-protein complex content is directly proportional, and the content of protein in sample is detected accordingly.
Brief description of the drawings
Fig. 1 detects the step flow chart of method of protein for the present invention based on graphene sensor.
Fig. 2 is the structural representation of graphene sensor of the present invention.
Fig. 3 obtains the structural representation of FITC-IgG processes to mark IgG using FITC in the embodiment of the present invention.
Fig. 4 is FITC-IgG occurs in the sensor surface based on graphene under ultraviolet light in the embodiment of the present invention
π-π stack the schematic diagram of reaction.
For FITC-IgG in the embodiment of the present invention Electronic Speculum of fluorescent quenching occurs in the sensor surface based on graphene for Fig. 5
Scanning figure.
Fig. 6 is sensitivity and the sensor resistance of the content based on graphene sensor measurement IgG in the embodiment of the present invention
The schematic diagram that variable quantity changes with FITC-IgG changes of contents.
Embodiment
In order that objects, features and advantages of the present invention are more clear, below in conjunction with drawings and Examples, to the present invention
Embodiment make more detailed description, in the following description, elaborate many concrete details to fill
The understanding present invention divided, but the present invention can be implemented with many other modes for being different from describing.Therefore, the present invention not by
The limitation of the specific implementation of following discloses.
One kind detects method of protein based on graphene sensor, as shown in figure 1, comprising the following steps：
(1) graphene sensor is made：As shown in Fig. 2 being provided with graphene film 2, the graphene on a substrate 1
The two ends of film 2 are provided with electrode 3；
(2) pretreatment of detected sample：Fluorescent dye is added in sample to be detected, for marking the egg in sample
White matter, obtains fluorescent dye-protein complex；
(3) in sample protein content detection：Detected sample by pretreatment is added drop-wise to graphene film table
Face, descends fluorescence to be quenched for a period of time in ultraviolet light, and graphene film resistance changes, resistance change and sample
The content of middle fluorescent dye-protein complex is directly proportional, and the content of protein in sample is detected accordingly.
Wherein, the substrate is PET, PC, PEN, PP, PS or PMMA.
Wherein, the graphene film is single-layer graphene film.
Wherein, the material of the electrode is Au, Ag, Cu or Ti.
Wherein, the fluorescent dye is fluorescein isothiocynate (FITC), Fluoresceincarboxylic acid (FAM) or phycoerythrin
(PE)。
Wherein, the ultraviolet light time is 1min-10min.
The present invention detects that the principle of method of protein is based on graphene sensor, due to the carbon hexagon of graphene
Structure and polycyclic aromatic hydrocarbon molecule are closely similar, are stacked using π-π and combine graphene and multiring aromatic hydrocarbon substance, and fluorescent dye
Mostly phenyl ring or heterocycle and with conjugated double bond compound, it can produce π-π heaps under ultraviolet light in graphenic surface
It is folded to cause electro transfer to be adsorbed onto graphenic surface, fluorescent quenching is produced, while also causing electro transfer to adsorb because π-π are stacked
Onto graphenic surface, increase the carrier concentration of graphene film, resistivity reduces, the reduction of graphene film resistance, stone
Black alkene film resistor variable quantity is directly proportional to fluorescent dye in sample-protein complex content, and egg in sample is detected accordingly
The content of white matter.
Illustrated below with the specific embodiment that is detected as of IgG content：FITC is added in the sample, as shown in figure 3,
IgG and FITC generation bonding reaction, obtains FITC-IgG fluorescent material solution；FITC-IgG fluorescent material solution is added drop-wise to base
In the graphene film surface of graphene sensor, reacted under the irradiation of ultraviolet light 0, as shown in figure 4, the carbon six of graphene
Side shape structure and polycyclic aromatic hydrocarbon molecule are closely similar, and FITC is phenyl ring and the compound with conjugated double bond, so FITC-
IgG can produce π-π and stack in graphenic surface causes electro transfer to be adsorbed onto graphenic surface, occurs fluorescent quenching, such as Fig. 5 institutes
Show.
Meanwhile, cause electro transfer to be adsorbed onto graphene also due to FITC-IgG can produce π-π and stack in graphenic surface
Surface, can increase the carrier concentration of graphene film, and resistivity reduces, the reduction of graphene film resistance, graphene film
Resistance change is directly proportional to fluorescent dye in sample-protein complex content, and containing for protein in sample is detected accordingly
Amount..
Based on method of the present invention, the FITC-IgG solution of various concentrations is measured, as a result as shown in fig. 6,
FITC-IgG solution concentrations are directly proportional to the resistance change based on graphene sensor, can be by based on graphene sensor
The change of resistance change obtain FITC-IgG solution concentrations, the content of IgG in sample is detected accordingly.Further, it is also possible to
Find out, the sensor based on graphene is about responded in 1min to FITC-IgG, resistance value rapid decrease, about 5min, pass
The resistance value of sensor drops to stationary value, thus the present invention based on graphene sensor detection method of protein have it is highly sensitive
Degree, precision, and method is simple, the fast advantage of measuring speed.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.
Claims (6)
1. one kind detects method of protein based on graphene sensor, it is characterised in that comprise the following steps：
(1) graphene sensor is made：Face is provided with graphene film on a substrate, and the graphene film two ends are provided with electricity
Pole；
(2) pretreatment of detected sample：Fluorescent dye is added in sample to be detected, for marking the albumen in sample
Matter, obtains fluorescent dye-protein complex；
(3) in sample protein content detection：Detected sample by pretreatment is added drop-wise to graphene film surface,
Ultraviolet light descends fluorescence to be quenched for a period of time, and graphene film resistance changes, resistance change with it is glimmering in sample
Photoinitiator dye-protein complex content is directly proportional, and the content of protein in sample is detected accordingly.
2. it is according to claim 1 a kind of based on graphene sensor detection method of protein, it is characterised in that described
Substrate is PET, PC, PEN, PP, PS or PMMA.
3. it is according to claim 1 a kind of based on graphene sensor detection method of protein, it is characterised in that described
Graphene film is single-layer graphene film.
4. it is according to claim 1 a kind of based on graphene sensor detection method of protein, it is characterised in that described
The material of electrode is Au, Ag, Cu or Ti.
5. it is according to claim 1 a kind of based on graphene sensor detection method of protein, it is characterised in that described
Fluorescent dye is fluorescein isothiocynate (FITC), Fluoresceincarboxylic acid (FAM) or phycoerythrin (PE).
6. it is according to claim 1 a kind of based on graphene sensor detection method of protein, it is characterised in that described
The ultraviolet light time is 1min-10min.
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| CN109212206B (en) * | 2017-07-05 | 2019-11-15 | 京东方科技集团股份有限公司 | Influenza virus detection chip and the method for detecting influenza virus using it |
| CN107402199B (en) * | 2017-07-31 | 2019-09-10 | 京东方科技集团股份有限公司 | Gene sequencing chip and its sequencing approach and gene sequencing device |
| CN108645824B (en) * | 2018-04-12 | 2020-05-22 | 中国科学院化学研究所 | Sensor array chip and preparation method and application thereof |
| CN110702649B (en) * | 2019-09-14 | 2022-09-16 | 济宁医学院 | Detection method for interaction between graphene-nano silver compound and double-stranded DNA |
| CN111678889B (en) * | 2020-07-13 | 2023-05-02 | 北京碳世纪科技有限公司 | Quantum dot and fluorescent molecule doped graphene biosensor for detecting trace viruses |
| CN112378974A (en) * | 2020-10-30 | 2021-02-19 | 哈尔滨烯芯科技有限公司 | Graphene film sensor based on protein specificity and preparation method thereof |
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