CN109459477A - A kind of assemble method of the biosensor of dual signal amplification and its application - Google Patents
A kind of assemble method of the biosensor of dual signal amplification and its application Download PDFInfo
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- CN109459477A CN109459477A CN201811130463.4A CN201811130463A CN109459477A CN 109459477 A CN109459477 A CN 109459477A CN 201811130463 A CN201811130463 A CN 201811130463A CN 109459477 A CN109459477 A CN 109459477A
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- biosensor
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- signal amplification
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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
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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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
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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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
- G01N27/416—Systems
Abstract
The invention discloses a kind of assemble method of the biosensor of dual signal amplification and its applications, belong to a nanometer field of new materials, by preparing gold nano-probe and assembling interlayer type biosensor two-step method, in gained sensor application and HER2 detection.Method for organizing of the present invention is simple to operation, and resulting biosensor can enhance photosignal；The AuNPs of surface plasma body resonant vibration, generates collective's concussion of free electron, and thermoelectron is directly transferred to WS from AuNPs2Conduction band realize dual signal amplification detection HER2 to further enhance photosignal.
Description
Technical field
The invention belongs to nanometer field of new materials, and in particular to a kind of assemble method of the biosensor of dual signal amplification
And its application.
Background technique
WS2With big surface area, fabulous flowability of carrier, high active site density, wider light absorption and
The advantages that being easy to functionalization is widely used in the fields such as hydrogen manufacturing, optical sensing, bio-imaging and treatment of cancer.However, being permitted
More results of study show, WS2With low-down photoelectric conversion efficiency, therefore limit its optical electro-chemistry application.Except this it
Outside, the building of pattern and surface texture is also a greatly challenge.
Therefore, consumption electron donor agent (H is introduced in system2O2) and local type surface plasma body resonant vibration noble metal
After nanoparticle (Au NPs), photoelectric characteristic, double expansion photosignal can be improved.Glucose in electrolyte can be golden
The glucose oxidase catalysis adsorbed on probe generates gluconic acid and H2O2, the latter can consume WS2Hole in valence band, because
This can enhance photosignal.Under the radiation of visible light, Au NPs becomes the Au NPs of local type surface plasma body resonant vibration,
Collective's concussion of free electron is generated, thermoelectron is directly transferred to WS from Au NPs2Conduction band, to further increase photoelectricity
Efficiency of transmission.And WS2The microstructure of nano-wire array three-dimensional makes catalysis material have bigger surface area, exposure more
Active site, reduce material impedance, increase the stability of material, promote the diffusion of electrolyte.This experiment, building
Dual signal amplification WS2The biosensor of NW/TM-aptamer-HER2- [peptide-Au NPs-GOx] interlayer type, with
This is as a kind of new means sensor for detecting HER2.
Summary of the invention
The object of the present invention is to provide a kind of assemble method of the biosensor of dual signal amplification and its applications, to solve
The above problem.
To achieve the goals above, the technical solution adopted by the present invention are as follows:
A kind of assemble method of the biosensor of dual signal amplification of the present invention, comprising the following steps:
1) it prepares gold nano-probe: 50 μ L polypeptide solutions being added in 2mLAuNPs solution and 50 μ L glucose oxidizing ferment are molten
Liquid is placed on oscillator and is protected from light oscillation 12 hours, 12000 turns of probe will obtained, after 4 DEG C of centrifugation 10min, is dispersed in 2mL and goes
In ionized water, and it is placed in 4 DEG C of refrigerator and saves；
2) interlayer type biosensor is assembled: by 0.5cm × 0.5cmWS2It is molten that nano-wire array material immerses 5 μ L aptamers
In liquid, to hatch 4 hours, 1% bovine serum albumin of electrode is closed into 30min, obtained electrode is rinsed with deionized water, then,
Electrode is added in 0.5,1,2,5,8,10ng/mL HER2 to be rinsed with deionized water, most after hatching 2 hours in 4 DEG C of refrigerators
Afterwards, the gold nano-probe 40 μ L being labeled is added above-mentioned electrode and hatches 2 hours, and assembled electrode is put into comprising glucose
PBS buffer solution in hatch 25min after, take out electrolyte and electrode, obtain WS2NW/TM-aptamer-HER2-
The biosensor of [peptide-AuNPs-GOx] interlayer type dual signal amplification.
The concentration of polypeptide and glucose oxidase is 0.4mg/mL in step 1).
The concentration of glucose is 10mM in step 2).
The concentration of PBS buffer solution is 0.1mol/L, pH=7.0 in step 2).
WS of the present invention2The preparation method of nano-wire array material, comprising the following steps:
1) 1.6493gNa is added into reaction vessel2WO4·2H240mL deionized water is added in O, stirs 30min；
2) 3MHCl is added dropwise, the pH of solution is adjusted to 1.2, solution becomes faint yellow from colourless at this time；
3) by 1.765gH2C2O4It is added in above-mentioned mixed solution, stirring is then diluted to 100mL and obtains H to dissolving2WO4
Forerunner；
4) 40mLH is taken2WO4Precursor solution sequentially adds 2g (NH4)2SO4With 0.411g thiocarbamide, stirring is transferred to dissolving
In autoclave；
5) titanium net of 2cm × 4cm is successively put into reaction kettle with concentrated hydrochloric acid, ethyl alcohol, deionized water processing afterwards for several times,
It 180 DEG C, reacts 16 hours.After reaction, titanium net taking-up is washed with deionized for several times, 60 DEG C dry 12 hours；
6) obtained sample is transferred in tube furnace, is roasted 1 hour for 450 DEG C in air atmosphere.
WS of the present invention2The amplification of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type dual signal
Application of the biosensor in HER2 detection.
The invention has the benefit that method for organizing of the present invention is simple to operation, resulting biosensor can enhance light
Electric signal；The AuNPs of surface plasma body resonant vibration, generates collective's concussion of free electron, and thermoelectron is directly transferred to from AuNPs
WS2Conduction band realize dual signal amplification detection HER2 to further enhance photosignal.
Detailed description of the invention
Fig. 1 is WS2The biosensor of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type is tested
Schematic diagram；
Fig. 2 is WS2The electricity of the biosensor of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type
Son transmitting schematic diagram；
Fig. 3 is WS prepared by embodiment 12The scanning electron microscope (SEM) photograph of nano-wire array；
Fig. 4 is the transmission electron microscope picture of nanogold prepared by embodiment 2；
Fig. 5 A is (a) titanium net of preparation, (b) WS2NW/TM,(c)WS2NW/TM-AuNPs,(d)WS2NW/TM-AuNPs packet
Containing 5mMH2O2Electrolyte photocurrent response；Fig. 5 B is photo-current intensity with HER2 concentration curve, interior illustration be with
Corresponding calibration curve；
Fig. 6 is that the sensor of embodiment preparation is used to detect the comparative diagram of HER2 selectivity；
The HER2 detection of Fig. 7 immunosensor and ELISA in human serum is compared figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment 1:
WS2The preparation method of nano-wire array material, comprising the following steps: be added into 200mL beaker
1.6493gNa2WO4·2H240mL deionized water is added in O, stirs 30min.3MHCl is added dropwise, the pH of solution is adjusted to 1.2,
Solution becomes faint yellow from colourless at this time.By 1.765gH2C2O4It is added in above-mentioned mixed solution, then stirring is diluted to dissolving
H is obtained to 100mL2WO4Forerunner.Take 40mLH2WO4Precursor solution sequentially adds 2g (NH4)2SO4With 0.411g thiocarbamide, stirring is extremely
Dissolution, is transferred in autoclave.After the titanium net of 2cm × 4cm is successively used concentrated hydrochloric acid, ethyl alcohol, deionized water processing for several times
It is put into reaction kettle, 180 DEG C, reacts 16 hours.After reaction, titanium net taking-up is washed with deionized for several times, 60 DEG C of bakings
It is 12 hours dry.Obtained sample is transferred in tube furnace, is roasted 1 hour for 450 DEG C in air atmosphere.
Fig. 3 is WS2The scanning electron microscope (SEM) photograph of nano-wire array, as can be observed from Figure WS2Nano wire covers entire titanium
Net.
Embodiment 2:
A kind of assemble method of the biosensor of dual signal amplification of the present invention, comprising the following steps: molten in 2mLAuNPs
50 μ L, 0.4mg/mL polypeptide solutions and 50 μ L, 0.4mg/mL glucose oxidase solutions are added in liquid, is placed on oscillator and is protected from light
Oscillation 12 hours.12000 turns of probe will obtained after 4 DEG C of centrifugation 10min, are dispersed in 2mL deionized water, and be placed on 4 DEG C
It is saved in refrigerator.By 0.5cm × 0.5cmWS2Nano-wire array immerses in 5 μ L adaptation liquid solution, hatches 4 hours.Electrode is used
1% bovine serum albumin closes 30min, and obtained electrode is rinsed with deionized water.Then, by 0.5,1,2,5,8,10 ng/mL's
Electrode is added in HER2, is rinsed after hatching 2 hours in 4 DEG C of refrigerators with deionized water.Finally, the gold nano that 40 μ L are labeled is visited
Needle is added above-mentioned electrode and hatches 2 hours.Assembled electrode is put into after hatching 25min in 10mM glucose solution, takes out electricity
It solves liquid and electrode carries out photoelectricity test.
Fig. 4 is the transmission electron microscope picture of nanogold particle, as shown, the diameter of nanogold is about in 14nm.
Fig. 1 is WS2The biosensor of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type is tested
Schematic diagram.As shown, nanogold particle is by the polypeptide marker of glucose oxidase and specificity.Glucose oxidase can urge
Change glucose and generates H2O2, it can consume WS2The hole enhancing WS generated in valence band2Photosignal.In addition, introducing local
The nanogold particle of type surface plasma body resonant vibration can enhance photoelectric conversion efficiency.WS2Specificity is wound on nano wire
Aptamers, be capable of specificity combination HER2 antigen, and the polypeptide on probe can specificity identification HER2 antigen.With electricity
The concentration that pole loads HER2 increases, and the amount of capture probe is consequently increased, photosignal enhancing.Therefore can be used to detect
HER2。
Fig. 2 is WS2The electricity of the biosensor of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type
Son transmitting schematic diagram.Under the radiation of visible light (420nm), WS2The energy of the photon of absorption is higher than the energy of its valence band, because
This can be that electronics transits to from valence band and generates electron-hole pair on conduction band, generate light on the electron transmission to titanium net on conduction band
Electric signal, the raw hole of valence band can be catalyzed the H that glucose generates by glucose oxidase2O2Consumption, so that photosignal increases
By force.In addition, the nanogold particle of local type surface plasma body resonant vibration can generate the collective oscillation of free electron, so that generating
Thermoelectron be transferred to WS from nanogold particle2Conduction band on, therefore can further enhance photosignal.
Fig. 5 A is (a) titanium net of preparation, (b) WS2NW/TM,(c)WS2NW/TM-AuNPs,(d)WS2NW/TM-AuNPs packet
Containing 5mMH2O2Electrolyte photocurrent response.As shown, titanium net does not have photoelectric respone, WS2Nano-wire array can generate
Weak photoelectric current.And after hatching nanogold particle on nano-wire array, photoelectric respone is remarkably reinforced, and is added into electrolyte
5mMH2O2Afterwards, photoelectric current is enhanced close to three times.Thus the feasibility of experimental principle is demonstrated.Fig. 5 B be photo-current intensity with
HER2 concentration curve, interior illustration are corresponding calibration curve.As schemed, as HER2 concentration increases from 0ng/mL
Photoelectric current to 10ng/mL, generation also linearly increases.Fig. 5 B is that interior illustration is corresponding calibration curve, linear to return
Returning equation is y=0.33598x+1.0328 (R2=0.99867), and wherein y (μ A) is photo-signal, and x (ng/mL) is HER2
Concentration.In S/N=3, Monitoring lower-cut is 0.36ng/mL.
Fig. 6 is that the sensor of embodiment preparation is used to detect the comparative diagram of HER2 selectivity.To the albumen coexisted with HER2
Matter carries out the analysis of selectivity, to analyze the WS of building2NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] is sandwich
The selectivity of the biosensor of type.When protein β-site amyloid precursor protein lyases 1 (BACE1), cancer coexists
Embryonal antigen (CEA), human immunoglobulin G (human IgG), human immunoglobulin M (mankind IgM), P53, protein kinase A
(PKA) when concentration is 1ng/mL, weaker photosignal is shown, thus proves that the sensor only has response to HER2,
With preferable selectivity.
In order to evaluate the immunosensor in actual sample to the potential clinical applicability of HER2 detection, 4 kinds are analyzed
Dilute serum sample from patient with breast cancer, and the result of itself and single goal business elisa assay is compared.Such as figure
Shown in 7, the HER2 testing result between both methods is not significantly different, this shows the sandwich immune sensing utensil of design
The reliable strategy for thering are very big potentiality to detect as HER2 in serum sample.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of assemble method of the biosensor of dual signal amplification, which comprises the following steps:
1) it prepares gold nano-probe: 50 μ L polypeptide solutions and 50 μ L glucoses oxidation enzyme solutions being added in 2mLAuNPs solution,
It is placed on oscillator and is protected from light oscillation 12 hours, 12000 turns of probe will obtained after 4 DEG C of centrifugation 10min, are dispersed in 2mL deionization
In water, and it is placed in 4 DEG C of refrigerator and saves；
2) interlayer type biosensor is assembled: by 0.5cm × 0.5cmWS2Nano-wire array material immerses in 5 μ L adaptation liquid solution,
Hatching 4 hours, by electrode 1% bovine serum albumin closing 30min, obtained electrode rinses with deionized water, then, by 0.5,
1,2,5,8,10ng/mL HER2 is added electrode and is rinsed with deionized water, after hatching 2 hours in 4 DEG C of refrigerators finally, by 40
μ L labeled gold nano-probe is added above-mentioned electrode and hatches 2 hours, and assembled electrode is put into the PBS comprising glucose
After hatching 25min in buffer solution, electrolyte and electrode are taken out, WS is obtained2NW/TM-aptamer-HER2-[peptide-
AuNPs-GOx] interlayer type dual signal amplification biosensor.
2. a kind of assemble method of the biosensor of dual signal amplification according to claim 1, which is characterized in that step
1) concentration of polypeptide and glucose oxidase is 0.4ng/mL in.
3. a kind of assemble method of the biosensor of dual signal amplification according to claim 1, which is characterized in that step
2) concentration of glucose is 10mM in.
4. a kind of assemble method of the biosensor of dual signal amplification according to claim 1, which is characterized in that step
2) concentration of PBS buffer solution is 0.1mol/L, pH=7.0 in.
5. a kind of WS as described in claim 12The preparation method of nano-wire array material, which is characterized in that including following step
It is rapid:
1) 1.6493gNa is added into reaction vessel2WO4·2H240mL deionized water is added in O, stirs 30min；
2) 3MHCl is added dropwise, the pH of solution is adjusted to 1.2, solution becomes faint yellow from colourless at this time；
3) by 1.765gH2C2O4It is added in above-mentioned mixed solution, stirring is then diluted to 100mL and obtains H to dissolving2WO4Forerunner；
4) 40mLH is taken2WO4Precursor solution sequentially adds 2g (NH4)2SO4With 0.411g thiocarbamide, stirring is transferred to high pressure to dissolving
In reaction kettle；
5) titanium net of 2cm × 4cm is successively put into reaction kettle with concentrated hydrochloric acid, ethyl alcohol, deionized water processing afterwards for several times, 180 DEG C,
Reaction 16 hours.After reaction, titanium net taking-up is washed with deionized for several times, 60 DEG C dry 12 hours；
6) obtained sample is transferred in tube furnace, is roasted 1 hour for 450 DEG C in air atmosphere.
6. a kind of application of the biosensor of the amplification of the dual signal as described in claim 1-4 is any, which is characterized in that gained
WS2The biosensor of NW/TM-aptamer-HER2- [peptide-AuNPs-GOx] interlayer type dual signal amplification is examined in HER2
Application in survey.
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