CN103940989A - Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor - Google Patents
Immunosensor based on hybrid chain reaction and single molecule counting and application of immunosensor Download PDFInfo
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- CN103940989A CN103940989A CN201410171532.1A CN201410171532A CN103940989A CN 103940989 A CN103940989 A CN 103940989A CN 201410171532 A CN201410171532 A CN 201410171532A CN 103940989 A CN103940989 A CN 103940989A
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/525—Tumor necrosis factor [TNF]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
- G01N2333/7151—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF]; for lymphotoxin [LT]
Abstract
The invention discloses an immunosensor based on hybrid chain reaction and single molecule counting. The immunosensor consists of a capturing antibody, a detection antibody, streptavidin, a biotinylated primer and a hairpin probe, wherein the capturing antibody is anti-TNF-alpha; the detection antibody is Bio-anti-TNF-alpha; a base sequence of the biotinylated primer (Bio-I) is 5'-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3'; the hairpin probe consists of a hairpin probe H1 and a hairpin probe H2; a base sequence of the hairpin probe H1 is 5'-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3'; a base sequence of the hairpin probe H2 is 5'-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3'. The invention also provides a preparation method of the immunosensor and application of the immunosensor in detection of the TNF-alpha. The immunosensor disclosed by the invention has the advantages of high detection sensitivity, small sample use amount, no need of enzyme amplification and the like, and quantitative detection on low-concentration TNF-alpha can be realized.
Description
Technical field
The present invention relates to a kind of immunosensor and application thereof based on cross chain reaction and monomolecular counting.
Background technology
Single Molecule Detection (SMD) is to take a kind of method that the analysis of individual molecule is fundamental construction, and up to the present, it receives much concern in the research of life science.The method is widely used in the research of cell imaging, protein interaction and the quantitative detection of DNA and protein.In the above-mentioned application of mentioning, quantitatively detecting is so that the counting one by one of object molecule is realized, and this quantitative detection means has represented the final limit detecting.Traditional average measurement method is to come quantitative with the relation between signal intensity and object concentration.Signal intensity is higher, and the concentration of determined object is higher.With average measurement method difference be, in SMD quantivative approach, signal intensity is no longer so important, and each molecule that can produce signal can be counted, and has more visuality and numerical, has guaranteed higher reappearance.Due to SMD quantitative detecting method have advantages of average measurement incomparable, at present, had work that SMD is applied among quantitative test.
In order to realize SMD, quantitatively detect, the signal intensity of single molecule is must be enough strong to such an extent as to can be detected.One of at present common SMD quantitative detecting method is exactly to use fluorescent marker, as organic fluorescent dye and quantum dot (QDs).TanDeng seminar successfully utilizes dye marker molecular beacon to monitor the reaction kinetics between molecule.But conventional fluorescent dye is mainly deposited problem both ways at present, one is, relatively low fluorescence intensity, and another is exactly photo-labile.In order to address the above problem, QDs has been widely used among Single Molecule Detection as a kind of brand-new nanometer label.Jin seminar utilizes the single QDs of functionalization to realize the quantitative detection to protein.Experimental result shows, QDs compares with traditional fluorescent dye, shows outstanding light stability and sensitivity.Although QDs has the characteristic of high quantum yield and outstanding light stability, yet the heavy metal toxicity of QDs self has limited its application in protein detection.In order to overcome the problems referred to above, another of appearance guarantees that the method for single molecular signal intensity is exactly, and utilizes the oligonucleotide sequence thing that serves as a mark.It is that basic DNA detects strategy that Echopf seminar has designed a kind of microballoon, in the method, by RCA, introduces a large amount of fluorescence probes, realized to single target thing molecule quantitatively.Yet the amplifying technique that RCA is fundamental construction needs the auxiliary of proteinase.Proteinase not only requires harsh to reaction conditions, and has increased complicacy and cost, and these have all limited with proteinase and have assisted as the application of basic method in reality detects.Therefore, in the urgent need to develop a kind of brand-new, without the method for enzyme, build the quantitative detection platform of SMD.
Tumor necrosis factor-alpha (TNF-α) is a kind of important peptide cell factor, meanwhile, is also the biomarker of cancer.TNF-α, as a kind of endogenic medium or accessory factor, can induce the hemorrhagic necrosis of some tumour.And many physiology and pathological process are all followed the variation of TNF-α concentration, such as HIV infection, systemic loupus erythematosus, endotoxin shock and rheumatic arthritis etc., therefore, the Sensitive Detection of TNF-α has important practical significance.The detection of TNF-α at present adopts enzyme linked immunosorbent assay (ELISA) more, but the method exists the problems such as repeatability is bad, and sensitivity is not high, and detectability is higher, and is prone to false positive, the authenticity that impact detects.
Summary of the invention
For the deficiencies in the prior art, the object of this invention is to provide a kind of immunosensor based on cross chain reaction and monomolecular counting, the detection by this immunosensor for TNF-α, has highly sensitive, high repeatability and other advantages, and there is high selectivity and low matrix effect.
Another object of the present invention is to provide the application of this immunosensor in TNF-α detects.
For achieving the above object, the present invention adopts following technical scheme:
An immunosensor based on cross chain reaction and monomolecular counting, is comprised of capture antibody, detection antibody, Streptavidin, biotinylated primer and hair fastener probe;
Described capture antibody is tumor necrosis factor-alpha capture antibody (anti-TNF-α);
Described detection antibody is the anti-human tumor necrosis factor-alpha of biotinylated monoclonal rabbit (Bio-anti-TNF-α);
The base sequence of described biotinylated primer (Bio-I) is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3 ', as shown in SEQ ID NO.1;
Described hair fastener probe is comprised of hair fastener probe H1 and hair fastener probe H2, and wherein the base sequence of hair fastener probe H1 is 5 '-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3 ', as shown in SEQ ID NO.2;
The base sequence of hair fastener probe H2 is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3 ', as shown in SEQ ID NO.3.
A preparation method for immunosensor based on cross chain reaction and monomolecular counting, comprises the following steps:
(1) structure of sandwich immunoassay course of reaction: tumor necrosis factor-alpha capture antibody (anti-TNF-α) is dripped in the substrate of glass of silanization processing, hatch 8-14h under the moisturizing condition of 37 ℃; Add sealer, under moisturizing condition, react 3-8h, the site that sealing exposes, to reduce non-specific adsorption; Add again the detection antibody (Bio-anti-TNF-α) of the different loci that can identify antigen, at 37 ℃, hatch 1-3h; After every single step reaction, all use PBS-T and pure water rinsing three times,, remove unconjugated reagent;
(2) structure of the immunosensor based on cross chain reaction: after immuning hybridization reaction, add Streptavidin (SA) in substrate of glass, hatch 1-3h; After PBS-T and pure water rinsing three times, add biotinylated primer (Bio-I), hatch 1-3h; After substrate of glass is cleaned, add the potpourri of hair fastener probe H1, H2, hatch 2-6h; Substrate of glass is used PBS-T and pure water rinsing three times again, removes unreacted hair fastener probe, obtains;
In step (1), the concentration of anti-TNF-α is 10-30pM, and the concentration of sealer is 20-60mM, and the concentration of Bio-anti-TNF-α is 20-40pM;
Described sealer is monoethanolamine or bovine serum albumin, preferably monoethanolamine;
The silanization of substrate of glass is processed and is comprised the following steps:
A: clean, soak 8-16h in the chromic acid solution that is 5%～8% in mass concentration by substrate of glass, rinse with a large amount of clear water, then use pure water rinsing; Afterwards, by substrate of glass successively ultrasonic cleaning in acetone, ethanol and pure water, each 10min, every kind of solution cleans twice, the solution more renewing after each cleaning; Afterwards, the substrate of glass cleaning up is added to ultrasonic 30min in the mixed solution that hydrochloric acid, pure water, hydrogen peroxide volume ratio are 1:1:1; Use again pure water ultrasonic cleaning twice, each 10min; Finally, the cover glass having activated is put into the thermostatic drying chamber of 120 ℃, to remove residual moisture above;
B: silanization, it is that 6% GOPS-anhydrous toluene solution carries out silanization processing that the substrate of glass that step a is handled well is put into volume fraction, this reaction is reacted 6h at 95 ℃; After reaction finishes, substrate of glass is taken out, successively ultrasonic cleaning 15min in toluene, ethanol and pure water; After nitrogen stream dries up, the cover glass of the silanization preparing is stored in exsiccator standby;
In step (2), the concentration of Streptavidin is 10-80pM, and the concentration of Bio-I is 0.1-0.5nM, and in the potpourri of hair fastener probe H1, H2, H1 is identical with H2 concentration, is 5-15nM;
In step (2), the base sequence of described Bio-I is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3 ';
The base sequence of hair fastener probe H1 is 5 '-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3 ';
The base sequence of hair fastener probe H2 is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3 ';
Preferably, hair fastener probe H1 and H2 be annealed step before using, and object is the stability that guarantees hair fastener, and annealing process is that then Slow cooling is to room temperature at 90 ℃ of heating 5min.
In step (1), (2), the volume ratio of the potpourri addition of anti-TNF-α, sealer, Bio-anti-TNF-α, Streptavidin, Bio-I, hair fastener probe H1, H2 is (1～2): (1～2): (1～2): (1～2): (1～2).
The application of this immunosensor in TNF-α detects.Its application process is:
(1) capture antibody anti-TNF-α is dripped in the substrate of glass of silanization processing, under the moisturizing condition of 37 ℃, hatch 8-14h; Add sealer, under moisturizing condition, react 3-8h, the site that sealing exposes, to reduce non-specific adsorption; , add TNF-α to be detected, hatch 1-3h, formed Antibody-antigen complex; Add again the detection antibody (Bio-anti-TNF-α) of the different loci that can identify antigen, at 37 ℃, hatch 1-3h; And form the immune complex of " sandwich " type;
(2) after immuning hybridization reaction, in substrate of glass, add Streptavidin (SA), hatch 1-3h; After PBS-T and pure water rinsing three times, add Bio-I, hatch 1-3h; After substrate of glass is cleaned, add the potpourri of hair fastener probe H1, H2, hatch 2-6h; Substrate of glass is used PBS-T and pure water rinsing three times again, removes unreacted hair fastener probe;
(3) fluorescence imaging detects: add SYBR Green I, under room temperature, hatch 10min; Suck excessive unreacted SYBRGreen I, with after PBS-T and pure water rinsing three times, add the PBS buffer solution of same volume, carry out fluorescence imaging experiment, the fluorescence picture obtaining is processed, go forward side by side line number according to one's analysis;
In step (1), (2), (3), anti-TNF-α, sealer, TNF-α, Bio-anti-TNF-α to be detected, Streptavidin, Bio-I, the potpourri of hair fastener probe H1, H2 are, the volume ratio of SYBR Green I addition is (1～2): (1～2): (1～2): (1～2): (1～2): (1～2): (1～2);
In step (3), the concentration of SYBR Green I is 5-15 μ M;
In step (3), the experiment that fluorescence imaging detects is set to: select 60 * object lens, it is 100ms that the time shutter is set, and yield value is 2000, and the cover glass of gained after experiment is fixed on objective table; Open mercury lamp, adjust excitation wavelength optical filter, select the excitation channel between 470～490nm, luminescent dye molecule SYBR Green I is excited and send fluorescence; Adjusting focal length, obtains fluorescence picture, with MetaMorph software, the fluorescence picture obtaining is processed, and goes forward side by side line number according to one's analysis.
Cross chain reaction (HCR) is to utilize hair fastener probe one group of complementation, that dynamics is limited to realize the chain reaction of hybridization, and whole reaction is that by base-pair, to be formed be that the free energy producing drives.HCR reaction is to carry out under at isothermal, without the condition of enzyme.The amplification method that the enzyme of take with other is fundamental construction is compared, and HCR is more stable, more inexpensive.Therefore, we take HCR signal amplification technique and SYBR Green I interpolation dyestuff is basis, has built a kind of brand-new, sensitive SMD quantitative detecting method.
The formation meeting catalysis hair fastener probe H1 of immune complex and the self assembly of H2, form a long double helix polymkeric substance, SYBRGreen I is the fluorescence signal labeled molecule using in the design, the characteristic of this molecule is, in solution or while running into single stranded DNA, fluoresce hardly, and after in the butt crack that is inserted into double-stranded DNA, can produce the fluorescence signal of obvious enhancing.Be inverted fall to penetrating microscopical auxiliary under, each bright spot is corresponding to a single object molecule, bright spot can be counted exactly and realize to TNF-α quantitatively.
The present invention has following beneficial effect with respect to prior art:
(1) to have detection sensitivity high for immunosensor of the present invention, and sample use amount is little, without advantages such as enzyme amplifications, can realize the quantitative detection to low concentration TNF-α.
(2) by the material that adds sealer to reduce to there is cycloalkyl groups in the suprabasil combination of silanized glass, reduced the interference of the non-specific adsorption of biomolecule to background, reduced the generation of false positive signal, improved the sensitivity of experiment.
(3) detection method that immunosensor of the present invention quantitatively detects for TNF-α has good reappearance and precision, and TNF-α is had to good selectivity, can realize the detection to TNF-α in complex matrices.
Accompanying drawing explanation
Fig. 1 is HCR-monomolecular counting experimental principle figure;
Fig. 2 is the linear relationship (N=3) between bright spot number and TNF-α concentration;
Fig. 3 is that immunosensor is investigated result figure, wherein 1:BSA to the selectivity of TNF-α; 2: fibrin ferment; 3: human IgG; 4:PDGF; 5:TNF-α; 6: mixing sample (TNF-α, 1pM; BSA, fibrin ferment, human IgG and PDGF, 10pM);
Fig. 4 is matrix interference test findings, and wherein 1 is normal serum; 2 is Na
+, 146mM; 3 is Cl
-, 180 μ M; 4 is GLU, 6.11mM; 5 is UA, 420 μ M; 6 is DA, 0.8nM;
Fig. 5 a is the negative group of replication experiment result;
Fig. 5 b is the positive group of replication experiment result;
Fig. 5 c is replication experiment control group result.
Embodiment
The present invention is further illustrated in conjunction with the embodiments, should be noted that following explanation is only in order to explain the present invention, does not limit its content.
The instrument using in experiment and reagent are: (1) instrument: K30 type dry-type thermostat (difficult to understand Sheng, Hangzhou); DHG-907A type electric heating constant temperature blast drier (essence is grand, Shanghai); Thunder magnetic pHS-3C type acidometer (China); Branson-200 type supersonic cleaning machine (sino-america joint-venture Bi Nengxinchao company limited, Shanghai); H-101 type turbine mixer (going up Haikang standing grain photoelectric instrument company limited, Shanghai).Epifluorescence microscope system (Olympus Optical Co.Ltd, Tokyo, Japan), mainly comprise inverted fluorescence microscope (Olympus IX81), electronics enhancement mode photoelectrical coupler (EMCCD) (Cascade, 512B, America), inverted fluorescence microscope control module (Olympus IX2-UCB), Halogen lamp LED (Olympus LG-PSs2); Precision optics vibration-isolating platform (Yiao Information Optical Science & Technology Co., Ltd., Shanghai, Shanghai); Precision purification AC voltage-stabilized source (JONCHN Co., Ltd., Shanghai); (2) reagent: human tumor necrosis factor-alpha (TNF-α), polyclone mouse-anti human tumor necrosis factor-alpha antibody (anti-TNF-α), the anti-human tumor necrosis factor-alpha of biotinylated monoclonal rabbit (Bio-anti-TNF-α), immunoglobulin G while (IgG) (Abcam, UK); Uric acid (UA), bovine serum albumin(BSA) (BSA), glucose (GLU) (the Shanghai biological company limited of raw work, Shanghai); 3-epoxy radicals the third oxygen propyl trimethoxy silicane (C
9h
20o
5si, GOPS), Tween-20 (Tween-20), fibrin ferment and PDGF (PDGF) (Sigma-Aldrich Co.St.Louis, MO, USA); Dopamine (DA) (Aladdin reagent company limited, Shanghai); Streptavidin (SA) and SYBR Green I (Beijing Bai Taike Bioisystech Co., Ltd, Beijing); Ethanol (analyze pure, Tianjin Fu Yu Fine Chemical Co., Ltd, Tianjin); Acetone (analyze pure, Tianjin Fu Yu Fine Chemical Co., Ltd, Tianjin); Dry toluene is that analytically pure toluene obtains through secondary decompression distillation preparation; Na
2hPO
412H
2o (analyze pure, Tianjin extensively becomes chemical reagent factory, Tianjin); NaH
2pO
42H
2o (analyze pure, Tianjin extensively becomes chemical reagent factory, Tianjin); NaCl (analyze pure, Tianjin City Tanggu chemical reagent factory, Tianjin); Mixed cellulose ester microporous membrane (aperture 0.22 μ m, upper Haixing County sub-scavenging material factory, Shanghai); Cover glass (22 * 22mm, thickness 0.17mm, Cole-parmer, Vernon Hills, I11., USA).
The formula of the various buffer solution that use in experiment is as follows:
PBS buffer solution: 0.15mol/L NaCl, 7.6 * 10
-3mol/L Na
2hPO
4, 2.4 * 10
-3mol/L NaH
2pO
4, pH7.4.
PBS-T buffer solution: 0.15mol/L NaCl, 7.6 * 10
-3mol/L Na
2hPO
4, 2.4 * 10
-3mol/L NaH
2pO
4, 0.05%Tween-20, pH7.4.
TE buffer solution: 10mmol/L Tris-HCl, 1mmol/L Na
2eDTA, pH8.0.
HCR reaction buffer: 50mM Na
2hPO
4, 0.5M NaCl, pH6.8.
Embodiment 1
An immunosensor based on cross chain reaction and monomolecular counting, detects antibody (Bio-anti-TNF-α), Streptavidin, biotinylated primer and hair fastener probe by tumor necrosis factor-alpha capture antibody (anti-TNF-α), the anti-human tumor necrosis factor-alpha of monoclonal rabbit and forms;
The base sequence of described biotinylated primer (Bio-I) is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3 ';
Described hair fastener probe is comprised of hair fastener probe H1 and hair fastener probe H2, and wherein the base sequence of hair fastener probe H1 is 5 '-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3 ';
The base sequence of hair fastener probe H2 is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3 '.
The preparation method of this immunosensor, comprises the following steps:
(1) structure of sandwich immunoassay course of reaction: the tumor necrosis factor-alpha capture antibody of 50 μ L (anti-TNF-α) (20pM) is dripped in the substrate of glass of silanization processing, hatch 12h under the moisturizing condition of 37 ℃; The sealer (50mM, monoethanolamine) that adds 50 μ l reacts the site that 5h seals exposure, to reduce non-specific adsorption under moisturizing condition.Add again the detection antibody (Bio-anti-TNF-α) of 50 μ l (30pM) at 37 ℃, to hatch 2h; After every single step reaction, be all to remove unconjugated reagent three times by PBS-T and pure water rinsing;
(2) structure of the immunosensor based on cross chain reaction: after immuning hybridization reaction, to the Streptavidin (SA) that adds 50 μ l in substrate of glass, hatch 2h.After use PBS-T and pure water rinsing three times, add the biotinylated primer (Bio-I) of 50 μ l (0.1nM), hatch 2h.After substrate is cleaned, add the potpourri (H1,10nM, H2,10nM) of hair fastener probe, hatch 4h.H1 and H2 be annealed step before using, and object is the stability that guarantees hair fastener, and annealing process is that then Slow cooling is to room temperature at 90 ℃ of heating 5min.Finally, whole substrate is removed unreacted hair fastener probe for three times by PBS-T and pure water rinsing again, obtains.
In step (1), the silanization disposal route of substrate of glass comprises cleans and silanization two steps, in the chromic acid solution that is first 6% by substrate of glass in mass concentration, soaks 12h, rinses, then use pure water rinsing with a large amount of clear water; Afterwards, by substrate of glass successively ultrasonic cleaning in acetone, ethanol and pure water, each 10min, every kind of solution cleans twice, the solution more renewing after each cleaning; Afterwards, the substrate of glass cleaning up is added to ultrasonic 30min in the mixed solution that hydrochloric acid, pure water, hydrogen peroxide volume ratio are 1:1:1; Use again pure water ultrasonic cleaning twice, each 10min; Finally, the cover glass having activated is put into the thermostatic drying chamber of 120 ℃, to remove residual moisture above; Then the substrate of glass of handling well is put into volume fraction and be 6% GOPS-anhydrous toluene solution and carry out silanization processing, this reaction is reacted 6h at 95 ℃; After reaction finishes, substrate of glass is taken out, successively ultrasonic cleaning 15min in toluene, ethanol and pure water; After nitrogen stream dries up, the cover glass of the silanization preparing is stored in exsiccator standby.
In step (2), the base sequence of Bio-I is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3 ';
The base sequence of hair fastener probe H1 is 5 '-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3 ';
The base sequence of hair fastener probe H2 is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3 '.
This immunosensor at the application process in TNF-α detects is:
(1) the tumor necrosis factor-alpha capture antibody of 50 μ L (anti-TNF-α) (20pM) is dripped in the substrate of glass of fresh preparation, under the moisturizing condition of 37 ℃, hatch 12h.The sealer (50mM, monoethanolamine) that adds 50 μ L reacts the site that 5h seals exposure, to reduce non-specific adsorption under moisturizing condition.Add the TNF-α of 50 μ L to hatch 2h, formed Antibody-antigen complex, then add the detection antibody (Bio-anti-TNF-α) of 50 μ L (30pM) at 37 ℃, to hatch 2h.The different loci of this antibody recognition antigen, and form the immune complex of " sandwich " type; After every single step reaction, be all to remove unconjugated reagent three times by PBS-T and pure water rinsing;
(2), after immuning hybridization reaction, to the streptavidin (SA) that adds 50 μ L in substrate of glass, hatch 2h.After use PBS-T and pure water rinsing three times, add the biotinylated primer (Bio-I) of 50 μ L (0.1nM), hatch 2h.After substrate is cleaned, add the potpourri (H1,10nM, H2,10nM) of hair fastener probe, hatch 4h.H1 and H2 be annealed step before using, and object is the stability that guarantees hair fastener, and annealing process is that then Slow cooling is to room temperature at 90 ℃ of heating 5min.Finally, whole substrate is removed unreacted hair fastener probe for three times by PBS-T and pure water rinsing again;
(3) add the SYBR Green I (10 μ M) of 50 μ L, under room temperature, hatch 10min.Suck excessive unreacted SYBR Green I, clean after three times, add the PBS buffer solution of same volume, carry out fluorescence imaging experiment.Select 60 * object lens, it is 100ms that the time shutter is set, and yield value is 2000, and the cover glass of gained after experiment is fixed on objective table.Open mercury lamp, adjust excitation wavelength optical filter, select the excitation channel between 470～490nm, luminescent dye molecule SYBRGreen I is excited and send fluorescence.Adjusting focal length, obtains fluorescence picture, with MetaMorph software, the fluorescence picture obtaining is processed, and goes forward side by side line number according to one's analysis.
Embodiment 2
An immunosensor based on cross chain reaction and monomolecular counting, in its preparation method, the concentration of anti-TNF-α is 10pM; Sealer is bovine serum albumin, and concentration is 60mM; The concentration of Bio-anti-TNF-α is 20pM, and the concentration of Streptavidin is 10pM, and the concentration of Bio-I is 0.5nM, and in the potpourri of hair fastener probe H1, H2, H1 is identical with H2 concentration, is 5nM;
The volume ratio of the potpourri addition of anti-TNF-α, sealer, Bio-anti-TNF-α, Streptavidin, Bio-I, hair fastener probe H1, H2 is 1:2:1:2:1; All the other are with embodiment 1.
Embodiment 3
An immunosensor based on cross chain reaction and monomolecular counting, in its preparation method, the concentration of anti-TNF-α is 30pM; Sealer is bovine serum albumin, and concentration is 20mM; The concentration of Bio-anti-TNF-α is 40pM, and the concentration of Streptavidin is 80pM, and the concentration of Bio-I is 0.1nM, and in the potpourri of hair fastener probe H1, H2, H1 is identical with H2 concentration, is 15nM;
The volume ratio of the potpourri addition of anti-TNF-α, sealer, Bio-anti-TNF-α, Streptavidin, Bio-I, hair fastener probe H1, H2 is 2:1:2:1:2; All the other are with embodiment 1.
Embodiment 4
Linear and detectability is investigated:
The TNF-α solution of preparation variable concentrations, detection method, with embodiment 1, is investigated the relation between fluorescence bright spot number and TNF-α concentration.Result shows: fluorescence bright spot number and TNF-α present good linear relationship at 50fM between the concentration range of 1pM, and equation of linear regression is Y=1.237+9.505C, and related coefficient is 0.998, and linear relationship chart is shown in Fig. 2.
Embodiment 5
Precision, reappearance, selectivity and the recovery are investigated:
In order to verify precision and the reappearance of this immunosensor, in a few days experiment and the investigation (N=5) of the relative standard deviation (RSD) of experiment have in the daytime been carried out.Experimental result shows, once, in experiment, the coefficient of alteration of five samples under 1pM and two variable concentrations of 0.1pM is respectively 3.6% and 3.2%.Under above-mentioned two concentration, the coefficient of alteration between continuous five experiments is respectively 4.2% and 3.8% equally.
Because the specificity of method occupies important effect in biological sample analysis, therefore, be necessary to investigate the method specific recognition capability to TNF-α in biological specimen, in experiment, select human IgG, fibrin ferment, PDGF (PDGF) and BSA as chaff interference.As can be seen from Figure 3, under identical antigen concentration (1.0pM), the signal intensity that chaff interference produces is well below TNF-α.In order further to investigate this design in mixing sample, specific selectivity to TNF-α, by the TNF-α of 1.0pM and the human IgG of 10.0pM, the PDGF of the fibrin ferment of 10.0pM, 10.0pM and the BSA of 10.0pM hatch simultaneously, under the fluorescence intensity and the same terms producing under this condition, simple TNF-α almost identical.These results show, this Fluoro-Immnnosensor has good selectivity to TNF-α.
Also carried out the investigation of TNF-α recovery in calf serum.Under two kinds of different TNF-α interpolation concentration, the recovery of (1.0pM and 0.1pM) is in the scope of 93%-95%.
Embodiment 6
Sample matrix components interference test:
Select Na+, Cl-, glucose (GLU), uric acid (UA) and five kinds of components of dopamine (DA) of in ordinary people's serum, coexisting, concentration of component is highest level in normal human serum close to it, under the condition that detection exists in disturbance component, the signal intensity of its fluorescence immunoassay experiment and normal sample (namely not adding any interference component) are compared, result as shown in Figure 4, under the condition existing in disturbance component, the signal intensity of fluorescence immunoassay experiment is basic identical with normal sample.The interference that proof complex matrices produces this immunosensor is very little.
Embodiment 7
Replication experiment
Bright spot in immunosensor of the present invention, is to be inserted in long duplex DNA and to be produced by a large amount of SYBR Green I, and in order to prove the importance of HCR in this design, we utilize typical fluorescence imaging to test to confirm.Wherein, using and add the immunosensor of object TNF-α as positive group, not containing the immunosensor of object TNF-α as negative group, add object TNF-α, but the immunosensor that does not add hair fastener probe is as a control group; Experimental result as shown in Figure 5, when having object TNF-α to exist, can be observed a large amount of fluorescence bright spots and exist.Otherwise, even if add after dyestuff, in the visual field, do not observe bright spot yet.Presentation of results, does not have the generation of double-stranded DNA just can not produce bright spot.In addition, in control group, add object TNF-α, but do not add hair fastener probe, the chain extension reaction of HCR can not occur in this case, now, almost there is no visible bright spot.Similarly, this result convincingly demonstrates the vital role of HCR reaction in SMD counting.
Embodiment 8
Adopt the immunosensor of the embodiment of the present invention 1 preparation, four parts of normal human serum samples are investigated.Testing result is in Table 1, and result shows: in four serum samples, the TNF-α concentration detecting is at 0.39pM in the scope of 0.52pM, and (0.29pM is to 0.58pM) TNF-α normal range of this and bibliographical information matches.
Table 1 sample determination result
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various modifications that creative work can make or distortion still in protection scope of the present invention.
Claims (9)
1. the immunosensor based on cross chain reaction and monomolecular counting, is characterized in that, capture antibody, detection antibody, Streptavidin, biotinylated primer and hair fastener probe, consists of;
Described capture antibody is anti-TNF-α;
Described detection antibody is Bio-anti-TNF-α;
The base sequence of described biotinylated primer is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA TTT TTT TTT-biotin-3 ';
Described hair fastener probe is comprised of hair fastener probe H1 and hair fastener probe H2, and wherein the base sequence of hair fastener probe H1 is 5 '-TTA ACC CAC GCC GAA TCC TAG ACT CAA AGT AGT CTA GGA TTC GGC GTG-3 ';
The base sequence of hair fastener probe H2 is 5 '-AGT CTA GGA TTC GGC GTG GGT TAA CAC GCC GAA TCC TAG ACT ACT TTG-3 '.
2. the preparation method of a kind of immunosensor based on cross chain reaction and monomolecular counting claimed in claim 1, is characterized in that, comprises the following steps:
(1) structure of sandwich immunoassay course of reaction: capture antibody anti-TNF-α is dripped in the substrate of glass of silanization processing, hatch 8-14h under the moisturizing condition of 37 ℃; Add sealer, under moisturizing condition, react 3-8h, the site that sealing exposes, to reduce non-specific adsorption; Add again and detect antibody Bio-anti-TNF-α, at 37 ℃, hatch 1-3h; After every single step reaction, all use PBS-T and pure water rinsing repeatedly, remove unconjugated reagent;
(2) structure of the immunosensor based on cross chain reaction: after immuning hybridization reaction, add Streptavidin in substrate of glass, hatch 1-3h; PBS-T and pure water rinsing repeatedly after, add Bio-I, hatch 1-3h; PBS-T and pure water rinsing repeatedly after, add the potpourri of hair fastener probe H1, H2, hatch 2-6h; Substrate of glass is used PBS-T and pure water rinsing repeatedly again, removes unreacted hair fastener probe, obtains;
In step (1), the concentration of anti-TNF-α is 10-30pM, and the concentration of sealer is 20-60mM, and the concentration of Bio-anti-TNF-α is 20-40pM;
Described sealer is monoethanolamine or bovine serum albumin;
In step (2), the concentration of Streptavidin is 10-80pM, and the concentration of Bio-I is 0.1-0.5nM, and in the potpourri of hair fastener probe H1, H2, H1 is identical with H2 concentration, is 5-15nM;
The volume ratio of the potpourri addition of anti-TNF-α, sealer, Bio-anti-TNF-α, Streptavidin, Bio-I, hair fastener probe H1, H2 is (1～2): (1～2): (1～2): (1～2): (1～2).
3. the preparation method of a kind of immunosensor based on cross chain reaction and monomolecular counting as claimed in claim 2, is characterized in that, in step (1), the silanization of substrate of glass is processed and comprised the following steps:
A: clean, soak 8-16h in the chromic acid solution that is 5%～8% in mass concentration by substrate of glass, rinse with a large amount of clear water, then use pure water rinsing; Afterwards, by substrate of glass successively ultrasonic cleaning in acetone, ethanol and pure water, each 10min, every kind of solution cleans repeatedly, the solution more renewing after each cleaning; Afterwards, the substrate of glass cleaning up is added to ultrasonic 30min in the mixed solution that hydrochloric acid, pure water, hydrogen peroxide volume ratio are 1:1:1; Use again pure water ultrasonic cleaning repeatedly, each 10min; Finally, the cover glass having activated is put into the thermostatic drying chamber of 120 ℃, removed residual moisture above;
B: silanization, it is that 6% GOPS-anhydrous toluene solution carries out silanization processing that the substrate of glass that step a is handled well is put into volume fraction, this reaction is reacted 6h at 95 ℃; After reaction finishes, substrate of glass is taken out, successively ultrasonic cleaning 15min in toluene, ethanol and pure water; After nitrogen stream dries up, the cover glass of the silanization preparing is stored in exsiccator standby.
4. the preparation method of a kind of immunosensor based on cross chain reaction and monomolecular counting as claimed in claim 2, is characterized in that, in described step (1), sealer is monoethanolamine, and concentration is 50mM.
5. the preparation method of a kind of immunosensor based on cross chain reaction and monomolecular counting as claimed in claim 2, is characterized in that, in described step (2), the concentration of Streptavidin is 30pM.
6. the preparation method of a kind of immunosensor based on cross chain reaction and monomolecular counting as claimed in claim 2, it is characterized in that, in described step (2), hair fastener probe H1 and H2 be annealed step before using, annealing process is at 90 ℃ of heating 5min, is then cooled to room temperature.
7. the application of the immunosensor based on cross chain reaction and monomolecular counting in TNF-α detects described in claim 1.
8. the application of the immunosensor based on cross chain reaction and monomolecular counting as claimed in claim 7 in TNF-α detects, is characterized in that, application process is:
(1) capture antibody anti-TNF-α is dripped in the substrate of glass of silanization processing, under the moisturizing condition of 37 ℃, hatch 8-14h; Add sealer, under moisturizing condition, react 3-8h, the site that sealing exposes, to reduce non-specific adsorption; , add TNF-α to be detected, hatch 1-3h, formed Antibody-antigen complex; Add again and detect antibody Bio-anti-TNF-α, at 37 ℃, hatch 1-3h; And form the immune complex of " sandwich " type;
(2) after immuning hybridization reaction, in substrate of glass, add Streptavidin, hatch 1-3h; PBS-T and pure water rinsing repeatedly after, add Bio-I, hatch 1-3h; After substrate of glass is cleaned, add the potpourri of hair fastener probe H1, H2, hatch 2-6h; Substrate of glass is used PBS-T and pure water rinsing repeatedly again, removes unreacted hair fastener probe;
(3) fluorescence imaging detects: add SYBR Green I, under room temperature, hatch 10min; Suck excessive unreacted SYBR Green I, after using PBS-T and pure water rinsing repeatedly, add the PBS buffer solution of same volume, carry out fluorescence imaging experiment, the fluorescence picture obtaining is processed, go forward side by side line number according to one's analysis;
In step (1), (2), (3), anti-TNF-α, sealer, TNF-α, Bio-anti-TNF-α to be detected, Streptavidin, Bio-I, the potpourri of hair fastener probe H1, H2 are, the volume ratio of SYBR Green I addition is (1～2): (1～2): (1～2): (1～2): (1～2): (1～2): (1～2);
In step (3), the concentration of SYBR Green I is 5-15 μ M.
9. application as claimed in claim 8, is characterized in that, in the step of application process (3), the experiment that fluorescence imaging detects is set to: select 60 * object lens, it is 100ms that time shutter is set, and yield value is 2000, and the cover glass of gained after experiment is fixed on objective table; Open mercury lamp, adjust excitation wavelength optical filter, select the excitation channel between 470～490nm, luminescent dye molecule SYBR Green I is excited and send fluorescence; Adjusting focal length, obtains fluorescence picture, with MetaMorph software, the fluorescence picture obtaining is processed, and goes forward side by side line number according to one's analysis.
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