CN104181292B - A kind of protein single molecules level ampere immunity analytical approach - Google Patents

A kind of protein single molecules level ampere immunity analytical approach Download PDF

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CN104181292B
CN104181292B CN201410426738.4A CN201410426738A CN104181292B CN 104181292 B CN104181292 B CN 104181292B CN 201410426738 A CN201410426738 A CN 201410426738A CN 104181292 B CN104181292 B CN 104181292B
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CN104181292A (en
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谢青季
覃晓丽
刘玲
徐爱贵
谭月明
傅迎春
陈超
姚守拙
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Hunan Normal University
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Abstract

A kind of protein single molecules level ampere immunity analytical approach, comprise the following steps: 1. with the immunoelectrode of nano gold mark for working electrode, on the working electrode (s centered by nano gold mark thing, carry out gold label silver stain and primary element displacement reaction, the primary element displacement reaction of layer and golden salt is contaminated by silver, optionally amplify the size of nano gold mark thing, silver-colored dye amount can be enlarged markedly and amplify metal marker thing electrochemical analysis signal; 2. the method for this multiple amplification is used for electrochemical immunoanalytical, the Anodic Stripping Currents signal of the metal level that this working electrode is enriched to is obtained by original position anodic stripping voltammetry, thus indirectly realize the quantitative test of target analytes in sample, make electrochemical method can detect the protein being low to moderate single molecules level.The present invention can be used for analyzing quality testing based on the single goal of biocompatible, metal marker and anodic stripping voltammetry and surveys and multiobjective analysis thing multi-channel detection.

Description

A kind of protein single molecules level ampere immunity analytical approach
Technical field
The present invention relates to a kind of protein single molecules level ampere immunity analytical approach, especially relate to a kind of protein single molecules level ampere immunity analytical approach based on metal marker and the multiple amplification of signal.
Background technology
The bioanalysis of reacting based on various biocompatible has attracted very big concern [Satori, the C.P. of academia and industrial community; Henderson, M.M.; Krautkramer, E.A.; Kostal, V.; Distefano, M.M.; Arriaga, E.A. chem.Rev. 2013, 113, 2733].In essence, biocompatible has high specificity, and therefore bioanalysis analyzes thing to it very high specificity and selectivity.So; improve the detection sensitivity based on the bioanalytical method of biocompatible; being the primary study content of field of bioanalysis, is also aspect urgent problem [Song, the Y.J. such as major disease early warning, food and drug safety, environmental analysis that detect based on disease marker; Zhang, Y.Q.; Bernard, P.E.; Reuben, J.M.; Ueno, N.T.; Arlinghaus, R.B.; Zu, Y.L.; Qin, L.D. nat.Commun. 2012, 3, 1283].Meanwhile, the quantitative test detection method of development single molecules level is extensively concerned analytical chemistry subject primary study direction [Weiss, S.Science1999,283,1676.] for a long time always.In most cases, be difficult to directly itself go to obtain very large bioanalysis signal from biocompatible reaction, so often adopt suitable biomarker method amplify and export analytic signal [Zhang, L.B.; Zhu, J.B.; Guo, S.J.; Li, T.; Li, J.; Wang, E.K. j.Am.Chem.Soc. 2013, 135, 2403].Up to now, the labeling method in bioanalysis can be divided into two large classes usually, molecular labeling (as radioactive label and enzyme labeling) method and nano material labelling method.In nano material labelling method, the nano materials such as nm of gold (AuNPs), Nano Silver (AgNPs), metal sulfide/selenide/telluride quantum dots (QDs), carbon nano-tube and Graphene are usually adopted to mark.
In the fundamental research and application and development of numerous ambits such as material, environment, the energy, the deficient salt (oxygenant) of active metal and primary element displacement reaction (GRR) technology comparatively between active metal (reductive agent) are widely applied.Such as, GRR technology, for developing the various metal materials (as Au, Pt, Pd) of structure-controllable and performance enhancement, comprising preparation and having the nano material of special shape for biomarker [Sun, Y.; Xia, Y. science 2002, 298, 2176.].In addition, gold label silver stain technology has been widely used in the biocompatible type analysis sensing that gold nano grain (AuNPs) marks, reliably can amplify the signal of bioanalysis, because conventional quinhydrones electronation Ag +silver dye phenomenon, can only optionally occur in catalytic gold nano grain (AuNP) surface [Taton, T.A.; Mirkin, C.A.; Letsinger, R.L. science 2000, 289, 1757.].
Application number is the Chinese invention patent application of 201310459224.4, disclose " a kind of original position Anodic stripping voltammetry method based on metal marker and biocompatible ", although make Monitoring lower-cut be effectively reduced, but sensitivity is high not enough, the Monitoring lower-cut of single molecules level can not be reached.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiencies in the prior art, in conjunction with gold label silver stain and GRR method, provides a kind of protein single molecules level ampere immunity analytical approach.The method can be amplified gold nano label in multiple times, and be converted into and can survey electrochemical signals, recycling application number is the original position Anodic stripping voltammetry in 201310459224.4 applications for a patent for invention, carries out quantitative test to the target analytes (as protein) in sample.The method versatility is good, easy and simple to handle, makes Monitoring lower-cut can be low to moderate single molecules level (Monitoring lower-cut can reach detection 5 immunoglobulin G while (hIgG) and human a-fetoprotein (hAFP)).
The technical scheme that the present invention solves the employing of its technical matters is that a kind of protein single molecules level ampere immunity analytical approach, comprises the following steps:
1. take immunoelectrode as working electrode, on the working electrode (s centered by nano gold mark thing, carry out gold label silver stain [see Taton, T.A.; Mirkin, C.A.; Letsinger, R.L. science 2000, 289, 1757.] and primary element displacement reaction [see Sun, Y.; Xia, Y. science 2002, 2982176.] repeatedly reaction (preferably 1 ~ 10 time), the primary element displacement reaction of layer and golden salt is contaminated by silver, optionally amplify the size of nano gold mark thing (in nm of gold after silver dye, the silver-colored simple substance that nm of gold Surface Creation is a large amount of, nm of gold is amplified, silver dye layer carries out primary element displacement reaction again, then generate again golden simple substance at the Surface Creation of nm of gold, nm of gold is amplified again), silver-colored dye amount can be enlarged markedly and amplify metal marker thing electrochemical analysis signal;
2. the method for this multiple amplification is used for electrochemical immunoanalytical, the Anodic Stripping Currents signal of the metal level that this working electrode is enriched to is obtained by original position anodic stripping voltammetry, thus indirectly realize the quantitative test of target analytes in sample, make electrochemical method can detect the protein being low to moderate single molecules level.
Further, described golden salt is the golden salt solusion replacing silver dye layer; Described golden salt is that then volume is different for 1uL ~ 10mL(concentration difference) volume can replace the silver-colored golden salt solusion contaminating layer.(golden salt is enough, and silver has reacted, and reacts and namely stops)
Further, the concentration of described golden salt is 1mmol/L lean solution ~ saturated solution.
Further, described golden salt is gold chloride, potassium chloroaurate, sodium chloraurate, gold sodium sulfide or sulphurous acid gold potassium.
Further, the concentration range of described golden salt is 1umol/L ~ 0.1mol/L; The preferred 5mmol/L of gold chloride concentration of displacement Nano silver grain.
Further, the preparation process of the biological composite of described nano gold mark thing is: be combined with bio-ligand by golden nanometer particle, and form biological composite, described biological composite has bioaffinity to target analytes in sample.
Further, described bio-ligand is antigen or antibody, or protein.Such as, golden nanometer particle can be combined with antibody or antigen, form biological composite; Again antibody to be measured or antigen are combined with this biological composite, form immunoelectrode.
Further, the cathode potential (application number is 201310459224.4, in application for a patent for invention) that can realize the metallic ion electro-deposition of metal marker thing applied in advance in described air is the cathode potential enough realizing the metal electrodeposition that diffusion controls; The described cathode potential enough realizing the metal electrodeposition that diffusion controls is 1.0V(vs.SCE) ~-2.0V(vs.SCE); " Vvs.SCE " means the current potential (volt) relative to saturated calomel electrode.
Further, use anodic stripping voltammetry when carrying out quantitative test to metallic ion, adopt 1 group, 2 groups or 2 groups, with top electrode, are often organized between electrode and are formed a Measurement channel; Often organizing electrode is two electrodes or three-electrode system.
Further, described target analytes can be protein.
The salt (oxygenant) of described deficient active metal and the primary element displacement reaction technology comparatively between active metal (reductive agent) are used for the silver dye layer reaction after gold label silver stain.
Beneficial effect of the present invention: (1) gold label silver stain technology is used for the biocompatible type analysis sensing of gold nano grain mark, reliably can amplify the signal of bioanalysis, because the silver dye phenomenon of conventional quinhydrones electronation silver ion, optionally can only occur in the gold nano grain surface of catalytic activity, the primary element displacement reaction of layer and golden salt is contaminated by silver, optionally amplify the size of gold nano grain label, the ampere immunity analytic signal of silver-colored dye amount and amplification metal marker thing can be enlarged markedly; (2) after the primary element displacement reaction of golden salt, on gold nano grain, nanogold particle is generated again, more substantial gold label silver stain can be carried out further, repeatedly repeat the signal that bioanalysis is farthest amplified in two kinds of reactions, the ampere immunity analysis of protein single molecules level can be realized.
The present invention is that a kind of, gold label silver stain, primary element displacement reaction and Anodic stripping voltammetry method affine based on immunity analyze to protein target in sample the method that thing carries out quantitative test, and is the electrochemical analysis method detecting target analytes at single molecules level.The present invention in conjunction with gold label silver stain and primary element displacement reaction (GRR) for the super quick analysis sensing of biocompatible type, by GRR method and gold label silver stain, multiple amplification is carried out to metal marker thing, carry out Electrochemical Detection, sensitivity is significantly improved, Monitoring lower-cut can be reduced to single molecules level.
The experiment proved that, patent of invention " based on the original position Anodic stripping voltammetry method of metal marker with the biocompatible " (application number: 201310459224.4) compare of the present invention and original application, the electrochemical immunoanalytical response signal that the present invention is based on metal marker thing and the multiple amplification of signal increases at least 5 times, the protein of single molecules level can be detected.The analysis test experience of human immunoglobulin(HIg) (hIgG) and alpha-fetoprotein (hAFP) is shown, the present invention has the wide range of linearity across at least 9 orders of magnitude, Monitoring lower-cut is low to moderate single molecules level (Monitoring lower-cut can reach detection 5 immunoglobulin G while (hIgG) and human a-fetoprotein (hAFP)), significantly be better than literature values (detection span 8 orders of magnitude of existing similar technique, can not carry out single molecules level detection).The present invention is used for actual sample detection and also obtains satisfactory result.
Accompanying drawing explanation
Fig. 1 is the experimental procedure schematic diagram of the inventive method;
On the hIgG immunoelectrode that Fig. 2 (A) marks at gold nano grain (AuNPs) for customary preparation methods in the embodiment of the present invention 1, adopt the former figure of Differential Pulse Anodic Stripping Voltammetry examination criteria sample;
The hIgG immunoelectrode that Fig. 2 (B) marks at gold nano grain (AuNPs) for customary preparation methods in embodiment 1 detects the canonical plotting obtained;
On the hIgG immunoelectrode that Fig. 2 (C) marks at AuNPs for the inventive method in embodiment 1, adopt the former figure of Differential Pulse Anodic Stripping Voltammetry examination criteria sample;
The hIgG immunoelectrode that Fig. 2 (D) marks at AuNPs for the inventive method in embodiment 1 detects the canonical plotting obtained;
The former figure of examination criteria sample on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (A) is customary preparation methods employing AuNPs mark in embodiment 2;
Examination criteria curve map on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (B) is customary preparation methods employing AuNPs mark in embodiment 2;
The former figure of examination criteria sample on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (C) is the inventive method employing AuNPs mark in embodiment 2;
Examination criteria curve map on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (D) is the inventive method employing AuNPs mark in embodiment 2.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Reference example 1
Below for embodiment 1 use hIgG immunoelectrode and nano material prepare in accordance with the following methods:
Two anti-, immunoelectrode that the golden nanometer particle (AuNPs) that embodiment 1 uses marks are prepared by the following method:
(1) the two preparation (Ab resisted of AuNPs mark 2-AuNPs): in 100mL boiling water, under vigorous stirring, add 250mL4%(mass concentration) HAuCl 4, more dropwise drip 1%(mass concentration) sodium citrate 2.5mL, after solution becomes claret, then heat 15min; After stopping heating, stir and be cooled to room temperature; Two anti-(the Ab of 30ug are added in the gold size of 1mL 2), attach overnight; After 4800rpm low-speed centrifugal 30min, phosphate buffer (PBS) cleans twice; Finally, bond is dispersed in the 1mL0.1mol/LPBS containing 1% bovine serum albumin(BSA) (BSA) again, increases the stability of immune gold size, reduces the non-specific adsorption in analyzing; When not using, the Ab of preparation 2-AuNPs is stored in refrigerator (4 oc) in;
(2) preparation of immunoelectrode: glass-carbon electrode (GCE) is sanding and polishing process in the aluminium oxide suspension of 0.5 micron (um) and 0.05um successively, electrode surface (being rinsed well by the alumina powder of electrode surface) is rinsed fully again, the alumina powder that next each ultrasonic 5min remains to remove electrode surface in ultrapure water, ethanol, ultrapure water respectively with ultrapure water; Then, dense H is dripped at electrode surface 2sO 4(dense H 2sO 4concentration be 18.4mol/L) washing lotion keep 15s, with ultrapure water; Finally with electrochemical cleaning thoroughly to remove pollutant; Electrochemical cleaning step is: at 10mL0.50mol/LH 2sO 4in sweep fast cyclic voltammetry scan to stable with-1.0V to 1.0V with 0.1V/s; The glass-carbon electrode handled well 50 ~ 500mL ultrapure water (H residual by electrode surface 2sO 4rinse well), then dry up for electro-deposition Au with nitrogen plate;
By bipotential step method by Au platedeposit on the glass-carbon electrode handled well, namely containing 2.0mmol/LHAuCl 40.50mol/LH 2sO 4in, the pulse plating 180s(Au from 1.1V to 0V plate/ GCE); Cleaned by electrode, nitrogen dries up, and rapidly 6.0 μ L is contained 1.0mg/mL primary antibodie (Ab 1) PBS solution drop to Au plateon/GCE electrode, refrigerator (4 oc) preserve, spend the night, to ensure the saturated adsorption of antibody at electrode surface, obtain Ab 1/ Au plate/ GCE modified electrode; By Ab 1/ Au plate/ GCE modified electrode successively with PBS solution cleaning, dry up after, PBS solution 6.0uL being contained 3wt%BSA drops on electrode, 4 DEG C keep 1h with closed non-specific adsorption sites, obtain BSA/Ab 1/ Au plate/ GCE modified electrode; When not using, electrode is kept at 4 oin the PBS of C.
By the BSA/Ab prepared 1/ Au plate/ GCE modified electrode, drips the PBS that 6.0uL contains variable concentrations antigen (hIgG, immunoglobulin G while), incubation 1h at 37 DEG C, then uses PBS solution cleaning electrode surface, obtains hIgG/BSA/Ab 1/ Au plate/ GCE modified electrode, this modified electrode drips again 6mL containing Ab 2pBS solution (the Ab of-AuNPs 2-AuNPs mass concentration 0.5mg/mL) incubation 40min at 37 DEG C, obtain Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode (Ab 1, Ab 2for goat anti-human immunoglobulin G);
The gold label silver stain step of immunoelectrode is as follows: Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode PBS solution is fully cleaned, and resists with remove non-specific adsorption two, after dry, drips silver enhancement solution (component: 1.0g p-dihydroxy-benzene, 35mg silver nitrate, the 50mLpH3.5 citrate buffer solution (0.243mol/LC of 6.0uL 6h 8o 7× H 2o+0.163mol/LNa 3c 6h 5o 7× 2H 2o) and 50mL deionized water), dark reaction 30min, with ultrapure water clean, obtain silver/Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode.
Reference example 2
Alpha-fetoprotein (hAFP) immunoelectrode of the AuNPs mark that embodiment 2 uses is prepared in accordance with the following methods:
(1) the two preparation (Ab resisted of AuNPs mark 2-AuNPs): in 100mL boiling water, under vigorous stirring, add 250uL4%(mass concentration) HAuCl 4, more dropwise drip 1%(mass concentration) sodium citrate 2.5mL, after solution becomes claret, then heat 15min; After stopping heating, stir and be cooled to room temperature; Two anti-(the Ab of 30ug are added in the gold size of 1mL 2), attach overnight; After 4800rpm low-speed centrifugal 30min, phosphate buffer (PBS) cleans twice; Finally, bond is dispersed in the 1mL0.1mol/LPBS containing 1% bovine serum albumin(BSA) (BSA) again, increases the stability of immune gold size, reduces the non-specific adsorption in analyzing; When not using, the Ab of preparation 2-AuNPs is stored in refrigerator (4 oc) in;
(2) preparation of immunoelectrode: glass-carbon electrode (GCE) successively sanding and polishing process in the aluminium oxide suspension of 0.5um and 0.05um, electrode surface (being rinsed well by the alumina powder of electrode surface) is rinsed fully again, the alumina powder that next each ultrasonic 5min remains to remove electrode surface in ultrapure water, ethanol, ultrapure water respectively with ultrapure water; Then, dense H is dripped at electrode surface 2sO 4(dense H 2sO 4concentration be 18.4mol/L) washing lotion keep 15s, with ultrapure water; Finally with electrochemical cleaning thoroughly to remove pollutant; Electrochemical cleaning step is: at 10mL0.50mol/LH 2sO 4in sweep fast cyclic voltammetry scan to stable with-1.0V to 1.0V with 0.1V/s; The glass-carbon electrode handled well 50 ~ 500mL water rinses (by the H that electrode surface remains 2sO 4rinse well), then dry up for electro-deposition Au with nitrogen plate;
By bipotential step method by Au platedeposit on the glass-carbon electrode handled well, namely containing 2.0mmol/LHAuCl 40.50mol/LH 2sO 4in, the pulse plating 180s(Au from 1.1V to 0V plate/ GCE); Cleaned by electrode, nitrogen dries up, and 6.0uL is contained 1.0mg/mL primary antibodie (Ab rapidly 1) PBS solution drop to Au plateon/GCE electrode, refrigerator (4 oc) preserve, spend the night, to ensure the saturated adsorption of antibody at electrode surface, obtain Ab 1/ Au plate/ GCE modified electrode; By Ab 1/ Au plate/ GCE modified electrode successively with PBS solution cleaning, dry up after, the PBS solution that 6.0 μ L contain 3wt%BSA is dropped on electrode, 4 DEG C keep 1h with closed non-specific adsorption sites, obtain BSA/Ab 1/ Au plate/ GCE modified electrode; When not using, electrode is kept at 4 oin the PBS of C;
By the BSA/Ab prepared 1/ Au plate/ GCE modified electrode, drips the PBS that 6.0uL contains variable concentrations antigen (hAFP, human a-fetoprotein), incubation 1h at 37 DEG C, then uses PBS solution cleaning electrode surface, obtains hAFP/BSA/Ab 1/ Au plate/ GCE modified electrode, this modified electrode drips again 6mL containing Ab 2pBS solution (the Ab of-AuNPs 2-AuNPs mass concentration is 0.5mg/mL) incubation 40min at 37 DEG C, obtain Ab 2-AuNPs/hAFP/BSA/Ab 1/ Au plate/ GCE electrode.(Ab in this embodiment 1, Ab 2for mouse-anti human a-fetoprotein).
The gold label silver stain step of immunoelectrode is as follows: except replacing immunoglobulin (Ig) hIgG with alpha-fetoprotein (hAFP), and other are identical with " the gold label silver stain step of immunoelectrode " in reference example 1.
Embodiment 1:hIgG immunity electroanalysis
The present embodiment is the electroanalysis of the detection target analytes hIgG of, metal marker affine based on immunity and anodic stripping voltammetry, compares the inventive method and the response of customary preparation methods in immunoelectrode.
The present embodiment comprises the following steps:
(1) the inventive method: 1. carry out gold label silver stain reaction (see reference example 1) on the immunoelectrode prepared, then carry out primary element displacement reaction (GRRs), the primary element displacement reaction step of immunoelectrode is as follows: silver/Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode drips the chlorauric acid solution (HAuCl of 6.0uL5.0mmol/L 4), react 10 minutes, dry up and clean with ultrapure water, obtain Au-silver/Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode; Make to generate nanogold particle again on the surface in former nm of gold, the amplification of size has been carried out to the nm of gold of mark.Also can drip 6.0uL5.0mmol/L potassium chloroaurate, sodium chloraurate, gold sodium sulfide or sulphurous acid gold potassium solution similarly, carry out the amplification that signal is carried out in same GRR reaction; 2. owing to generating more nm of gold, so after primary element displacement reaction, gold label silver stain reaction can be carried out at the electrode surface again, generate more Nano silver grain, the amount making it possible to dissolve the silver be enriched on electrode surface increases, and electrochemical response signal increases; Hocket after 4 times by gold label silver stain reaction and primary element displacement reaction, last gold label silver stain obtains more Nano Silver, thus obtains larger electrochemical response signal.
(2) customary preparation methods: on the immunoelectrode in reference example 1, only carries out a gold label silver stain reaction.
(3) detection method: the i.e. Chinese invention patent of original application " the original position Anodic stripping voltammetry method based on metal marker and biocompatible " (application number: 201310459224.4), 1. in three-electrode system (working electrode is the immunoelectrode of the gold label silver stain in above-mentioned reference example), cathode potential-the 0.3V(vs.SCE that electrochemical apparatus also applies fully to realize the metal A g cationic electrodeposition of gold label silver stain is in advance connected in air), then by 8uL1mol/LHNO 3add to immunoelectrode surface and conducting electrolytic cell, make metal marker thing be dissolved as Ag +, and simultaneously by Ag +electroreduction becomes atomic state metal A g, thus on immunoelectrode surface enrichment argent.2. direct at former working electrode surface, Differential Pulse Anodic Stripping Voltammetry (DPV) is used to detect the signal that argent Anodic Stripping is silver ion, testing conditions is :-0.3V (vs.SCE(saturated calomel electrode)) deposition 10min, sweep interval is-0.3 ~ 0.7V, obtain the Anodic Stripping Currents signal of the argent that this working electrode is enriched to, thus indirectly realize the quantitative test of target analytes hIgG in sample.
Measurement result is analyzed: composition graphs 2 is discussed, Fig. 2 gives the immunoelectrode of AuNPs label structure in two kinds of methods to the signal response of variable concentrations antigen, on the hIgG immunoelectrode that Fig. 2 (A) marks at gold nano grain (AuNPs) for customary preparation methods in the embodiment of the present invention 1, adopt the former figure of Differential Pulse Anodic Stripping Voltammetry examination criteria sample; The hIgG immunoelectrode that Fig. 2 (B) marks at gold nano grain (AuNPs) for customary preparation methods in embodiment 1 detects the canonical plotting obtained; On the hIgG immunoelectrode that Fig. 2 (C) marks at AuNPs for the inventive method in embodiment 1, adopt the former figure of Differential Pulse Anodic Stripping Voltammetry examination criteria sample; The hIgG immunoelectrode that Fig. 2 (D) marks at AuNPs for the inventive method in embodiment 1 detects the canonical plotting obtained; As we know from the figure, along with antigen concentration increases, current-responsive also increases thereupon; For same antigen concentration, the response signal of method of the present invention is obviously greater than customary preparation methods; In the present invention, electric current and the antigen concentration range of linearity are 0.4fg/mL ~ 400ng/mL, the detection of AuNPs mark is limited to 0.2fg/mL(and is equivalent in 6uL sample, only have 5 protein molecules, signal to noise ratio (S/N ratio) is 3), customary preparation methods detection line is then 1.2fg/mL; The inventive method range of linearity is wide, detectability is low, in conjunction with national inventing patent " the original position Anodic stripping voltammetry method based on metal marker and the biocompatible " (application number: electrochemical detection method 201310459224.4) makes Monitoring lower-cut reach single molecules level of original application.
Embodiment 2 is based on the alpha-fetoprotein immunity electroanalysis of the inventive method
The present embodiment is the electroanalysis of the detection alpha-fetoprotein of, metal marker affine based on immunity and anodic stripping voltammetry, obtains the response in alpha-fetoprotein electrode by technical scheme of the present invention.
The present embodiment comprises the following steps:
Sample determination:
1. the alpha-fetoprotein analysis of gold label silver stain:
(1) except replacing immunoglobulin (Ig) hIgG with alpha-fetoprotein (hAFP), other are identical with the implementation step of " the inventive method " in embodiment 1.Namely on the immunoelectrode prepared, 1. carry out gold label silver stain reaction (see reference example 2), then carry out primary element displacement reaction (GRRs), the primary element displacement reaction step of immunoelectrode is as follows: silver/Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode drips the chlorauric acid solution (HAuCl of 6.0uL5.0mmol/L 4), react 10 minutes, dry up and clean with ultrapure water, obtain Au-silver/Ab 2-AuNPs/hIgG/BSA/Ab 1/ Au plate/ GCE electrode; Make to generate nanogold particle again on the surface in former nm of gold, the amplification of size has been carried out to the nm of gold of mark; Also can drip 6.0uL5.0mmol/L potassium chloroaurate, sodium chloraurate, gold sodium sulfide or sulphurous acid gold potassium solution similarly, carry out the amplification that signal is carried out in same GRR reaction; 2. owing to generating more nm of gold, so after primary element displacement reaction, gold label silver stain reaction can be carried out at the electrode surface again, generate more Nano silver grain, the amount making it possible to dissolve the silver be enriched on electrode surface increases, and electrochemical response signal increases; Hocket after 4 times by gold label silver stain reaction and primary element displacement reaction, last gold label silver stain obtains more Nano Silver, thus obtains larger electrochemical response signal.
(2) customary preparation methods: except replacing immunoglobulin (Ig) hIgG with alpha-fetoprotein (hAFP), other are identical with the implementation step of " customary preparation methods " in embodiment 1.Namely, on the immunoelectrode in reference example 2, a gold label silver stain reaction is only carried out.
(3) detection method: except replacing immunoglobulin (Ig) hIgG with alpha-fetoprotein (hAFP), other are identical with the implementation step of " detection method " in embodiment 1.
Measurement result is analyzed: composition graphs 3 is discussed, Fig. 3 gives immunoelectrode that AuNPs label in two kinds of methods builds to the signal response of variable concentrations antigen, and Fig. 3 (A) is the former figure of examination criteria sample on alpha-fetoprotein (hAFP) immunoelectrode that in embodiment 2, customary preparation methods adopts AuNPs mark; Examination criteria curve map on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (B) is customary preparation methods employing AuNPs mark in embodiment 2; The former figure of examination criteria sample on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (C) is the inventive method employing AuNPs mark in embodiment 2; Examination criteria curve map on alpha-fetoprotein (hAFP) immunoelectrode that Fig. 3 (D) is the inventive method employing AuNPs mark in embodiment 2; As we know from the figure, along with antigen concentration increases, current-responsive also increases thereupon; For same antigen concentration, the response signal of method of the present invention is obviously greater than conventional method; In the present invention, electric current and the antigen concentration range of linearity are 0.5fg/mL ~ 500ng/mL, the detection of AuNPs mark is limited to 0.1fg/mL(and is equivalent in 6uL sample, only have 5 protein molecules, signal to noise ratio (S/N ratio) is 3), the Monitoring lower-cut of customary preparation methods is then 1.6fg/mL; The inventive method range of linearity is wide, detectability is low, in conjunction with patent of invention " the original position Anodic stripping voltammetry method based on metal marker and the biocompatible " (application number: electrochemical detection method 201310459224.4) makes to have lower Monitoring lower-cut of original application.
In sum, the sensitivity of analytical method that the present invention relates to is high, easy and simple to handle, versatility is good, with " the original position Anodic stripping voltammetry method based on metal marker and biocompatible " patent of invention (application number: the 201310459224.4) coupling of method, achieve metal marker thing detects single molecules level protein for electrochemical immunoanalytical, and the testing result in actual blood sample is satisfactory, the single goal that can be widely used in based on biocompatible, metal marker and anodic stripping voltammetry analyzes quality testing survey and multiobjective analysis thing multi-channel detection.

Claims (17)

1. a protein single molecules level ampere immunity analytical approach, is characterized in that, comprise the following steps:
1. be working electrode with immunoelectrode, on the working electrode (s centered by nano gold mark thing, carry out the repeatedly reaction of gold label silver stain and primary element displacement reaction, the primary element displacement reaction of layer and golden salt is contaminated by silver, optionally amplify the size of nano gold mark thing, silver-colored dye amount can be enlarged markedly and amplify metal marker thing electrochemical analysis signal;
2. the method for this multiple amplification is used for electrochemical immunoanalytical, the Anodic Stripping Currents signal of the metal level that this working electrode is enriched to is obtained by original position anodic stripping voltammetry, thus indirectly realize the quantitative test of target analytes in sample, make electrochemical method can detect the protein being low to moderate single molecules level.
2. protein single molecules level ampere immunity analytical approach according to claim 1, is characterized in that, described golden salt is the golden salt solusion replacing silver dye layer.
3. protein single molecules level ampere immunity analytical approach according to claim 2, is characterized in that, the concentration of described golden salt is 1 μm of ol/L lean solution ~ saturated solution.
4. the protein single molecules level ampere immunity analytical approach according to Claims 2 or 3, is characterized in that, described golden salt is gold chloride, potassium chloroaurate, sodium chloraurate, gold sodium sulfide or sulphurous acid gold potassium.
5. protein single molecules level ampere immunity analytical approach according to claim 4, is characterized in that, the concentration of described golden salt is 1 μm of ol/L ~ 0.1mol/L.
6. protein single molecules level ampere immunity analytical approach according to claim 1, is characterized in that, described golden salt is the solution replacing silver dye layer of 1 μ L ~ 10mL volume.
7. the protein single molecules level ampere immunity analytical approach according to claim 1,2,3,5 or 6, it is characterized in that, the preparation process of described nano gold mark thing is: golden nanometer particle and bio-ligand are combined into biological composite, and described bio-ligand is antigen or protein.
8. protein single molecules level ampere immunity analytical approach according to claim 4, it is characterized in that, the preparation process of described nano gold mark thing is: golden nanometer particle and bio-ligand are combined into biological composite, and described bio-ligand is antigen or protein.
9. protein single molecules level ampere immunity analytical approach according to claim 7, it is characterized in that, described protein is antibody.
10. protein single molecules level ampere immunity analytical approach according to claim 8, it is characterized in that, described protein is antibody.
11. protein single molecules level ampere immunity analytical approachs according to claim 1,2,3,5 or 6, it is characterized in that, when using anodic stripping voltammetry to carry out quantitative test to metallic ion, adopt 1 group, 2 groups or 2 groups, with top electrode, are often organized between electrode and are formed a Measurement channel; Often organizing electrode is two electrodes or three-electrode system.
12. protein single molecules level ampere immunity analytical approachs according to claim 4, it is characterized in that, when using anodic stripping voltammetry to carry out quantitative test to metallic ion, adopt 1 group, 2 groups or 2 groups, with top electrode, are often organized between electrode and are formed a Measurement channel; Often organizing electrode is two electrodes or three-electrode system.
13. protein single molecules level ampere immunity analytical approachs according to claim 7, it is characterized in that, when using anodic stripping voltammetry to carry out quantitative test to metallic ion, adopt 1 group, 2 groups or 2 groups, with top electrode, are often organized between electrode and are formed a Measurement channel; Often organizing electrode is two electrodes or three-electrode system.
14. protein single molecules level ampere immunity analytical approachs according to claim 1,2,3,5,6,8 or 9, it is characterized in that, the number of times of described gold label silver stain and primary element displacement reaction is 1 ~ 10 time.
15. protein single molecules level ampere immunity analytical approachs according to claim 4, it is characterized in that, the number of times of described gold label silver stain and primary element displacement reaction is 1 ~ 10 time.
16. protein single molecules level ampere immunity analytical approachs according to claim 7, it is characterized in that, the number of times of described gold label silver stain and primary element displacement reaction is 1 ~ 10 time.
17. protein single molecules level ampere immunity analytical approachs according to claim 11, it is characterized in that, the number of times of described gold label silver stain and primary element displacement reaction is 1 ~ 10 time.
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