CN105651840A - Mimic electrochemical immunosensor for detecting beta-amyloid protein oligomers and preparation method thereof - Google Patents
Mimic electrochemical immunosensor for detecting beta-amyloid protein oligomers and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a mimic electrochemical immunosensor for detecting beta-amyloid protein (ABeta) oligomers and preparation method thereof and belongs to the technical field of biosensing and electroanalytical chemistry. gold nanoparticles jointly act with sulfhydryl-modified aptamer and thionine to form a stable nano conjugate; an antibody is fixed to the surface of a carboxylic graphene oxide modified electrode; after an ABeta oligomer is recognized, the oligomer is further connected with the aptamer modified gold nanoparticle conjugate, thus forming a sandwich mimic immunosensor; the ABeta oligomer is measured through thionine electrochemical signals. The immunosensor has high sensitivity, good specificity and high stability; compared with traditional immunosensors, the immunosensor features preparation simplicity and avoidance of the use of enzyme-labeled antibody reagents, is useful in the detection of ABeta oligomers in cerebrospinal fluid to promote early diagnosis of Alzheimer's disease and has clinical applicable value.
Description
Technical field
The present invention relates to a kind of for para-immunity electrochemical sensor detecting amyloid-beta oligomer and preparation method thereof, it is specifically related to a kind of with antibody and the aptamer para-immunity electrochemical sensor forming sandwich-type structure identification amyloid-beta oligomer and preparation method thereof, belongs to bio-sensing and technical field of electroanalytical chemistry detection.
Background technology
Along with China steps into aging society, the sickness rate of Alzheimer (AD) gradually steps up, and the physical and mental health of serious harm old people also affects life quality. The detection of AD mark is to realize early diagnosis and understand the important channel of PD. Research shows, in the generation of AD and brain, the formation of amyloid plaques is closely related. Caused by the excessive amyloid-beta (A ��) that speckle is formed as secretase complex abnormal cutting amyloid precursor protein and produces. Compared with monomer and fiber aggregate A ��, the specific surface area that A beta oligomers is bigger makes it contain abundant ��-pleated sheet conformation, thus hydrophobic residue be extruded out from protein surface cause flow of calcium ions, dopamine to leak outside, mitochondrial depolarization and cause apoptosis. Therefore, oligomer is considered to have more toxicity than fiber aggregate, and solubility A beta oligomers has been taken as the mark of AD.
The method of conventional detection A beta oligomers mainly has euzymelinked immunosorbent assay (ELISA), nuclear magnetic resonance method, spectrographic method, colorimetry and electrochemical process. in these methods, euzymelinked immunosorbent assay (ELISA) has high reliability, but ratio is relatively time-consuming and the enzyme label antibody reagent of needs costliness, nuclear magnetic resonance method needs complicated main equipment, the interference that spectrographic method generally requires the complicated probe molecule of synthesis and signal is easily subject in living things system other materials, nanogold colorimetric method achieves the quick mensuration of A ��, but selectivity need nonetheless remain for improving. electrochemical process is owing to possessing quickly, the advantages such as easy and easy microminiaturization, researcher utilizes electrochemical method and biomolecule (to include antibody, albumen and polypeptide) in detection A ��, obtain good selectivity and sensitivity [(a) YuY.Y. with the strong interaction of A ��, ZhangL., LiC.L., SunX.Y., TangD.Q., ShiG.Y., Angew.Chem.Int.Ed.2014, 53:12832-12835. (b) LiH., XieH.N., CaoY., DingX.R., YinY.M., LiG.X., Anal.Chem.2013, 85:1047-1052. (c) YuY.Y., SunX.Y., TangD.Q., LiC.L., ZhangL., NieD.X., YinX.X., ShiG.Y., Biosens.Bioelectron.2015, 68:115-121. (d) LiuL., HeQ.G., ZhaoF., XiaN., LiuH.J., LiS.J., LiuR.L., ZhangH., Biosens.Bioelectron.2014, 51:208-212. (e) LiuL., ZhaoF., MaF.J., ZhangL.P., YangS.L., XiaN., Biosens.Bioelectron.2013, 49:231-235. (f) RamaE.C., Gonz �� lez-Garc �� aM.B., Costa-Garc �� aA., Sensor.Actuat.B2014, 201:567-571.].But due in body fluid A beta oligomers concentration relatively low and easy occur assemble formed fiber characteristic so that actual sample detect in have sizable difficulty. Realize the detection of the A beta oligomers in body fluid (such as cerebrospinal fluid) early diagnosis and the Morbidity control of AD are had great importance.
Selectivity is the performance indications that biosensor is important. It is capable of the molecule to albumen high specific recognition except expensive antibody, also has aptamer. The effect essence of aptamer is that one section of single stranded nucleic acid molecule is folded to form specific three dimensional structure and specific binding with biological target. The aptamer filtered out not only has high specific, high-affinity and the high stability that antibody possesses, and also has traditional immunization and the unrivaled advantages of chemical molecular identification such as being easy to chemical modification, functionalization, a large amount of low cost and repeatable synthesis. The aptamer of A beta oligomers can coexist lower specific binding with it [TsukakoshiK., AbeK., SodeK., IkebukuroK., Anal.Chem.2012,84:5542-5547.] at A beta monomers and corpus fibrosum. The successful screening of aptamer provides a kind of possibility for selective enumeration method A beta oligomers. It addition, the susceptiveness for improving test is provided opportunity by controlled the constructing of nano material.
This method utilizes aptamer that the high specific of target molecule is combined, design construction antibody and aptamer sandwich type para-immunity electrochemical sensor; And then utilize the excellent properties of nano material, introduce electrochemical signals and realize the amplification of signal; The mensuration being applied in cerebrospinal fluid mark A beta oligomers, promotes the early diagnosis of AD. Have no relevant report at present.
Summary of the invention
It is an object of the invention to provide a kind of para-immunity electrochemical sensor detecting A beta oligomers, it is achieved the highly sensitive and high selective enumeration method of marker molecules A beta oligomers in AD body fluid. Another object is in that to provide its preparation method.
For realizing the object of the invention, the present invention is using aptamer as recognition group, thionine is as labelling groups, and golden nanometer particle amplifies group as signal, and the aptamer of golden nanometer particle and sulfydryl modification and thionine are cooperatively formed stable nanometer conjugate; The electrode surface simultaneously modified at carboxylated graphene oxide carries out antibody to be fixed, identify the golden nanometer particle conjugate that after A beta oligomers, bind nucleic acid aptamers is modified further, form sandwich type para-immunity sensor, realize the mensuration to A beta oligomers by the electrochemical signals of thionine.
The described para-immunity electrochemical sensor for detecting A beta oligomers is prepared from especially by following method:
1, aptamer
Selection and A beta oligomers have the aptamer of strong binding ability, and its sequence is: GCCTGTGTTGGGGCGGGTGCG, and carries out the modification of 5'SHC6.
2, AuNPs solution is prepared: citric acid solution is joined the HAuCl boiled4��4H2In O solution, stirring reaction, then it is cooled to room temperature and obtains AuNPs solution, be placed in refrigerator standby. Take the AuNPs prepared and the mixing of thionine solution, stir under room temperature, be centrifuged, take off layer, dissolve with culture fluid; It is eventually adding above-mentioned aptamer, reacts under stirring at room temperature, by centrifugation, washing, dissolve, namely obtain aptamer and AuNPs conjugate that thionine is modified jointly.
Citric acid and HAuCl4��4H2The mol ratio of O is 2:1; Volume/the mol ratio of AuNPs and thionine solution is 7:0.22mL/mM;Aptamer concentration in the solution is 0.9 ��M.
3, the synthesis of graphene oxide and carboxyl-functional
Adopt the Hummers method improved to prepare graphene oxide: (1) low-temp reaction stage, in 0-4 DEG C of ice bath, add concentrated sulphuric acid, add graphite powder and sodium nitrate, take out from ice bath after stirring reaction, under 10-20 DEG C of condition, be slowly added to potassium permanganate continuously stirred reaction; (2) in, the temperature stage of reaction, moves on in the water-bath of 37-40 DEG C by above-mentioned solution, adds deionized water, continues stirring, and reaction is cooled to room temperature after stopping; (3) mixed solution is moved in about 90-95 DEG C of water-bath, continues reaction when stirring, be subsequently added deionized water and terminate reaction; Add H2O2, react under stirring condition; It is eventually adding HCl, after scrubbed, vacuum drying, namely obtains graphene oxide solid.
The mol ratio of concentrated sulphuric acid, graphite powder, sodium nitrate and potassium permanganate is 1:0.20:0.014:0.045.
4, take the above-mentioned graphene oxide solid prepared to be dissolved in ultra-pure water and be configured to solution, it is added thereto to NaOH and chloroacetic acid subsequently, react under Ultrasonic Conditions, the hydroxyl and the epoxy radicals that make surface of graphene oxide become carboxyl, through filtration, washing, vacuum drying, weighing, re-dissolved, obtain carboxylated graphene oxide solution.
The carboxylated preferred 0.5mg/mL of graphene oxide solution concentration.
5, the constructing of electrochemical sensor: carboxylated graphene oxide is modified on glass-carbon electrode by the mode of drop coating by (1), dries, as host material under infrared lamp; (2) this electrode is activated in amido link activating reagent NHS/EDC, and with deionized water wash, (NHS/EDC mol ratio is 1:15, and concentration is 2mM and 30mM respectively); (3) then this electrode is immersed in antibody-solutions and reacts, connect antibody, wash followed by PBS buffer solution, remove the material of surface physics absorption; (4) process by bovine serum albumin lock solution, eliminate the impact of non-specific binding and close remaining avtive spot; (5) electrode is immersed in the A beta oligomers solution of variable concentrations, by the specific binding identification A beta oligomers of antibody with antigen; (6) this electrode is put in the solution of the AuNPs conjugate containing above-mentioned preparation and cultivate, form the para-immunity sensor of " sandwich " type.
The optium concentration of described antibody-solutions is 10 �� g/mL. NHS is N-hydroxy-succinamide, and EDC is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride.
Compared with prior art, the present invention has following beneficial effect:
(1) para-immunity sensor of the present invention has good selectivity, A beta oligomers can be selectively measured under the forms such as A beta monomers and corpus fibrosum exist, specificity is good, selectivity is high, and this is the high binding ability decision of the specific recognition effect by antibody and aptamer.
(2) para-immunity sensor construction of the present invention substitutes two anti-molecules in traditional immunization sensor with aptamer, thus avoiding the enzyme label antibody reagent using two anti-molecules expensive.
(3) present invention utilizes the signal amplification of nano material, improves sensitivity, and detection is limited to 45nM, can be used for the mensuration of A beta oligomers in AD cerebrospinal fluid, promotes the early diagnosis of AD. Compared with traditional immunization sensor, have and prepare feature that is simple and that avoid using enzyme label antibody reagent, there is clinical value.
Accompanying drawing explanation
Fig. 1 is principle of the invention figure.
Fig. 2 is the ac impedance spectroscopy in para-immunity sensor preparation process of the present invention, in figure, a is naked glass-carbon electrode, b is the electrode after graphene oxide is modified, c is the electrode after amido link activating reagent is modified, d is the electrode after antibody modification, and e is the electrode after identifying A beta oligomers, and f is the modified electrode after connecting AuNPs conjugation group further.
Fig. 3 is the para-immunity sensor of the present invention response curve to variable concentrations A beta oligomers.
Fig. 4 is the selectivity block diagram of para-immunity sensor of the present invention, and in figure, a-f represents A �� respectively1-42Corpus fibrosum, A ��1-42Monomer, A ��1-40Corpus fibrosum, A ��1-40Monomer, A ��1-42/A��1-40Oligomer.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme being described further, following example will assist in those skilled in the art and are further appreciated by the present invention, but do not limit the present invention in any form.
The preparation method that embodiment 1 detects the para-immunity biosensor of A beta oligomers
(1) aptamer and the thionine conjugated on AuNPs
Selection and A beta oligomers have the aptamer of strong binding ability, and its sequence is: GCCTGTGTTGGGGCGGGTGCG, and carries out the modification of 5'SHC6.
Prepare the AuNPs solution that diameter is 20nm: under rapid stirring the sodium citrate solution of 3.75mL1% (mass percent) is joined 250mL0.01% (mass percent) HAuCl of boiling4��4H2In O solution, it was observed that the color of solution can be become darkviolet by light yellow within 2min. Keep mixed solution constantly boiling and stir 15min, being cooled to room temperature, AuNPs solution can be obtained, be placed in the refrigerator of 4 DEG C standby. Then, the thionine solution of 7mLAuNPs and the 2mL1mM prepared is mixed, stir 24h, centrifugal (8000r/min, 15min) under room temperature, take off layer, then be dissolved to 5mL with culture fluid; It is eventually adding the aptamer of 45 �� L100 ��M, the reaction 5h when stirring for 25 DEG C, centrifugal (8000r/min) 15min, washing, dissolving, namely obtain aptamer and AuNPs conjugate that thionine is modified jointly.
(2) synthesis of graphene oxide and carboxyl-functional
The Hummers method improved is adopted to prepare graphene oxide: to be broadly divided into three processes, (1) the low-temp reaction stage, in the beaker lower than 4 DEG C, add 46mL concentrated sulphuric acid, add 2g graphite powder and 1g sodium nitrate, take out from ice bath after stirring 1h, maintain temperature and be not less than 10 DEG C, be slowly added to 6g potassium permanganate continuously stirred 1h; (2) in, the temperature stage of reaction, moves on in the water-bath of 37 DEG C by above-mentioned solution, adds the deionized water of 160mL, continues stirring, and reaction is cooled to room temperature after stopping; (3) mixed solution is moved in about 95 DEG C of water-baths, the reaction 30min when stirring, it is subsequently added 90mL deionized water and terminates reaction; Add the H of 30mL30% (mass percent)2O2, under stirring condition, react 15min; It is eventually adding 180mL1% (mass percent) HCl, after washing, vacuum drying, namely obtains graphene oxide solid.
Take the above-mentioned graphene oxide solid prepared of 0.1mg to be dissolved in 100mL ultra-pure water and be configured to 1mg/mL solution, it is added thereto to 5.0gNaOH and 4.0g chloroacetic acid subsequently, ultrasonic response 4h, the hydroxyl and the epoxy radicals that make surface of graphene oxide become carboxyl, filtration, washing, vacuum drying, weighing, re-dissolved, namely obtain the carboxylated graphene oxide solution of 0.5mg/mL.
(3) the constructing of electrochemical sensor
A beta oligomers sequence if not otherwise indicated, all uses 1-42 aminoacid sequence, and selectivity experiment is simultaneously used 1-42 and 1-40 aminoacid sequence.
Electrochemical sensor to construct process specific as follows: the graphene oxide that 10 is carboxylated for �� L is modified on glass-carbon electrode by the mode of drop coating by (1), dries, as host material under infrared lamp; (2) activation 1h in amido link activating reagent NHS/EDC (NHS/EDC mol ratio is 1:15, and concentration is 2mM and 30mM respectively), and wash by secondary deionized water; (3) this electrode is immersed in the solution of 10mg/mL antibody, reacts 4h, connect antibody, wash followed by 0.1M phosphate (PBS) buffer solution (pH=7.4), remove the material of surface physics absorption; (4) process by the bovine serum albumin lock solution of 1% (mass percent), to eliminate the impact of non-specific binding and to close remaining avtive spot; (5) electrode is immersed in the A beta oligomers solution of variable concentrations, by the specific binding identification A beta oligomers of antibody with antigen; (6) this electrode is put into cultivation 3h in the solution containing conjugate, forms the para-immunity sensor of " sandwich " type.
The application examples 1 mensuration to A beta oligomers
This type of immunosensor is transferred in 0.1MPBS buffer solution (pH=7.4), utilizes the electrochemical signals of Differential Pulse Voltammetry thionine. Measure and adopt three-electrode system: the glass-carbon electrode of modification is working electrode, and Ag/AgCl electrode is reference electrode, and platinum filament is to electrode. Further optimization experiment condition, including the concentration (10 �� g/mL) of antibody, sensor and A beta oligomers incubation time (60min) and with the incubation time (3h) of AuNPs conjugate. Under the experiment condition optimized, set up working curve according to the peak current of thionine and A beta oligomers concentration. This peak current is directly proportional in the scope of 0.2 ��M to 20 ��M to the concentration of A beta oligomers, and its linear equation is Ipa(�� A)=4.738+0.059C (��M), correlation coefficient is 0.990, and detection is limited to 45nM.
Application examples 2 performance of electrochemical sensors of the present invention is investigated
In order to study the stability of inventive sensor, it is respectively adopted 7 electrodes, makes to construct in the same way electrochemical sensor simultaneously, measure the A beta oligomers with concentration. Obtaining 7 measurement results, its relative standard deviation is 3.28%. The repeatability that this sensor has had is described. Additionally, also the stability of electrochemical sensor has been investigated. By this sensor after 4 DEG C store 48h, measure obtain peak current is initial current 89.58%, this further illustrates the stability that the biosensor constructed has had.
When A beta body and A beta monomers exist, peak current is less compared with A beta oligomers, and this is specific binding mainly due to antibody and A beta oligomers, and not binding fiber body and monomer, so the change of electrochemical signals will not be produced. Thus describing this type of immunosensor have significantly high selectivity.
The mensuration of A beta oligomers in application examples 3 cerebrospinal fluid
Configure according to the method for synthetic cerebrospinal fluid: 150mMNaCl, 3.0mMKCl, 1.4mMCaCl2��2H2O��0.8mMMgCl2��6H2O, 1mM phosphate forms. Adopt the method that mark-on reclaims, add the standard sample of variable concentrations, be analyzed. Obtaining recovery of standard addition is 90.00%-108.45%, and this result substantially conforms to requirement. Thus illustrate that the method has potential using value for the mensuration of A beta oligomers in actual sample.
The recovery testu of A beta oligomers in table 1 cerebrospinal fluid
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Claims (6)
1. it is used for the preparation method detecting the para-immunity electrochemical sensor of amyloid-beta (A ��) oligomer, it is characterised in that realize by the following method:
(1), aptamer is selected
Selection and A beta oligomers have the aptamer of strong binding ability, and its sequence is: GCCTGTGTTGGGGCGGGTGCG, and carries out the modification of 5'SHC6;
(2) AuNPs solution, is prepared: citric acid solution is joined the HAuCl boiled4��4H2In O solution, stirring reaction, then it is cooled to room temperature, obtains AuNPs solution, be placed in refrigerator standby; Take the AuNPs prepared and the mixing of thionine solution, stir under room temperature, centrifugal, take off layer, dissolve with culture fluid; It is eventually adding above-mentioned aptamer, reacts under stirring at room temperature, by centrifugation, washing, dissolve, namely obtain aptamer and AuNPs conjugate that thionine is modified jointly;
(3), the synthesis of graphene oxide and carboxyl-functional
Adopt the Hummers method improved to prepare graphene oxide: A, low-temp reaction stage, in 0-4 DEG C of ice bath, add concentrated sulphuric acid, add graphite powder and sodium nitrate, take out from ice bath after stirring reaction, under 10-20 DEG C of condition, be slowly added to potassium permanganate continuously stirred reaction; B, the middle temperature stage of reaction, move on in the water-bath of 37-40 DEG C by above-mentioned solution, adds deionized water, continues stirring, and reaction is cooled to room temperature after stopping; C, mixed solution is moved in about 90-95 DEG C of water-bath, continues reaction when stirring, be subsequently added deionized water and terminate reaction; Add H2O2, react under stirring condition; It is eventually adding HCl, after scrubbed, vacuum drying, namely obtains graphene oxide solid;
(4), take the above-mentioned graphene oxide solid prepared to be dissolved in ultra-pure water and be configured to solution, it is added thereto to NaOH and chloroacetic acid subsequently, react under Ultrasonic Conditions, the hydroxyl and the epoxy radicals that make surface of graphene oxide become carboxyl, through filtration, washing, vacuum drying, weighing, re-dissolved, obtain carboxylated graphene oxide solution;
(5), the constructing of electrochemical sensor: A, by the mode of drop coating, carboxylated graphene oxide is modified on glass-carbon electrode, dry under infrared lamp, as host material; B, this electrode is activated in amido link activating reagent NHS/EDC, and with deionized water wash; C, then this electrode is immersed in antibody-solutions react, connect antibody, followed by PBS buffer solution wash, remove surface physics absorption material; D, process by bovine serum albumin lock solution, eliminate the impact of non-specific binding and close remaining avtive spot; E, electrode is immersed in the A beta oligomers solution of variable concentrations, by the specific binding identification A beta oligomers of antibody with antigen; F, this electrode is put in the solution of the AuNPs conjugate containing above-mentioned preparation cultivate, formed " sandwich " type para-immunity sensor.
2. the preparation method of the para-immunity electrochemical sensor for detecting A beta oligomers as claimed in claim 1, it is characterised in that in step (2), citric acid and HAuCl4��4H2The mol ratio of O is 2:1; Volume/the mol ratio of AuNPs and thionine solution is 7:0.22mL/mM; Aptamer concentration in the solution is 0.9 ��M.
3. the preparation method of the para-immunity electrochemical sensor for detecting A beta oligomers as claimed in claim 1, it is characterised in that in step (3), the mol ratio of concentrated sulphuric acid, graphite powder, sodium nitrate and potassium permanganate is 1:0.20:0.014:0.045.
4. the preparation method of the para-immunity electrochemical sensor for detecting A beta oligomers as claimed in claim 1, it is characterised in that in step (5), NHS/EDC mol ratio is 1:15.
5. the preparation method of the para-immunity electrochemical sensor for detecting A beta oligomers as claimed in claim 1, it is characterised in that in step (5), antibody-solutions concentration is 10 �� g/mL;Graphene oxide solution concentration carboxylated in step (4) is 0.5mg/mL.
6. for detecting the para-immunity electrochemical sensor of A beta oligomers, it is characterised in that be prepared from by one of them described method of claim 1-5.
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