CN104777318A - C-reactive protein immune sensor and use method thereof - Google Patents

C-reactive protein immune sensor and use method thereof Download PDF

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CN104777318A
CN104777318A CN201510217349.5A CN201510217349A CN104777318A CN 104777318 A CN104777318 A CN 104777318A CN 201510217349 A CN201510217349 A CN 201510217349A CN 104777318 A CN104777318 A CN 104777318A
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reactive protein
crp
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戴坤
郑丽
许娜艳
杨志鹏
郭涛
杨云慧
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Yunnan Normal University
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Abstract

The invention discloses a C-reactive protein immune sensor and a use method thereof and belongs to immunodetection technologies. Hollow silver and platinum nanoparticles are used as markers to mark C-reactive protein antibodies, amination graphene serves as fixing substrates to fix the C-reactive protein antibodies on electrodes, and then the sandwich type immune sensor is manufactured. Hollow silver and platinum nano material mimic enzymes catalyze reduction of H2O2, and then a current response signal is generated. The current response signal is in direct proportion to bred CRP antigen concentration, and through the proportional relation, quantitative determination of the CRP is achieved. The sensor serves as working electrodes in a 0.009-0.011 mol/LPBS buffered solution, and 99-101MuLmol/L H2O2 is added under -0.19-0.21V constant potentials for chronoamperometry determination. The C-reactive protein immune sensor is high in detection flexibility, accurate and quick, good in stability and easy to manufacture.

Description

A kind of C reactive protein immunosensor and using method thereof
Technical field
The invention belongs to immunoassay technology, be specifically related to the C reactive protein immunosensor based on amination Graphene and silver-colored platinum nano material and using method thereof.
Background technology
Since entering 21 century, angiocardiopathy has replaced infectious disease in the past, becomes the first killer of human health, and occupy that human death leads 1/3 more than, and coronary heart disease occupies wherein more than 50%.For the pathogenesis of coronary heart disease, the coefficient result of multiple paathogenic factor is thought in research.Atherosclerotic is the pathogenetic pathologic basis of coronary disease, and in atherosclerotic generation, evolution, inflammatory reaction is carried throughout, and inflammatory factor act as important role.C reactive protein (C-reactive protein, CRP) is the coronary heart disease related inflammatory factor of latest find, plays an important role in atherosclerotic generation evolution.Mankind's c reactive protein (CRP) is the main acute phase albumen that plasma concentration fast, sharply raises when infection and tissue damage.The research of nearly ten years discloses, and CRP participates in directly and result in inflammation and cause the angiocardiopathies such as atherosclerotic, and is the indication factor and risk factor that angiocardiopathy is the strongest.At present, CRP is widely used in clinical early diagnosis and the antidiastole of angiocardiopathy, in addition the mensuration of CRP also has positive effect for the treatment of tumour and prognosis, therefore develop sensitive, CRP detection method is significant clinically accurately and rapidly.
In current serum, the detection of CRP is mainly based on immunology principle; Clinical conventional detection method has immune turbidimetry and turbidimetry, and the method such as Enzyme-linked Immunosorbent Assay, fluorescence, radiation, chemiluminescence.Because said method is general responsive not, length consuming time, easily produce false negative result or cost high, benefit is low, and complex operation, instrument are huge, the field screening that is not suitable for the potential crowd of coronary heart disease.Developing CRP field fast detection method that is easy, inexpensive, accurate and that be easy to promote is solve extensive sample examination, realizes one of key of coronary heart disease early diagnosis, significant.It is low that immunosensor has cost, the strong and high sensitivity of dirigibility, fast and the advantage such as portable, most with prospects, applies more and more extensive in clinical class sample detection.The sandwich method type Enzyme linked immunosensor of current report is commonly used horseradish peroxidase and to be made marks thing, and there is easy in inactivation, experiment condition is harsh, not easily preserves, and preparation difficulty, high in cost of production shortcoming, cannot meet the needs of field quick detection.The objects such as the key solved the problem is to seek novel antibody labeling material, realizes stability high, easily obtains, and sensitivity for analysis is high.
The carbon atomic layer that Graphene is arranged by one deck two dimensional surface forms, and gives the electric conductivity that Graphene is good.The concept of nano composite material is proposed in early 1980s by Rey and Komarneni.There is synergy between two kinds of metals due to formation composite nanometer particle, composite nanometer particle shows various function and special performance.Bimetal nano alloy particle can be regulated the physics and chemistry character of compounding ingredients by adding of the second metal, can be widely used in the various fields such as catalysis, optoelectronics, information storage, biomarker.2013, Wu etc. prepared the Pt-Fe of dumbbell shape 3o 4nano material, with its mark squamous cell anticancrin, utilizes Pt-Fe 3o 4the Mimetic Peroxidase of nano material is active, catalysis H 2o 2, prepared a kind of overdelicate electrochemical immunosensor and detected squamous cell carcinoma.Niu etc. have prepared alabastrine Pt/Pd bimetallic alloy, modify serigraphy gold electrode, prepare hydrogen peroxide sensor.Experiment shows Pt/Pd bimetallic alloy to the catalytic capability of hydrogen peroxide far away higher than independent Pt and Pd.2014, Chang etc. found that Pt/Ir composite nano materials also has Mimetic Peroxidase activity, have prepared hydrogen peroxide sensor based on Pt/Ir composite nano materials.Wu Xiao spring project team system the is standby Ag/M of different-shape and structure (M=Au, Pd, Pt) alloy nano particle, these nano particles have the Mimetic Peroxidase catalytic activity of similar HRP character.So far less than the report of the C reactive protein immunosensor based on amination Graphene and silver-colored platinum nano material.
Summary of the invention
The object of the present invention is to provide a kind of C reactive protein immunosensor and using method thereof, realize the quantitative detection of CRP, thus provide reliable technology platform for the early detection of the diseases such as coronary heart disease, antidiastole and curative effect monitoring.
The feature of C reactive protein immunosensor of the present invention obtains as follows: using hollow silver Pt nanoparticle as label mark C reactive protein antibody, for fixing matrix, C reactive protein antibody is fixed on electrode with amination Graphene, makes the immunosensor of sandwich type.Further optimization feature is: in hollow silver Pt nanoparticle, the massfraction of silver-colored platinum is than being 5:1.9 ~ 2.1, and the mass concentration that antibody is fixed is 9 ~ 11 μ g/mL.The synthetic method of hollow silver platinum nano material is prior art.
The principle of work of C reactive protein immunosensor of the present invention: the catalytic activity utilizing hollow silver platinum nano material, detects c reactive protein by sandwich method.Specifically, be exactly hollow silver platinum nano material mimetic enzyme catalysis H 2o 2reduction, generation current response signal, this current responsing signal is directly proportional to the CRP antigen concentration of cultivation, by this proportionate relationship, realizes the quantitative detection of CRP.
The using method of sensor of the present invention: in 0.009 ~ 0.011mol/LPBS buffer solution, to work electrode with this sensor, adds the H of 99 ~ 101 μ L 1mol/L under-0.19 ~ 0.21V constant potential 2o 2carry out the mensuration of chrono-amperometric, the relation that current value when reaching stable according to this sensor timing response is directly proportional to CRP concentration in sample, realize the quantitative measurement to CRP.Under optimum experiment condition, the sensing range of this sensor is 0.5mg/L-140mg/L, and linearly dependent coefficient is 0.9934, and Monitoring lower-cut is 0.17mg/L.
Beneficial effect of the present invention: detection sensitive highly sensitive, accurately fast, good stability, sensor is easily prepared.
Accompanying drawing explanation
Fig. 1 is embodiment sensor preparation and determination methods schematic flow sheet.
Fig. 2 is the transmission electron microscope picture of the silver-colored platinum nano material of the different silver-colored platinum massfraction ratio of embodiment.
The response current that Fig. 3 shows the nanometer material modified electrode of the different silver-colored platinum massfraction ratio of embodiment compares.
Fig. 4 shows embodiment different modifying electrode pair H 2o 2the catalytic effect of reduction current.
Fig. 5 is the cyclic voltammetry curve of embodiment sensor in different solutions.
Fig. 6 shows the impact of embodiment sweep velocity on electrode response electric current.
Fig. 7 is that embodiment different modifying electrode is at 5mmol/L K 3fe (CN) 4/ K 4fe (CN) 6aC impedance figure in solution.
Fig. 8 shows the different sessile antibody concentration of embodiment to the impact of sensor response current.
Fig. 9 shows the impact that Embodiment C RP antigen cultivates the time.
Figure 10 shows embodiment labelled antibody and cultivates the time to the impact of sensor response effect electric current.
Figure 11 is the working curve that embodiment sensor responds the CRP of variable concentrations.
Figure 12 shows the selectivity of embodiment immunosensor.
Embodiment
See following experiment:
1, instrument and reagent
(antibody titer is 1.6 × 10 to C reactive protein monoclonal antibody (anti-CRP) -10moL), antigen (CRP) is purchased from Shanghai Linc-Bio Science Co., Ltd., silver nitrate (AgNO 3) be purchased from the Shanghai rising sun and reach Fine Chemical Works, chloroplatinic acid (H 2ptCl 6) be purchased from Kunming Boren Precious Metals Co., Ltd., polyvinylpyrrolidone (PVP, K-30) Chemical Reagent Co., Ltd., Sinopharm Group is purchased from, sodium sulphide (Na2S) is purchased from Xilong Chemical Co., Ltd, sodium chloride (NaCl) is purchased from Guangdong brilliance chemical reagent company limited, ethylene glycol, absolute ethyl alcohol are all purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd., shitosan (CHIT), glutaraldehyde (GA), bovine serum albumin (BSA), phosphate buffer solution (PBS, pH 7.4) are all purchased from Sigma Co., USA.Amination Graphene (the NH that tetramino is modified 2-rGO) be purchased from Nanjing Xian Feng Nono-material Science & Technology Ltd., eluent buffer solution used in experiment is: 20mM Na 3pO 4.12H 2o (0.076g), 10%Sucrose (1g), 5%BSA (0.5g), 0.25%Tween20 (22.7 μ L), be settled to 10mL.In experiment agents useful for same be analyze pure, water used is redistilled water.
CHI660D electrochemical workstation is Chinese Shanghai Chen Hua instrument company product; Experiment uses three-electrode system, and saturated calomel electrode is contrast electrode, and immunosensor is working electrode, and platinum electrode is to electrode; JEM2100 transmission electron microscope is Jeol Ltd.'s product; TGL16 hydro-extractor is Changsha Xiang Zhi hydro-extractor Instrument Ltd. product; CS501 ultra thermostat is Chongqing experimental facilities factory product.
2, the synthesis of hollow silver platinum nano material
By prior art [such as document: WU Rong, HU Guang-Qi, HU Jian-Qiang.J.LightScattering, 2012,24 (3): 316-320 Wu's suedes, Hu Guanqi, Hu Jianqiang. light scattering journal, 2012,24 (3): 316-320] hollow silver platinum nano material has been synthesized.First add 60mL (0.01M) sodium sulphide, the ethylene glycol (EG) of 3mL and 0.167g polyvinylpyrrolidone in round-bottomed flask, and heat five minutes at 145 DEG C, magnetic agitation.Then the ethylene glycol solution of the silver nitrate of (0.1M) of 3mL is instilled, at 160 DEG C, react 1.5h, to be cooled after uniform temperature, appropriate chloroplatinic acid liquid is slowly added drop-wise in flask, the reaction regular hour, obtains hollow Ag/Pt nano material.Centrifugal 20min under 8000rpm.Taking precipitate adds saturated NaCl solution and dissolves, and reaction generates AgCl with it, then under 8000rpm centrifugal 20min.Taking precipitate respectively washs 3 times with ethanol, water respectively.Finally, by sediment ultrasonic and distributed and saved in redistilled water.
3, hollow silver platinum nano material mark C reactive protein antibody
Get the hollow silver Pt nanoparticle that 1mL is concentrated, use 200mM Na 2cO 3pH is regulated to be 9.Add 1.2mg/mL antibody 10 μ L, add 2 μ L at every turn, divide and add for five times, every minor tick 3min.2h is cultivated in jolting.Add BSA (1%) about 25 μ L to close, react half an hour.Finally, low-temperature centrifugation 15min, abandons supernatant liquor, and lower sediment material PBS washs 2 times, is dispersed in 1mL eluent buffer 4 DEG C degree and preserves after washing.
4, the preparation of C reactive protein immunosensor
By glass-carbon electrode with after abrasive paper for metallograph polishing, use the Al of three kinds of different-grain diameters successively again 2o 3powder is polished, and then uses HNO successively 3(1:1), absolute ethyl alcohol and distilled water ultrasonic cleaning 10min.The amination Graphene getting 2mg/mL mixes with the ratio of the shitosan 1:1 by volume of 0.5%, and get 10 μ L and be added drop-wise to electrode surface, 4 DEG C are spent the night.Then on electrode, drip the glutaraldehyde (GA) of 10 μ L 0.5mg/mL after 4 DEG C of reaction 0.5h, electrode PBS solution rinses 3 times, naturally dries.Finally, electrode drips the CRP antibody of 10 μ L 10 μ g/mL, spends the night under 4 DEG C of environment.The glass-carbon electrode PBS solution of having modified is dripped and is washed three times, naturally dries the nonspecific binding site 1h on rear 1%BSA (bovine serum albumin) enclosed-electrode surface.Rinse well by PBS solution after taking-up, namely can be used for the mensuration of C reactive protein in testing.
5, detection method
The CRP antigen of variable concentrations is dripped on immunosensor, cultivates 50min in 37 DEG C of constant temperature, rinse with PBS.Then drip the CRP antibody of the hollow Ag/Pt mark of 10 μ L, constant temperature rinses with PBS after cultivating 45min.In 0.1mol/LPBS buffer solution, to work electrode with this sensor, under-0.2V constant potential, add the H of 100 μ L 1mol/L 2o 2carry out the mensuration of chrono-amperometric.According to this sensor timing is corresponding reach stable time the relation that is directly proportional to CRP concentration in sample of current value, realize the quantitative measurement to CRP.Fig. 1 is the sensor preparation and determination methods schematic flow sheet.
6, results and analysis
(1) material characterizes
Three kinds of hollow Ag/Pt nano materials have been synthesized in experiment, and the amount difference adding platinum precursor solution can obtain the hollow Ag/Pt nano material of different-shape.In Fig. 2, the left side, centre and the right adopt identical synthetic method, adds different platinum precursor solutions, the hollow silver platinum nano material electron microscopic observation of the difference silver platinum ratio of synthesis.The massfraction of the silver-colored platinum on the left side is than 3:2, and the massfraction of middle silver-colored platinum is than 4:2, and the massfraction of the silver-colored platinum on the right compares 5:2.Can find out: when the massfraction of silver-colored platinum is than 3:2, Ag/Pt appearance of nano material disunity, disperses uneven; When the massfraction of silver platinum is than 4:2, Ag/Pt nano material has a small amount of hollow cystic structures, but dispersed poor; When the massfraction of silver platinum is than 5:2, the pattern of hollow silver platinum nano material is best, and uniform particles, dispersiveness is also best, and its particle diameter is approximately 70nm.
(2) catalytic effect compares
A. different silver-colored platinum nano material is to H 2o 2catalytic performance compare
Investigate different silver-colored alloy platinum material to H 2o 2catalytic performance compare.Get after 10 μ L silver alloy platinum materials mix with 10 μ L chitosan solutions and modify on glass-carbon electrode, after 5 hours, glass-carbon electrode is immersed supporting electrolyte BS) in, add 100 μ L 1mol/L H 2o 2the mensuration of chrono-amperometric is carried out under-0.2V.A, b, c represent the nano material of three kinds of silver-colored platinum massfraction ratios of difference, measurement result as shown in Figure 3: curve a, b, c represent that the hollow silver platinum nano material of the massfraction of silver-colored platinum than 4:2,5:2 and 3:2 is to H respectively 2o 2the current-responsive of catalytic action, find out from figure, the massfraction of silver-colored platinum than the hollow silver platinum nano material of 4:2 to H 2o 2catalytic effect best, stronger current responsing signal can be produced.Although the massfraction of silver-colored platinum than the hollow silver platinum nano material of 5:2 to H 2o 2catalytic action do not have massfraction better than the hollow silver platinum nano material of 4:2, but consider the pattern of hollow silver platinum nano material and smoothness that is dispersed and curve, the massfraction that this experiment still have selected silver-colored platinum than the silver-colored alloy platinum material of 5:2 as marker material.
The synergy of B.Ag and Pt nano material
In order to investigate Ag and Pt nano material to H 2o 2the synergy of catalytic effect, fixes silver-colored platinum, silver, the alloy platinum material of equivalent under the same conditions respectively by GCE, it immersed respectively in supporting electrolyte (PBS), adds 100 μ L 1mol/L H 2o 2under-0.2V, carry out the mensuration of chrono-amperometric, the results are shown in Figure 4: curve a, b, c represent different modifying electrode pair H respectively 2o 2the catalytic effect of reduction current, a is Ag/Pt NPs/GCE, b be Ag NPs/GCE, c is Pt NPs/GCE.As can be seen from Figure 4, the catalytic capability of the electrode pair hydrogen peroxide that Ag/Pt is nanometer-material-modified is the strongest, illustrates that silver-colored platinum has synergic catalytic effect.
C. the cycle voltammetry behavior of sensor
Fig. 5 is the immunosensor of cultivating equal in quality density antigen with or without H 2o 2buffer solution in cycle voltammetry behavior.Curve a is containing 1mol/L H 2o 2phosphate buffered solution in the background current observed; The background current that curve b observes in for phosphate buffered solution.Visible, when adding H 2o 2after, electric current sharply increases, and demonstrates silver-colored platinum nano material to H 2o 2reduction there is catalytic action.
D. test and also investigated the impact of sweep velocity on peak current, when sweep velocity changes from 20-100mV/s, sensor is containing 1mol/L H 2o 2pH is that cycle voltammetry behavior in the buffer solution of 7.4 is as Fig. 6.As can be seen from Figure 6, peak current increases along with the increase of sweep velocity.Simultaneously in order to investigate the characteristic of electric current, by square root mapping (see built-in figure) of peak current to scanning speed, as can be seen from built-in figure, peak current is directly proportional to the square root of sweep velocity, illustrates that this electric current is that diffusion controls.The cycle voltammetry behavior of to be sweep velocity from inside to outside respectively in Fig. 6 be 20,40,60,80,100mV/s, built-in figure is the square root v of redox current and sweep velocity 1/2linearity curve, a: reduction current; B: oxidation current.
E. the AC impedance behavior of different modifying electrode interface
Electrochemical AC impedance EIS is the common method characterizing electrode modification process.Fig. 7 is that different modifying electrode is at 5mmol/L K 3fe (CN) 4/ K 4fe (CN) 6aC impedance figure in solution.Curve a is the impedance curve of naked GCE, approximate straight line, shows that electron transmission only controls by diffusion.Curve b is the impedance curve of the glass-carbon electrode by chitosan-modified amination Graphene, and due to the resistance to mass tranfer of shitosan, resistance value increases to some extent (Ret=250 Ω).Curve c is for securing the impedance curve of the glass-carbon electrode of 10 μ L 10 μ g/mLCRP antibody (anti-CRP), and impedance obviously increases (Ret=490 Ω), and this is because antibody hinders the transmission of electronics at electrode surface.Curve d is the electrode after closing with bovine serum albumin (BSA), the further AC impedance curve cultivated after 10 μ L 30ng/mLCRP antigens, half circular diameter enlarges markedly (Ret=1000 Ω), and this is because the covering of BSA also has the specific binding of antibody and antigen all to hinder the transmission of electronics.Curve e has cultivated the electrode AC impedance figure after silver-colored platinum nano material labelled antibody (labeled anti-CRP), and resistance value increases further (Ret=1780 Ω), and this illustrates that electrode modification film strengthens electron impeding effect.
F. the optimization of experiment condition
A. different sessile antibody mass concentration is on the impact of sensor response current
Fig. 8 illustrates the impact of different antibodies concentration on sensor response current.When other conditions are constant, change the concentration of modified electrode sessile antibody solution used, respectively do a working curve (a:10 μ g/mL, b:20 μ g/mL, c:50 μ g/mL, d:100 μ g/mL).As can be seen from Figure, along with the increase of antibody mass concentration, the sensitivity of electrode response reduces.When antibody mass concentration is 10 μ g/mL, electrode response is the sensitiveest, therefore, determines that the best in quality concentration that antibody is fixing is in an experiment 10 μ g/mL.
B. antigen cultivates the time to the impact of sensor response effect electric current
Under the condition of 37 DEG C, adopt different cultivation time (10min to 70min) cultivate the antigen of same concentrations (30mg/L) and adopt chronoamperometry to measure the response current of immunosensor respectively the immunosensor being modified with antibody, result is as Fig. 9.As seen from the figure, along with the increase of time, electric current first increases rear reduction.When the cultivation time is 50min, current responsing signal is maximum, therefore selects 50min to be that best antigen cultivates the time.
C. labelled antibody cultivates the time to the impact of sensor response effect electric current
Another factor affecting immunosensor is the cultivation time of labelled antibody.Under the condition of 37 DEG C, adopted by the immunosensor being modified with antibody same antigen to cultivate the antigen (30mg/L) of time cultivation same concentrations, change labelled antibody and cultivate the time, in 10min to 60min scope, investigated labelled antibody and cultivated the time to the impact of sensor, result as shown in Figure 10.As we can see from the figure, during 40min to 50min, current-responsive is maximum and occur platform, so experimental selection 45min is the optimum mark antibody development time.
(3) calibration curve of sensor and detection limit
Under optimum experimental condition, obtain the working curve (Figure 11) that sensor responds the CRP of variable concentrations.As can be seen from Figure 11, the response current of sensor has good linear relationship with CRP concentration within the scope of 0.5mg/L-140mg/L, and linear equation is: I (μ A)=1.14C (ng/mL)+7.41, correlation coefficient r 2=0.9934.According to 3 σ rules, the Monitoring lower-cut of CRP is 0.17mg/L.The CRP proposed in the guide issued with reference to a large amount of perspective study result according to disease prevention and control centers of the U.S. in 2004 and American Heart Association is determined at the point of contact of diagnosis of coronary heart disease and future event early warning, i.e. CRP concentration < 1mg/L, relative risk level is low, CRP concentration is at 1-3mg/L, relative risk level is moderate, > 3mg/L is height, > 10mg/L is high risk, the incidence of disease of cardiovascular event in following 6 months of patient that CRP concentration obviously raises, mortality ratio significantly increases, the possibility developed complications is larger.Linear sensor wider range of the present invention, is applicable to the detection that coronary heart disease relative risk level is CRP concentration in the blood samples of patients of minuent, moderate, height and high degree.
(4) selectivity of sensor
In order to investigate the selectivity of sensor, under the optimal conditions of selected property, have selected bovine serum albumin (BSA, 20ng/mL), glutamic acid (Glu, 20ng/mL), glycocoll (20ng/mL, Gly), 1 human chorionic's glandular hormone (50MIU/mL, HCG), the response current of the CRP antigen of totally 4 kinds of interfering materials and 10mg/L carries out contrast test, and result as shown in figure 12.Under the same conditions, the response current of bovine serum albumin, glutamic acid, glycocoll, human chorionic's glandular hormone is less than the electric current of CRP antigen.This shows that this sensor has good selectivity.
(5) mensuration of the recovery and the mensuration of authentic sample
In order to investigate the practicality of this sensor, adopt standard addition method, be standard [the Algarra M. of height by relative risk level in the human serum of dilution 10 times, Gomes D., Esteves da Silva J.C.G., ClinicaChimica Acta 2013,415 (1): 1 – 9] add 3 kinds of CRP antigens without mass concentration, carry out the mensuration of the CRP recovery by this experimental technique, 3 times are detected average recovery rate is 100.4% (table 1), and result is satisfactory.By the people's whole blood sample gathered, leave standstill a hours, then adopt centrifugal 10 minutes of low temperature low speed (2000rpm), get the serum that upper strata yellow liquid must be collected.The CRP in the human serum sample of 10 times is diluted, replicate determination 3 times by this determination of experimental method.Average CRP concentration when calculating undiluted after considering extension rate in blood serum sample is 58.5mg/L (table 2).Illustrate that this sensor can be used for the detection of CRP content in authentic sample.
The recovery of table 1. immunosensor
In the true blood serum sample of table 2., CRP measures
(6) reappearance of sensor and stability
Carry out 4 times to the CRP antigen of 60mg/L to measure, relative standard deviation is only 3.8%, illustrate that the mensuration of electrode pair CRP antigen has good reappearance only to reduce by 4.6% through the cyclic voltammetry scan sensor peak point current of continuous 100 circles, illustrate that this sensor has good stability.The used time is not kept in the buffer solution of 4 DEG C sensor.
The present embodiment utilizes amination Graphene fixation of C RP antibody and adopts silver-colored Pt nanoparticle labelled antibody to construct a kind of novel CRP immunosensor.This sensor combines the excellent electric conductivity of amination Graphene and the good biocompatibility of silver-colored Pt nanoparticle and catalytic action, pass through optimal conditions, significantly improve the sensitivity of CRP sensor, selectivity, sensor is made to have the wider range of linearity, lower Monitoring lower-cut, the detection for CRP provides a new way.

Claims (3)

1. a C reactive protein immunosensor, it is characterized in that obtaining as follows: using hollow silver Pt nanoparticle as label mark C reactive protein antibody, for fixing matrix, C reactive protein antibody is fixed on electrode with amination Graphene, makes the immunosensor of sandwich type.
2. C reactive protein immunosensor as claimed in claim 1, is characterized in that: in hollow silver Pt nanoparticle, the massfraction of silver-colored platinum is than being 5:1.9 ~ 2.1, and the mass concentration that antibody is fixed is 9 ~ 11 μ g/mL.
3. the using method of C reactive protein immunosensor as claimed in claim 1, it is characterized in that: in 0.009 ~ 0.011mol/LPBS buffer solution, to work electrode with this sensor, under-0.19 ~ 0.21V constant potential, add the H of 99 ~ 101 μ L 1mol/L 2o 2carry out the mensuration of chrono-amperometric, the relation that current value when reaching stable according to this sensor timing response is directly proportional to CRP concentration in sample, realize the quantitative measurement to CRP.
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