CN104777318B - C-reactive protein immune sensor and use method thereof - Google Patents
C-reactive protein immune sensor and use method thereof Download PDFInfo
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- CN104777318B CN104777318B CN201510217349.5A CN201510217349A CN104777318B CN 104777318 B CN104777318 B CN 104777318B CN 201510217349 A CN201510217349 A CN 201510217349A CN 104777318 B CN104777318 B CN 104777318B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
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
Technical field
The invention belongs to immunoassay technology, be specifically related to C-reaction based on amination Graphene and silver platinum nano material
Protein immunization sensor and using method thereof.
Background technology
Since entering 21 century, cardiovascular disease has replaced infectious disease in the past, becomes the first killer of human health,
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, grind
Study carefully and be considered the coefficient result of multiple paathogenic factor.Atherosclerosis is the pathogenetic pathologic basis of coronary disease, and dynamic
In the generation of pulse atherosclerosis, 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, at Atherosclerosis
The generation evolution changed plays an important role.Mankind's c reactive protein (CRP) is that plasma concentration is fast when infecting with tissue injury
Speed, the main acute phase albumen drastically raised.The research of last decade discloses, and CRP directly participates in result in inflammation and causing moving
The cardiovascular disease such as pulse atherosclerosis, and be the indication factor and risk factor that cardiovascular disease is the strongest.At present, CRP is by extensively
The general clinical early diagnosis being applied to cardiovascular disease and Differential Diagnosis, in addition the measuring for the treatment of tumor and prognosis of CRP
Also have positive effect, therefore develop sensitive, accurately and rapidly CRP detection method the most significant.
In serum, the detection of CRP is based primarily upon immunology principle at present；Clinical conventional detection method has immune turbidimetry
And turbidimetry, and the method such as Enzyme-linked Immunosorbent Assay, fluorescence, radiation, chemiluminescence.Owing to said method is universal sensitive not,
Time-consuming long, easily produce false negative result or cost is high, benefit is low, and complex operation, instrument is huge, be not suitable for coronary disease
The field screening of sick potential crowd.Developing CRP field fast detection method easy, inexpensive, accurate and easy to spread is to solve
Extensive sample examination, it is achieved one of key of coronary heart disease early diagnosis is significant.Immunosensor has low cost, spirit
Activity is strong and high sensitivity, quickly and the advantage such as portable, most with prospects, applies more and more wider in clinical class sample detection
General.At present the sandwich assay type Enzyme linked immunosensor of report is commonly used horseradish peroxidase and is made marks thing, there is easy in inactivation, experiment
Condition is harsh, is difficult to preserve, preparation difficulty, high in cost of production shortcoming, it is impossible to meet the needs of field quick detection.Solve above-mentioned asking
The purposes such as novel antibody labeling material is sought in it is critical only that of topic, it is achieved stability is high, easily prepares, and sensitivity for analysis is high.
The carbon atomic layer that Graphene is arranged by one layer of two dimensional surface forms, and gives the electric conductivity that Graphene is good.Nanometer is multiple
The concept of condensation material is proposed in early 1980s by Rey and Komarneni.Owing to forming the two of composite nanometer particle
Planting and there is synergism between metal, composite nanometer particle shows various function and special performance.Bimetal nano alloy
Particle can be regulated by add physics and the chemical property to compounding ingredients of the second metal so that it is can extensively apply
In various fields such as catalysis, optoelectronics, information storage, biomarkers.2013, Wu etc. was prepared for the Pt-of dumbbell shape
Fe3O4Nano material, with its labelling squamous cell anticancrin, utilizes Pt-Fe3O4The Mimetic Peroxidase of nano material is lived
Property, it is catalyzed H2O2, it is prepared for a kind of overdelicate electrochemical immunosensor detection squamous cell carcinoma.Niu etc. are prepared for flakes
Pt/Pd bimetallic alloy, modify silk screen printing gold electrode, prepare hydrogen peroxide sensor.Experiment shows that Pt/Pd bimetallic closes
Gold is significantly larger than single Pt and Pd to the catalytic capability of hydrogen peroxide.2014, Chang etc. found Pt/Ir composite Nano material
Material also has Mimetic Peroxidase activity, is prepared for hydrogen peroxide sensor based on Pt/Ir composite nano materials.Wu Xiao spring class
Topic group is prepared for Ag/M (M=Au, Pd, Pt) alloy nano particle of different-shape and structure, and these nanoparticles have similar
The Mimetic Peroxidase catalysis activity of HRP character.So far the C-being not based on amination Graphene and silver platinum nano material is anti-
Answer the report of protein immunization sensor.
Summary of the invention
It is an object of the invention to provide a kind of C reactive protein immunosensor and using method thereof, it is achieved CRP determines
Amount detection, thus early discovery, Differential Diagnosis and the curative effect monitoring for diseases such as coronary heart disease provides reliable technology platform.
The feature of C reactive protein immunosensor of the present invention is to prepare as follows: with hollow silver Pt nanoparticle
As label labelling C reactive protein antibody, C reactive protein antibody is fixed on amination Graphene for fixing substrate
On electrode, make the immunosensor of sandwich type.Further optimizing feature is: the quality of silver platinum in hollow silver Pt nanoparticle
Score ratio is 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
For prior art.
The operation principle of C reactive protein immunosensor of the present invention: utilize the catalysis activity of hollow silver platinum nano material,
C reactive protein is detected by sandwich assay.Specifically, it is simply that hollow silver platinum nano material mimetic enzyme catalysis H2O2Reduction, produce electricity
Stream response signal, this current responsing signal is directly proportional to the CRP antigen concentration of cultivation, by this proportionate relationship, it is achieved CRP's
Detection by quantitative.
The using method of inventive sensor: in 0.009～0.011mol/LPBS buffer solution, make of this sensor
Working electrode, adds the H of 99～101 μ L 1mol/L under-0.19～0.21V constant potential2O2Carry 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, it is achieved to CRP's
Quantitative determination.Under optimum experiment condition, the detection range of this sensor is 0.5mg/L-140mg/L, and linearly dependent coefficient is
0.9934, Monitoring lower-cut is 0.17mg/L.
Beneficial effects of the present invention: detection sensitive highly sensitive, the most quickly, good stability, sensor is easily prepared.
Accompanying drawing explanation
Fig. 1 is the preparation of embodiment sensor and testing process schematic diagram.
Fig. 2 is the transmission electron microscope picture of the silver-colored platinum nano material of embodiment difference silver platinum mass fraction ratio.
Fig. 3 shows that the response current of the nanometer material modified electrode of embodiment difference silver platinum mass fraction ratio compares.
Fig. 4 shows that embodiment different modifying electrode is to H2O2The catalytic effect of reduction current.
Fig. 5 is embodiment sensor cyclic voltammetry curve in different solutions.
Fig. 6 shows the impact on electrode response electric current of the embodiment scanning speed.
Fig. 7 is that embodiment different modifying electrode is at 5mmol/L K3Fe(CN)4/K4Fe(CN)6AC impedance in solution
Figure.
Fig. 8 shows the impact on sensor response current of the embodiment difference sessile antibody concentration.
Fig. 9 shows that embodiment CRP antigen cultivates the impact of time.
Figure 10 shows that embodiment traget antibody cultivates the time impact on sensor response effect electric current.
Figure 11 is the working curve that the CRP of variable concentrations is responded by embodiment sensor.
Figure 12 shows the selectivity of embodiment immunosensor.
Detailed description of the invention
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
In Shanghai Linc-Bio Science Co., Ltd., silver nitrate (AgNO3) be purchased from the Shanghai rising sun and reach Fine Chemical Works, chloroplatinic acid (H2PtCl6)
Being purchased from Kunming Boren Precious Metals Co., Ltd., polyvinylpyrrolidone (PVP, K-30) is purchased from traditional Chinese medicines group chemical reagent to be had
Limit company, sodium sulfide (Na2S) is purchased from Xilong Chemical Co., Ltd, and sodium chloride (NaCl) is purchased from Guangdong brilliance chemical reagent
Company limited, ethylene glycol, dehydrated alcohol be all purchased from Tianjin Fengchuan Chemical Reagent Science & Technology Co., Ltd., chitosan (CHIT), penta
Dialdehyde (GA), bovine serum albumin (BSA), phosphate buffer solution (PBS, pH 7.4) are all purchased from Sigma Co., USA.Tetramino is repaiied
Amination Graphene (the NH of decorations2-rGO) it is purchased from Nanjing Xian Feng Nono-material Science & Technology Ltd., eluent used in experiment
Buffer solution is: 20mM Na3PO4.12H2O (0.076g), 10%Sucrose (1g), 5%BSA (0.5g), 0.25%
Tween20 (22.7 μ L), is settled to 10mL.In experiment, agents useful for same is analytical pure, and water used is redistilled water.
CHI660D electrochemical workstation is Chinese Shanghai Chen Hua instrument company product；Experiment uses three-electrode system, saturated
Calomel electrode is reference electrode, and immunosensor is working electrode, and platinum electrode is to electrode；JEM2100 transmission electron microscope is Japan
Electronics Co., Ltd's product；TGL16 centrifuge is Changsha Xiang Zhi centrifuge Instrument Ltd. product；CS501 ultrathermostat
For 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.Light
Scattering, 2012,24 (3): 316-320 Wu's flosss, Hu Guanqi, Hu Jianqiang. light scattering journal, 2012,24 (3): 316-
320] hollow silver platinum nano material has been synthesized.First 60mL (0.01M) sodium sulfide, the ethylene glycol (EG) of 3mL and 0.167g are added poly-
Vinylpyrrolidone is in round-bottomed flask, and heats five minutes at 145 DEG C, magnetic agitation.Then by the nitre of (0.1M) of 3mL
The ethylene glycol solution of acid silver instills, and reacts 1.5h at 160 DEG C, to be cooled after uniform temperature, by appropriate chloroplatinic acid liquid slowly
It is added drop-wise in flask, reacts the regular hour, obtain hollow Ag/Pt nano material.Centrifugal 20min under 8000rpm.It is heavy to take
Shallow lake thing adds saturated NaCl solution and dissolves, and reacts generation AgCl, more centrifugal 20min under 8000rpm.Taking precipitate divides
Respectively do not wash 3 times with ethanol, water.Finally, by precipitate ultrasonic and distributed and saved in redistilled water.
3, hollow silver platinum nano material labelling C reactive protein antibody
Take the hollow silver Pt nanoparticle that 1mL concentrates, use 200mM Na2CO3Regulation pH is 9.Add 1.2mg/mL antibody 10
μ L, adds 2 μ L every time, adds in five times, every minor tick 3min.2h is cultivated in shaking.Add BSA (1%) about 25 μ L to close, reaction
Half an hour.Finally, low-temperature centrifugation 15min, abandon the supernatant, lower sediment material PBS washs 2 times, is dispersed in 1mL after washing
Eluent buffer 4 DEG C spends preservation.
4, the preparation of C reactive protein immunosensor
After glass-carbon electrode is polished with abrasive paper for metallograph, the most again with the Al of three kinds of different-grain diameters2O3Powder is polished, the most successively
Use HNO3(1:1), dehydrated alcohol and distilled water ultrasonic cleaning 10min.Take the amination Graphene of 2mg/mL and the shell of 0.5% gathers
The ratio mixing of sugar 1:1 by volume, takes 10 μ L and is added drop-wise to electrode surface, and 4 DEG C overnight.Then on electrode, drip 10 μ L
The glutaraldehyde (GA) of 0.5mg/mL is after 4 DEG C of reaction 0.5h, and electrode PBS solution is rinsed 3 times, naturally dries.Finally, at electrode
The CRP antibody of upper dropping 10 μ L 10 μ g/mL, under 4 DEG C of environment overnight.The glass-carbon electrode PBS solution modified is dripped and is washed three times,
Naturally with the nonspecific binding site 1h on 1%BSA (bovine serum albumin) enclosed-electrode surface after drying.Use PBS molten after taking-up
Liquid is rinsed well, i.e. can be used for the mensuration of C reactive protein in experiment.
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 dripping the CRP antibody of the hollow Ag/Pt labelling of 10 μ L, constant temperature rinses with PBS after cultivating 45min.Delay at 0.1mol/LPBS
In dissolved liquid, work electrode with this sensor, under-0.2V constant potential, add the H of 100 μ L 1mol/L2O2Carry out timing electricity
The mensuration of stream.The relation that current value when reaching stable accordingly according to this sensor timing is directly proportional to CRP concentration in sample, real
The now quantitative determination to CRP.Fig. 1 is the sensor preparation and testing process schematic diagram.
6, result and analysis
(1) material characterization
Experiment has synthesized three kinds of hollow Ag/Pt nano materials, adds the available not similar shape of amount difference of platinum precursor solution
The hollow Ag/Pt nano material of looks.In Fig. 2, the left side, centre and the right are to use identical synthetic method, before adding different platinum
Drive liquid solution, the hollow silver platinum nano material electron microscopic observation of the different silver platinum ratios of synthesis.The mass fraction ratio of the silver-colored platinum on the left side
3:2, the mass fraction of middle silver-colored platinum compares 5:2 than 4:2, the mass fraction of the silver-colored platinum on the right.It can be seen that the quality of silver platinum is divided
When number is than 3:2, Ag/Pt appearance of nano material disunity, disperses uneven；When the mass fraction of silver platinum is than 4:2, Ag/Pt nanometer
Material has a small amount of hollow cystic structures, but dispersibility is poor；When the mass fraction of silver platinum is than 5:2, hollow silver platinum nano material
Pattern best, granule is uniform, and dispersibility is preferably also, and its particle diameter is about 70nm.
(2) catalytic effect compares
A. different silver platinum nano materials are to H2O2Catalytic performance compare
Investigate different silver alloy platinum material to H2O2Catalytic performance compare.Take 10 μ L silver alloy platinum materials and 10 μ L chitosan solutions
Modify after mixing on glass-carbon electrode, after 5 hours, glass-carbon electrode immersed supporting electrolyte BS) in, add 100 μ L 1mol/L
H2O2The mensuration of chrono-amperometric is carried out under-0.2V.A, b, c represent the nano material of three kinds of different silver platinum mass fraction ratios, survey
Determining result as shown in Figure 3: curve a, b, c represent the mass fraction of the silver platinum hollow silver platinum nanometer material than 4:2,5:2 and 3:2 respectively
Material is to H2O2The current-responsive of catalytic action, find out from figure, the mass fraction of the silver platinum hollow silver platinum nanometer than 4:2
Material is to H2O2Catalytic effect best, it is possible to produce stronger current responsing signal.Although the mass fraction of silver platinum is than 5:2's
Hollow silver platinum nano material is to H2O2Catalytic action do not have the mass fraction hollow silver platinum nano material than 4:2 good, it is contemplated that
To the pattern of hollow silver platinum nano material and dispersibility and the smoothness of curve, this experiment still have selected the quality of silver platinum
The silver-colored alloy platinum material of score ratio 5:2 is as marker material.
The synergism of B.Ag and Pt nano material
In order to investigate Ag and Pt nano material to H2O2The synergism of catalytic effect, GCE is the most solid
Determine the silver-colored platinum of equivalent, silver, alloy platinum material, it is immersed respectively in supporting electrolyte (PBS), add 100 μ L 1mol/L H2O2In-
Carrying out the mensuration of chrono-amperometric under 0.2V, result is shown in Fig. 4: curve a, and b, c represent that different modifying electrode is to H respectively2O2Reduction current
Catalytic effect, a is Ag/Pt NPs/GCE, and b is Ag NPs/GCE, and c is Pt NPs/GCE.Figure 4, it can be seen that Ag/Pt receives
The electrode that rice material is modified is the strongest to the catalytic capability of hydrogen peroxide, illustrates that silver platinum has synergic catalytic effect.
C. the cycle voltammetry behavior of sensor
Fig. 5 is that the immunosensor cultivating equal in quality density antigen is with or without H2O2Buffer solution in cyclic voltammetric
Behavior.Curve a is containing 1mol/L H2O2Phosphate buffered solution in the background current observed；Curve b is for phosphoric acid
The background current observed in salt buffer solution.Visible, when adding H2O2After, electric current sharply increases, it was demonstrated that silver-colored platinum nanometer material
Material is to H2O2Reduction there is catalytic action.
D. test the impact also having investigated scanning speed to peak current, when scanning speed changes from 20-100mV/s, pass
Sensor is containing 1mol/L H2O2PH is the such as Fig. 6 of the cycle voltammetry behavior in the buffer solution of 7.4.From fig. 6 it can be seen that peak
Electric current increases along with the increase of scanning speed.Simultaneously in order to investigate the characteristic of electric current, by the peak current square root to scanning speed
Mapping (see built-in figure), it can be seen that peak current is directly proportional to the square root of scanning speed from built-in figure, illustrates that this electric current is
Diffusion controls.Fig. 6 is the most respectively scanning speed be the cycle voltammetry behavior of 20,40,60,80,100mV/s, built-in
Figure is the square root v of redox current and scanning speed1/2Linearity 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 exists
5mmol/L K3Fe(CN)4/K4Fe(CN)6AC impedance figure in solution.Curve a is the impedance curve of naked GCE, approximates one
Straight line, shows that electron transmission is only controlled by diffusion.Curve b is by the chitosan-modified glass-carbon electrode of amination Graphene
Impedance curve, due to the resistance to mass tranfer of chitosan, resistance value increased (Ret=250 Ω).Curve c is for securing 10 μ L 10
The impedance curve of the glass-carbon electrode of μ g/mLCRP antibody (anti-CRP), impedance substantially increases (Ret=490 Ω), this be due to
Antibody hinders the electronics transmission at electrode surface.Curve d is the electrode after closing with bovine serum albumin (BSA), trains further
Having educated the AC impedance curve after 10 μ L 30ng/mLCRP antigens, half circular diameter significantly increases (Ret=1000 Ω), and this is
Owing to covering of BSA also has antibody and the specific binding transmission all hindering electronics of antigen.Curve e is to have cultivated silver-colored platinum to receive
Electrode AC impedance figure after rice material marking antibody (labeled anti-CRP), resistance value increases (Ret=further
1780 Ω), electron impeding effect is strengthened by this explanation electrode modification film.
F. the optimization of experiment condition
A. the different sessile antibody mass concentration impacts on sensor response current
Fig. 8 illustrates the impact on sensor response current of the different antibodies concentration.In the case of other conditions are constant, change
The concentration of change sessile antibody solution used by modified electrode, is respectively working curve (a:10 μ g/mL, b:20 μ g/mL, a c:50 μ
g/mL、d:100μg/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, accordingly, it is determined that the best in quality that antibody is fixing in an experiment
Concentration is 10 μ g/mL.
B. antigen cultivates the time impact on sensor response effect electric current
The immunosensor being modified with antibody is used under conditions of 37 DEG C different cultivation time (10min to 70min)
Cultivating the antigen of same concentrations (30mg/L) and use chronoamperometry to measure the response current of immunosensor respectively, result is such as
Fig. 9.As seen from the figure, increasing over time, electric current first increases and reduces afterwards.When the cultivation time is 50min, current-responsive
Signal is maximum, and therefore selecting 50min is that optimal antigen cultivates the time.
C. traget antibody cultivates the time impact on sensor response effect electric current
Another factor affecting immunosensor is the cultivation time of traget antibody.The immune sensing of antibody will be modified with
Device uses same antigen to cultivate the time under conditions of 37 DEG C and cultivates the antigen (30mg/L) of same concentrations, changes traget antibody training
Educate the time, in the range of 10min to 60min, investigated traget antibody and cultivated the time impact on sensor, result such as Figure 10 institute
Show.From the figure, it can be seen that during 40min to 50min, current-responsive is maximum and platform occurs, so experimental selection 45min is
The optimum mark antibody development time.
(3) calibration trace of sensor and detection limit
Under optimum experimental condition, it is thus achieved that the working curve (Figure 11) that the CRP of variable concentrations is responded by sensor.From figure
It can be seen that the response current of sensor has good linear in the range of 0.5mg/L-140mg/L with CRP concentration in 11
Relation, linear equation is: I (μ A)=1.14C (ng/mL)+7.41, correlation coefficient r2=0.9934.According to 3 σ rules, CRP's
Monitoring lower-cut is 0.17mg/L.The most perspective according to disease prevention and control centers of the U.S. in 2004 and American Heart Association's reference
The CRP proposed in guide that result of study is issued measures at diagnosis of coronary heart disease and the point of contact of future event early warning, i.e. CRP concentration
＜ 1mg/L, relative risk level is low, and CRP concentration is at 1-3mg/L, and relative risk level is moderate, and ＞ 3mg/L is high
Degree, ＞ 10mg/L is high risk, the patient's sickness rate of cardiovascular event, death in following 6 months that CRP concentration is significantly raised
Rate significantly increases, and the probability developed complications is bigger.The sensor of the invention range of linearity is wider, is applicable to coronary heart disease phase
It it is the detection of CRP concentration in the blood samples of patients of minuent, moderate, height and high degree to danger level.
(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), glycine (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 is as shown in figure 12.In phase
Under the conditions of Tong, bovine serum albumin, glutamic acid, glycine, the response current of human chorionic's glandular hormone are less than the electricity of CRP antigen
Stream.This shows that this sensor has good selectivity.
(5) mensuration of the response rate and the mensuration of authentic sample
In order to investigate the practicality of this sensor, use standard addition method, by danger relatively in the human serum of dilution 10 times
Danger level is standard [Algarra M., Gomes D., Esteves da Silva J.C.G., the Clinica of height
Chimica Acta 2013,415 (1): 1 9] add 3 kinds without the CRP antigen of mass concentration, carry out CRP by this experimental technique
The mensuration of the response rate, 3 times detection average recovery rate is 100.4% (table 1), and result is satisfactory.The people's whole blood that will gather
Product, stand about one hour, then use low temperature low speed (2000rpm) centrifugal 10 minutes, take what upper strata yellow liquid must be collected
Serum.The CRP in the human serum sample of 10 times, parallel assay 3 times is diluted by this determination of experimental method.Count after considering extension rate
The average CRP concentration calculated when obtaining undiluted in blood serum sample is 58.5mg/L (table 2).Illustrate that this sensor can be used for true sample
The detection of CRP content in product.
The response rate of table 1. immunosensor
In the true blood serum sample of table 2., CRP measures
(6) repeatability of sensor and stability
The CRP antigen of 60mg/L is carried out 4 mensuration, and relative standard deviation is only 3.8%, illustrates that electrode is to CRP antigen
Mensuration have preferable repeatability through continuous 100 circle cyclic voltammetry scan sensor peak point currents only reduce by 4.6%, explanation
This sensor has good stability.The sensor not used time is saved in the buffer solution of 4 DEG C.
The present embodiment utilizes amination Graphene fix CRP antibody and use silver Pt nanoparticle traget antibody to construct one
Plant novel CRP immunosensor.This sensor combines the excellent electric conductivity of amination Graphene and silver Pt nanoparticle
Good biocompatibility and catalytic action, by optimal conditions, significantly improve the sensitivity of CRP sensor, selectivity, make
Sensor has the wider range of linearity, relatively low Monitoring lower-cut, and the detection for CRP provides a new way.
Claims (2)
1. a C reactive protein immunosensor, it is characterised in that prepare as follows: make with hollow silver Pt nanoparticle
For label labelling C reactive protein antibody, for fixing substrate, C reactive protein antibody is fixed on electricity with amination Graphene
Extremely go up, make the immunosensor of sandwich type.
2. C reactive protein immunosensor as claimed in claim 1, it is characterised in that: silver in hollow silver Pt nanoparticle
The mass fraction of platinum ratio is for 5:1.9～2.1, and the fixing mass concentration of antibody is 9～11 μ g/mL.
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