CN102253194B - Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor - Google Patents
Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor Download PDFInfo
- Publication number
- CN102253194B CN102253194B CN 201110174132 CN201110174132A CN102253194B CN 102253194 B CN102253194 B CN 102253194B CN 201110174132 CN201110174132 CN 201110174132 CN 201110174132 A CN201110174132 A CN 201110174132A CN 102253194 B CN102253194 B CN 102253194B
- Authority
- CN
- China
- Prior art keywords
- concentration
- magnetic
- probes
- antigen
- peak current
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention relates to the field of biological monitoring, and specifically relates to a secondary antibody probe for a sandwich immune reaction by using an amperometric immunosensor, wherein, the secondary antibody probe is composed of calf thymus DNA molecular chains and nanospheres adhering to the calf thymus DNA molecular chains, and the surfaces of the nanospheres are covered by enzyme labeled antibody proteins while magnetic nanometer particles, which are formed by depositing a layer of amorphous zirconia on the surfaces of nanometer ferriferrous oxide particles, are filled in the nanospheres. The secondary antibody probe provided in the invention is suitable for measurring concentration of antigens by a sandwich immune reaction employing an amperometric immunosensor, and has the advantages of wide linear range of detection and low detection limits.
Description
Technical field
The invention belongs to field of biological detection, particularly relate to the probe of molecular recognition, this probe is suitable for adopting ampere immunity sensor to carry out the concentration of sandwich immunoassay response measurement antigen.
Background technology
Nineteen ninety Henry etc. has proposed the concept of immunosensor, immunosensor utilizes antigen, antibody as the molecular recognition original paper, the signal that produces in the Ag-Ab specific reaction process is transformed into electric signal by transducer, thereby measured object is carried out quantitative test.Compare with traditional detection technique, have high sensitivity, high specific, easy and simple to handle, analysis speed is fast, advantage such as cheap, and easily be automated operation, demonstrate good prospects for application in fields such as clinical diagnosis, health care, environmental monitoring, food securities.Yet the galvanochemistry amperometric sensor of having reported in use also has the following disadvantages: 1. the immune complexs that adopt " single stage method " namely to adopt antigen-antibody to form in conjunction with the back cause the electrode surface electric current to descend more, carry out quantitatively according to drop-out current, so just inevitably be difficult to overcome the influence of non-specific adsorption; 2. product is difficult to remove after the immunity combination takes place the antigen/antibody of electrode surface, and electrode can't upgrade, and uses and mostly is disposable; Although 3. ampere immunity sensor has plurality of advantages, in the face of the very complicated concrete detected object (as blood serum sample) of background, often need by detecting again after certain sample purification processes, influenced its ease for use.So the problems referred to above have limited the advantage of immunosensor to a certain extent.
In recent years, the nano-modified electrochemical sensor based on sandwich immunoassay causes people's extensive concern owing to its remarkable sensitivity.Because the sandwich immunoassay sensor is compared with " single stage method " sensor and had higher susceptibility and specificity, what prepare this type of sensor most critical just is the enrichments that resist two.There has been report to adopt multiple nano material to resist two and carried out enrichment, as Nano carbon balls, silicon dioxide, nanometer Pt, manganese dioxide, Graphene (GS), carbon nano-tube (CNTs), nm of gold (nano-Au), quantum dot etc.Yet these enrichment process are loaded down with trivial details, need centrifugal could separate probe and binding antibody not; Have research to use magnetic Nano material (as tri-iron tetroxide, the golden magnetic nano particle) enriched antibody of etc.ing, avoided loaded down with trivial details centrifugal process, but its magnetic material that synthesizes is reunited poor stability, and the concentration effect of single nanoparticle antagonist is limited easily.Yuan Ruo etc. have reported that the reducing action by ascorbic acid forms a kind of " chain gold thread ", form beading by a plurality of nm of gold atoms, enriched antibody in a large number thus, the building-up process of this but " chain gold thread " needs 5 day time at least, and it is nonmagnetic, still need to separate by centrifugal, the detachment process of probe is loaded down with trivial details.Therefore researching and developing a kind of new having the antibody enrichment material that separation is easy, accumulation rate is high and be applied in the electrochemical immunosensor has great importance.
ZrO
2As a kind of Lewis strong acid, by with protein molecular on carboxylic acid and the combination of the groups such as phosphoric acid on the DNA (Lewis highly basic) specificity orientation, can directly be used for fixing enzyme and antibody.Because antibody and enzyme are coated on superfine (1～2 μ m) and the very big nanometer ZrO of surface area
2On, labelled amount significantly increases and can keep biologically active for a long time.But single nanoparticle is limited to the concentration effect of two antiantibodys.
Summary of the invention
The object of the present invention is to provide a kind of two anti-probes, this probe is suitable for adopting ampere immunity sensor to carry out the concentration of sandwich immunoassay response measurement antigen, and has the excellent and low advantage of detectability of enriched antibody effect.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
A kind of two anti-probes that adopt ampere immunity sensor to carry out the sandwich immunoassay reaction, this probe is made of calf thymus DNA strand and the nanosphere that is attached on the calf thymus DNA strand, the surface of wherein said nanosphere is the antibody protein of enzyme labeling, inside is magnetic nanoparticle, and this particle is to form at the amorphous oxidation zirconium of ferroferric oxide nano granules surface deposition one deck; Described two anti-probes are " beading ", have 10 above-described magnetic nanoparticles on each probe molecule.
Of the present invention two anti-probes, wherein said magnetic nanoparticle are well known materials, and concrete preparation method can be that the described scheme of application for a patent for invention of CN101302361A is carried out with reference to publication number.
The preparation method of two anti-probes of the present invention is made up of following steps:
1) magnetic nanoparticle is added in the pH7.0 phosphate buffer, adding weight again is the horseradish peroxidase-labeled antibody of 0.1 times of magnetic nano-particle, and 4 ℃ are reacted 6h down, separate the Nano microsphere that obtains load antibodies;
2) resulting Nano microsphere is added in the pH7.0 phosphate buffer, the peroxidase and the weight that add weight again and be 0.5 times of described Nano microsphere are the bovine serum albumin(BSA) of 1.0 times of described Nano microspheres, and 4 ℃ of reaction 6h down separate obtaining nanosphere;
3) nanosphere is added in the pH7.0 phosphate buffer, adding weight again is the calf thymus DNA of 1.2 times of nanospheres, reacts 6h under the room temperature, separates to obtain two anti-probes.
In the probe of the present invention, at first prepared the Fe with nucleocapsid structure
3O
4(nuclear)/ZrO
2(shell) magnetic nano-particle, this magnetic nano particle sub-surface contains ZrO
2Can with the antibody combination, antagonist carries out enrichment.This probe has the following advantages: the 1) use of magnetic material, and make probe have magnetic, under the effect of externally-applied magnetic field, can realize separating, saved centrifugal and washing process loaded down with trivial details in the probe building-up process; 2) this probe is " beading ", has 10 above magnetic beads on each probe molecule, increases detecting probe surface marker enzyme antibody concentration, therefore improves the susceptibility of immunosensor.3) zirconium dioxide has good biocompatibility, can keep the activity of antibody for a long time, makes antibody be difficult for inactivation, and this probe can once be prepared and long preservation.
Of the present invention two anti-probes can be used for detecting antigen concentration, and concrete the detection is made up of following steps:
(a) preparation of quantitative criterion curve:
(1) the antigen standard solution of a series of variable concentrations of preparation;
(2) respectively the antigen standard solution of each concentration is joined in the reaction chamber of ampere immunity sensor by identical volume, 37 ℃ of incubation 30min wash earlier; Again two prepared anti-probes are dripped in the reaction chamber of ampere immunity sensor 37 ℃ of incubation 30min, washing; Then, add in the reaction chamber and contain the 0.1mol/L phosphate buffered solution that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol, behind the reaction 3min, record reduction peak current at electrochemical workstation with cyclic voltammetry (CV) or differential pulse voltammetry (DPV);
(3) with one group of data of the concentration of the resulting antigen standard solution of step (2) and corresponding reduction peak current thereof, carrying out linear regression with reduction peak current antagonism primary standard product solution concentration just obtains, the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve;
(b) detection of sample:
Testing sample is joined in the reaction chamber of ampere immunity sensor, method and the reaction conditions same with step (a) adopt cyclic voltammetry (CV) record reduction peak current, then, can calculate the concentration of antigen in the testing sample in the resulting straight-line equation of reduction peak current value substitution step (a) that test sample is obtained.
Employed ampere immunity sensor can prepare by known method voluntarily according to determined antigen in the method for above-mentioned detection antigen concentration.
Of the present invention two anti-probes are applicable to and adopt ampere immunity sensor to carry out the concentration of sandwich immunoassay response measurement antigen, because of the present invention two anti-probes have one dimension " beading " structure, improve the charge capacity that detecting probe surface two resists greatly, because the range of linearity and sensitivity and the two anti-concentration effect positive correlations of electrochemical immunosensor, therefore to have the range of linearity wide for the method for detection antigen concentration of the present invention, the advantage that detectability is low.
Description of drawings
Fig. 1 is detection principle and the two anti-probe building-up process synoptic diagram of two anti-probes.It is the building-up process synoptic diagram of two anti-probes in the square frame of right side.
Fig. 2 is that the probe that synthesizes utilizes externally-applied magnetic field to carry out separating resulting figure.
Fig. 3 is the transmission electron microscope picture of the two anti-probes that synthesized of following embodiment 1;
Galvanochemistry CV response diagram when Fig. 4 is variable concentrations AFP detection.Interior illustration is the bioassay standard curve.
Galvanochemistry DPV response diagram when Fig. 5 is variable concentrations HIV p24 detection.Interior illustration is the bioassay standard curve.
Embodiment
1, the preparation of two anti-probes and sign
(1) preparation of magnetic nanoparticle
The preparation of magnetic nanoparticle: concrete preparation method can be the application for a patent for invention of CN101302361A with reference to publication number.
The sign of magnetic nanoparticle: adopt X-ray fluorescence spectra (XRF) to Fe
3O
4/ ZrO
2Characterize, Zr-K occurred
β(17.8keV), Zr-K
α(15.8keV), Zr-L
β(2.1ke), Zr-L
α(2.0keV) peak and Fe-K
β(7.1KeV), Fe-K
α(6.4keV) peak illustrates to have Zr and Fe element in this magnetic particle.
(2) preparation of the Nano microsphere of load antibodies and sign
The preparation of the Nano microsphere of load antibodies: the 10mg magnetic nano-particle is dispersed in the 5mL pH7.0 phosphate buffer, the alpha-fetoprotein two anti-(HRP-anti-AFP) that adds 1mg horseradish peroxidase mark, after stirring 6h, add magnetic condition magnetic and separate, obtain the Nano microsphere of load antibodies.
(3) preparation of nanosphere
The Nano microsphere of 10mg load antibodies is dispersed in the pH7.0 phosphate buffer of 5mL, add 5mg horseradish peroxidase, 10mg bovine serum albumin(BSA) (BSA) successively, 4 ℃ are stirred 6h, utilize externally-applied magnetic field to separate magnetic nanoparticle, washing obtains nanosphere.
The preparation of (4) two anti-probes
The 10mg nanosphere is dispersed in the pH7.0 phosphate buffer of 5mL, adds the 12mg calf thymus DNA, stir 6h under the room temperature, magnetic resolution namely obtains two anti-probes.
The sign of two anti-probes: adopt transmission electron microscope that the probe preparation process has been carried out characterizing (Fig. 3).Two anti-probes have one dimension " bead chain shape " linear structure as seen from Figure 3.
(measuring elemental range is to adopt X-ray fluorescence spectra
9F～
92U) two anti-probes are characterized, Zr-k α peak 2.1keV, Fe-k α peak 6.4keV, P-k α peak 1.13keV and S-k α peak 2.3keV have been shown, because DNA contains a large amount of phosphate groups, has coated DNA so the appearance at P peak further illustrates the magnetic nano particle sub-surface.
Ultraviolet spectrum after mensuration magnetic nanoparticle and the DNA effect, discovery dna characteristics absorption peak to 524nm, illustrates that interaction has taken place for DNA and magnetic nanoparticle, makes absorption peak generation red shift by the 520nm red shift.As seen from Figure 2, two anti-probe magnetic probes have good superparamagnetism under the effect of 0.3mTde external magnetic field.
2. the preparation of ampere immunity sensor
Commercial screen printing electrode is modified:
(a) Graphene/shitosan (GS/CS) is in screen printing electrode finishing film forming
0.2% of adding 2mL pH5.0 shitosan (CS) acetum and the GS of 1mg mix obtaining suspension in the 5mL centrifuge tube; The 5mL suspension is dropped in working electrode surface in the screen printing electrode, and air dry under the room temperature is removed in conjunction with insecure nano particle with the washing of pH7.0 phosphate buffered solution then.
(b) adopt the current-time curvel method at the working electrode surface Direct Electroplating gold of screen printing electrode: with the HAuCl of 20 μ L1%
4Dropping applies-0.2V constant potential 30S at working electrode surface.Electroplating the back fully washs with PBS.
(c) in the reaction chamber of screen printing electrode, add 10 μ L AFP monoclonal primary antibodies after, screen printing electrode puts into that 4 ℃ of refrigerator low temperature incubations fully washed not combination with PBS after 12 hours and in conjunction with insecure antibody.
(d) with the 3mg/mL bovine serum albumin(BSA) (BSA) sealing not in conjunction with electrode surface not in conjunction with avtive spot, obtain ampere immunity sensor.
3. the detection of antigen concentration
Be to utilize the sensor that fixes primary antibodie to carry out incubation with two anti-probes of sample and preparation successively to the testing process of antigen, if contain determined antigen to some extent in the sample, then Zhi Bei two anti-probes are fixed on electrode surface by the form of immune complex, adding behind the substrate can catalytic substrate generation redox reaction, produces electric signal.It detects principle such as accompanying drawing 1.
(1) quantitative criterion curve preparation:
(a) AFP antigenic solution standard items are diluted to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml adds the AFP antigenic solution standard items of 10 μ L variable concentrations respectively in the reaction chamber of ampere immunity sensor, and at 37 ℃ of following incubation 30min.Reacted back with the careful flush away unreacted of PBS antigen.
(b) two step 1 is synthetic anti-probes are made into the 1mg/ml suspension, drip 10mL two anti-probe suspensions in the reaction chamber of ampere immunity sensor, 37 ℃ of incubation 30min, and PBS carefully washs;
(c) adding contains the 0.1mol/L phosphate buffered solution reaction 3min that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol in the reaction chamber of ampere immunity sensor; On electrochemical workstation, with cyclic voltammetry (CV) record reduction peak current I(sweep limit from-0.3V to-0.8V).
(d) one group of data of concentration of the resultant reduction peak current of step (C) and corresponding antigen standard solution, adopt the average gradient method to carry out linear regression analysis with reduction peak current antagonism primary standard product solution concentration, just obtain the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve.CV reduction peak current and AFP antigen concentration numerical value are seen illustration in Fig. 4.Linear equation is logI=0.66log[C
AFP]-5.15.
Testing sample is joined in the reaction chamber of ampere immunity sensor, method and the reaction conditions same with step (1) adopt cyclic voltammetry (CV) record reduction peak current, then, can calculate the concentration of antigen in the testing sample in the resulting straight-line equation of reduction peak current value substitution step (a) that test sample is obtained.Adopt 10 blank samples to detect calculating mean value
With standard deviation s, with
As detectability.
In this example, be 0.01～200ng/mL to the sensing range of AFP, detect and be limited to 4pg/mL.
Embodiment 2(contrast experiment 1)
One, experiment material
1. antigen and antibody: alpha-fetoprotein polyclonal antibody (HRP-anti-AFP) and the AFP ELISA kit of AFP antigen standard items, alpha-fetoprotein monoclonal primary antibodie (anti-AFP), horseradish peroxidase-labeled are all purchased Zhengzhou Bosai Biotechnology Co., Ltd.
2. ampere immunity sensor: make by embodiment 1 described method.
3. two anti-probes
3.1, sample: the two anti-probes that make by embodiment 1 described method.
3.2, reference substance
Reference substance 1:
(1) preparation of magnetic nanoparticle
The preparation of magnetic nanoparticle: concrete preparation method can be the application for a patent for invention of CN101302361A with reference to publication number.
(2) preparation of the Nano microsphere of load antibodies and sign
The preparation of the Nano microsphere of load antibodies: the 10mg magnetic nano-particle is dispersed in the 5mL pH7.0 phosphate buffer, the alpha-fetoprotein two anti-(HRP-anti-AFP) that adds 1mg horseradish peroxidase mark, after stirring 6h, externally-applied magnetic field separates unconjugated antibody, add magnetic condition magnetic and separate, obtain the Nano microsphere of load antibodies.
(3) preparation of magnetic probe
The Nano microsphere of 10mg load antibodies is dispersed in the pH7.0 phosphate buffer of 5mL, add 5mg horseradish peroxidase, 10mg bovine serum albumin(BSA) (BSA) successively, 4 ℃ are stirred 6h, utilize externally-applied magnetic field to separate magnetic nanoparticle, washing obtains magnetic probe and represents with the HRP-anti-AFP-ZMPs probe.
Reference substance 2: the method with reference to following reported literature prepares: Tang J, Su B, Tang D, et al.Conductive carbon nanoparticles-based electrochemical immunosensor with enhanced sensitivity for[alpha]-fetoprotein using irregular-shaped gold nanoparticles-labeled enzyme-linked antibodies as signal improvement[J] .Biosensors and Bioelectronics, 2010,25 (12): 2657-2662.Gained two anti-probes are represented with code name HRP-anti-AFP-GNGs.
Reference substance 3: the method with reference to following reported literature prepares: Zhuo Y, Yi W J, Lian W B, et al.Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification[J] .Biosens Bioelectron.2011,26 (5): 2188-2193.Gained two anti-probe code name AuNCs-HRP-Ab
2Expression.
Reference substance 4: with the anti-product 4 in contrast of the AFP polyclone of the horseradish peroxidase-labeled in the AFP ELISA kit (available from Zhengzhou Bosai Biotechnology Co., Ltd).Represent with HRP-anti-AFP.
Two, experimental technique
1. experiment is divided into 5 groups, and experiment is except the two anti-probe differences that add in every group, and all the other operations are identical.
2. experimentation
2.1 quantitative criterion curve preparation:
(a) AFP antigenic solution standard items are diluted to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, the ampere immunity sensor of preparation is divided into 5 groups, every group contains 10 sensors, adds the AFP antigenic solution standard items of 10 μ L variable concentrations in the reaction chamber of ampere immunity sensor respectively, and at 37 ℃ of following incubation 30min.Reacted back with the careful flush away unreacted of PBS antigen.
What (b) sample for preparing in this example and reference substance are fitted in is made into the 1mg/ml suspension, drip 10 μ L samples in the group 1 in the reaction chamber of ampere immunity sensor, add reference substance 1～4 probe suspension 10 μ L respectively, 37 ℃ of incubation 30min in the reaction chamber of group 2～group 5 sensors.
(c) adding contains the 0.1mol/L phosphate buffered solution reaction 3min that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol in the reaction chamber of ampere immunity sensor; On electrochemical workstation, with cyclic voltammetry (CV) record reduction peak current I(sweep limit from-0.3V to-0.8V).
(d) one group of data of concentration of the resultant reduction peak current of step (C) and corresponding antigen standard solution, adopt the average gradient method to carry out linear regression analysis with reduction peak current antagonism primary standard product solution concentration, just obtain the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve; Adopt 10 blank samples to detect calculating mean value
With standard deviation s, with
As detectability.
Table 1 is the comparison of different two anti-probe in detecting AFP antigen effects, can be drawn by table 1, when two prepared anti-probes of the application carry out Electrochemical Detection, compares with two anti-probes of additive method preparation and to have wide, the highly sensitive advantage of the range of linearity.
The effect of the different two anti-probe in detecting AFP antigens of table 1 relatively
Embodiment 3
1, the preparation of two anti-probes and sign
(1) preparation of magnetic nanoparticle and sign
Shown in embodiment 1 step 1.
(2) preparation of the Nano microsphere of load antibodies and sign
The preparation of the Nano microsphere of load antibodies: the 10mg magnetic nanoparticle is dispersed in the 5mL pH7.0 phosphate buffer, adds the HIV p24 antibody (HRP-anti-HIV p24) of 1mg horseradish peroxidase-labeled.After stirring 6h, externally-applied magnetic field separates unconjugated antibody, obtains the Nano microsphere of load antibodies.
(3) preparation of nanosphere
Shown in embodiment 1 step 3.
The preparation of (4) two anti-probes
Shown in embodiment 1 step 4.
2. adopt two anti-probe in detecting antigen concentrations
(1) preparation of ampere immunity sensor
(a) Graphene/shitosan (GS/CS) is in screen printing electrode finishing film forming
With (a) in the step 2 among the embodiment 1.
(b) electrogilding
With (b) in the step 2 among the embodiment 1
(c) in the reaction chamber of screen printing electrode, add 10 μ L HIV p24 monoclonal primary antibodies after, screen printing electrode was put into 4 ℃ of refrigerator low temperature incubations after 12 hours, fully washed not combination and in conjunction with insecure antibody with PBS.
(d) BSA sealing
With (d) in the step 2 among the embodiment 1
(2) quantitative criterion curve preparation:
(a) HIV antigenic solution standard items are diluted to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml, the HIV antigenic solution standard items that in the reaction chamber of ampere immunity sensor, add 10 μ L variable concentrations, and at 37 ℃ of following incubation 30min.Reacted back with the careful flush away unreacted of PBS antigen.
(b) two step 1 is synthetic anti-probes are made into the 1mg/ml suspension, drip 10mL two anti-probe suspensions, 37 ℃ of incubation 30min in the reaction chamber of ampere immunity sensor;
(c) adding contains the 0.1mol/L phosphate buffered solution reaction 3min that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol in the reaction chamber of ampere immunity sensor; On electrochemical workstation, with differential pulse voltammetry (DPV) record reduction peak current I, (sweep limit from 0.2V extremely-0.5V).
(d) one group of data of concentration of the resultant reduction peak current of step (C) and corresponding antigen standard solution, adopt the average gradient method to carry out linear regression analysis with reduction peak current antagonism primary standard product solution concentration, just obtain the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve.DPV reduction peak current and HIV antigen concentration numerical value are seen illustration in Fig. 5, and its linear equation is: I=15+0.97Log[C
P24].
Testing sample is joined in the reaction chamber of ampere immunity sensor, method and the reaction conditions same with step (1) adopt DPV record reduction peak current, then, can calculate the concentration of antigen in the testing sample in the resulting straight-line equation of reduction peak current value substitution step (a) that test sample is obtained.Adopt 10 blank samples to detect calculating mean value
With standard deviation s, with
As detectability.
In this example, be 0.01～200ng/mL to the sensing range of AFP, detect and be limited to 4pg/mL.
Embodiment 4(contrast experiment 2)
One, experiment material
1. antigen and antibody: the HIV p24 antibody (HRP-anti-HIV p24) of HIV p24 antigen standard items, HIV p24 monoclonal antibody (anti-HIV p24), horseradish peroxidase-labeled and HIV p24ELIS kit are all available from U.S. Abcam company.
2. ampere immunity sensor: make by embodiment 3 described methods.
3. two anti-probes
3.1 sample: the two anti-probes that make by embodiment 3 described methods.
3.2 reference substance
Reference substance 1:
(1) preparation of magnetic nanoparticle
The preparation of magnetic nanoparticle: concrete preparation method can be the application for a patent for invention of CN101302361A with reference to publication number.
(2) preparation of the Nano microsphere of load antibodies and sign
The preparation of the Nano microsphere of load antibodies: the 10mg magnetic nano-particle is dispersed in the 5mL pH7.0 phosphate buffer, add 1mg HRP-anti-HIV p24, after stirring 6h, externally-applied magnetic field separates unconjugated antibody, add magnetic condition magnetic and separate, obtain the Nano microsphere of load antibodies.
(3) preparation of magnetic probe
The Nano microsphere of 10mg load antibodies is dispersed in the pH7.0 phosphate buffer of 5mL, add 5mg horseradish peroxidase, 10mg bovine serum albumin(BSA) (BSA) successively, 4 ℃ are stirred 6h, utilize externally-applied magnetic field to separate magnetic nanoparticle, washing obtains magnetic probe and represents with HRP-anti-HIV p24-ZMPs probe.
ELIAS secondary antibody in the reference substance 2:HIV p24ELISA kit is product 2 in contrast.Be called HRP-anti-HIV p24.
Two, experimental technique
1. experiment is divided into 3 groups, and experiment is except the two anti-probe differences that add in every group, and the operating process of experimental technique is identical.
2. experimentation
2.1 quantitative criterion curve preparation:
(a) p24 antigen standard items are diluted to 0.01ng/ml, 0.05ng/ml, 0.1ng/ml, 0.5ng/ml, 1ng/ml, 5ng/ml, 10ng/ml, 15ng/ml, 25ng/ml is divided into 3 groups with the ampere immunity sensor for preparing, and every group contains 9 sensors, the p24 antigen standard items that add 10 μ L variable concentrations in the reaction chamber of ampere immunity sensor respectively, and at 37 ℃ of following incubation 30min.Reacted back with the careful flush away unreacted of PBS antigen.
(b) the 1mg/ml suspension that the sample for preparing in this example and reference substance are fitted in, drip 10 μ L samples in the group 1 in the reaction chamber of ampere immunity sensor, drip reference substance 1～2 probe suspension 10 μ L respectively in the reaction chamber of group 2～group 3 sensors, 37 ℃ of incubation 30min, washing.
(c) adding contains the 0.1mol/L phosphate buffered solution reaction 3min that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol in the reaction chamber of ampere immunity sensor; On electrochemical workstation, with differential pulse voltammetry (DPV) record reduction peak current I(sweep limit from 0.2V extremely-0.5V).
(d) one group of data of concentration of the resultant reduction peak current of step (C) and corresponding antigen standard solution, adopt the average gradient method to carry out linear regression analysis with reduction peak current antagonism primary standard product solution concentration, just obtain the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve; Adopt 10 blank samples to detect calculating mean value
With standard deviation s, with
As detectability.
Table 2 is comparisons of different two anti-probe in detecting HIV p24 antigen effects, can be drawn by table 2, when two prepared anti-probes of the application carry out Electrochemical Detection, compares with two anti-probes of additive method preparation and to have wide, the highly sensitive advantage of the range of linearity.
The effect of the different two anti-probe in detecting HIV p24 antigens of table 2 relatively
Embodiment 5(actual sample detects)
The sensor of preparation carries out the electro-chemistry immunity detection to three unknown samples in the two anti-probes that adopt in the above-mentioned example 1 preparation and the example 1, and utilizes AFP ELISA kit that these three actual samples are detected simultaneously.Process is as follows:
1, the preparation of two anti-probes and sign
Shown in step 1 shown in the embodiment 1
2, the preparation of ampere immunity sensor
Shown in step 2 shown in the embodiment 1
3, the ampere immunity sensor method detects three unknown concentration test sample
Shown in step 3 shown in the embodiment 1
4, the ELISA method detects three unknown concentration samples in the unknown above-mentioned steps 3
With reference to the operation of AFP ELISA kit instructions, comprising:
(a) prepare: take out kit, room temperature rewarming balance 30 minutes from refrigerator.
(b) dosing: 20 times of concentrated cleaning solutions are diluted to former times cleansing solution with distilled water.
(c) add standard items and sample to be tested: get the enzyme mark bag of sufficient amount by plate, be fixed on the framework, each hole site is recorded in the product hole that sets up standard respectively, sample to be tested hole and blank hole, adds standard items 50 μ L in the standard items hole; Add sample to be tested 10 μ L in the sample to be tested hole earlier, add sample dilution 40 μ L (being 5 times of sample dilutions) again; The blank hole does not add.
(d) incubation: 37 ℃ of water-baths or constant temperature oven incubation 30min.
(e) wash plate: discard liquid, pat dry on the thieving paper, cleansing solution is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, pats dry on the thieving paper, so repeats to wash plate 4 times.
(f) enzyme-added mark working fluid: every hole adds enzyme mark working fluid 50 μ L, and the blank hole does not add.
(g) incubation: 37 ℃ of water-baths or constant temperature oven incubation 30min.
(h) wash plate: discard liquid, pat dry on the thieving paper, cleansing solution is filled it up with in every hole, leaves standstill 1min, gets rid of cleansing solution, pats dry on the thieving paper, so repeats to wash plate 4 times (also available wash plate machine by specification operation wash plate).
(i) colour developing: every hole adds developer A liquid 50 μ L earlier, adds developer B liquid 50 μ L again, dull and stereotyped vortex mixer mixing 30s (or gently shake mixing 30s with have gentle hands), 37 ℃ of lucifuges colour developing 15min.
(j) stop: take out ELISA Plate, every hole adds stop buffer 50 μ L, cessation reaction (color changes yellow by blueness is upright).
(k) measure: with the blank well zeroing, in back 15 minutes of termination, with the light absorption value (OD value) in each hole of 450nm wavelength measurement.
(l) calculate: according to concentration and the corresponding OD value of standard items, calculate the linear regression equation of typical curve, according to the OD value of sample, calculate corresponding sample concentration at regression equation again, also can use various application software to calculate.Ultimate density multiply by extension rate for practical measurement concentration.
The 5 actual sample recovery
In three samples in the above-mentioned steps 3, add variable concentrations AFP standard items respectively, and utilize AFP concentration in the sample that adopts after above-mentioned steps 3 detects the adding standard items, and calculate recovery rate, recovery computing formula is the recovery=100%* (this law Jian Ce Zhi – this method)/addition.
Testing result sees Table 3 in this example, can be drawn by table 3, and this method testing result and ELISA have accordance preferably, and it reclaims number percent between 95%-101%.Conclusion in 1 can draw in conjunction with the embodiments, compares with additive method, utilizes this method to detect AFP detection accuracy height, and sensitivity is good.
AFP testing result in table 3 human serum
A. the recovery=100%* (recovery Zhi – this method)/addition
Claims (3)
1. one kind is adopted ampere immunity sensor to carry out the two anti-probes that sandwich immunoassay reacts, this probe is made of calf thymus DNA strand and the nanosphere that is attached on the calf thymus DNA strand, the surface of wherein said nanosphere is the antibody protein of enzyme labeling, inside is magnetic nanoparticle, and this particle is to form at the amorphous oxidation zirconium of ferroferric oxide nano granules surface deposition one deck; Described two anti-probes are " beading ", have 10 above-described magnetic nanoparticles on each probe molecule.
2. the preparation method of the described two anti-probes of claim 1, this method is made up of following steps:
1) the 10mg magnetic nano-particle is dispersed in the 5mL pH7.0 phosphate buffer, the alpha-fetoprotein two that adds the 1mg horseradish peroxidase-labeled is anti-, behind the stirring 6h, adds magnetic condition magnetic and separates, and obtains the Nano microsphere of load antibodies;
2) Nano microsphere with the 10mg load antibodies is dispersed in the pH7.0 phosphate buffer of 5mL, adds 5mg horseradish peroxidase, 10mg bovine serum albumin(BSA) successively, and 4 ℃ are stirred 6h, utilizes externally-applied magnetic field to separate magnetic nanoparticle, and washing obtains nanosphere;
3) the 10mg nanosphere is dispersed in the pH7.0 phosphate buffer of 5mL, adds the 12mg calf thymus DNA, stir 6h under the room temperature, magnetic resolution namely obtains two anti-probes.
3. method that adopts claim 1 described two anti-probe measurement antigen concentrations, this method is made up of following steps:
(a) preparation of quantitative criterion curve:
(1) the antigen standard solution of a series of variable concentrations of preparation;
(2) respectively the antigen standard solution of each concentration is joined in the reaction chamber of ampere immunity sensor by identical volume, earlier 37 ℃ of incubation 30min; Again two prepared anti-probes are dripped the working electrode surface at ampere immunity sensor, 37 ℃ of incubation 30min; Then, add in the reaction chamber and contain the 0.1mol/L phosphate buffered solution that concentration is 5mmol/L urea peroxide solution and 1mmol/L catechol, behind the reaction 3min, record reduction peak current at electrochemical workstation with cyclic voltammetry (CV) or differential pulse voltammetry (DPV);
(3) with one group of data of the concentration of the resulting antigen standard solution of step (2) and corresponding reduction peak current thereof, carrying out linear regression with reduction peak current antagonism primary standard product solution concentration just obtains, the straight-line equation of reduction peak current and antigen standard solution concentration corresponding relation, i.e. typical curve;
(b) detection of sample:
Testing sample is joined in the reaction chamber of ampere immunity sensor, method and the reaction conditions same with step (a) adopt cyclic voltammetry (CV) record reduction peak current, then, can calculate the concentration of antigen in the testing sample in the resulting straight-line equation of reduction peak current value substitution step (a) that test sample is obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110174132 CN102253194B (en) | 2011-06-25 | 2011-06-25 | Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110174132 CN102253194B (en) | 2011-06-25 | 2011-06-25 | Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102253194A CN102253194A (en) | 2011-11-23 |
| CN102253194B true CN102253194B (en) | 2013-08-28 |
Family
ID=44980572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110174132 Active CN102253194B (en) | 2011-06-25 | 2011-06-25 | Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102253194B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102980925B (en) * | 2012-11-26 | 2013-11-13 | 济南大学 | Sandwich type electrochemical immunosensor, preparation method and application thereof |
| CN103344754B (en) * | 2013-05-09 | 2014-12-31 | 中国科学院宁波材料技术与工程研究所 | Magnetically labeled biological detection system and detection method thereof |
| CN103558396B (en) * | 2013-10-29 | 2015-08-26 | 宁波大学 | A kind of quantitative detecting method of alpha-fetoprotein |
| CN104181292B (en) * | 2014-08-27 | 2015-12-02 | 湖南师范大学 | A kind of protein single molecules level ampere immunity analytical approach |
| CN112689759A (en) * | 2018-08-09 | 2021-04-20 | Bbb有限公司 | Biosensor using magnetic nanoparticles, and detection apparatus and detection method using biosensor |
| CN110927238B (en) * | 2019-12-12 | 2022-10-11 | 山东理工大学 | Preparation method and application of sandwich type photoelectrochemical sensor for detecting prostate specific antigen |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6773928B1 (en) * | 1999-09-22 | 2004-08-10 | The United States Of America As Represented By The Secretary Of The Army | Compositions and methods for enhancing bioassay performance |
| CN1943560A (en) * | 2005-10-09 | 2007-04-11 | 北京师范大学 | Sandwich structure magnetic composite micro ball with functional shell layer, its preparing method and use |
| CN101168781A (en) * | 2007-10-17 | 2008-04-30 | 李越希 | In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof |
| CN101400800A (en) * | 2005-09-21 | 2009-04-01 | 康比麦崔克斯有限公司 | Process to detect binding events on an electrode microarray using enzymes |
| CN102072954A (en) * | 2010-10-29 | 2011-05-25 | 济南大学 | Research and application of electrochemiluminescent immunosensor for detecting tumor markers |
| CN102087283A (en) * | 2009-12-08 | 2011-06-08 | 华中科技大学 | Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004011672A1 (en) * | 2002-07-26 | 2004-02-05 | Ntera Limited | Porous nanostructured film sensor |
| US20050250094A1 (en) * | 2003-05-30 | 2005-11-10 | Nanosphere, Inc. | Method for detecting analytes based on evanescent illumination and scatter-based detection of nanoparticle probe complexes |
| KR20100042082A (en) * | 2008-10-15 | 2010-04-23 | 삼성전자주식회사 | Solid support with enhanced density of a signal material, kit containing the same and method for detecting target material using the same |
| WO2011042564A1 (en) * | 2009-10-09 | 2011-04-14 | Universite De Strasbourg | Labelled silica-based nanomaterial with enhanced properties and uses thereof |
-
2011
- 2011-06-25 CN CN 201110174132 patent/CN102253194B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6773928B1 (en) * | 1999-09-22 | 2004-08-10 | The United States Of America As Represented By The Secretary Of The Army | Compositions and methods for enhancing bioassay performance |
| CN101400800A (en) * | 2005-09-21 | 2009-04-01 | 康比麦崔克斯有限公司 | Process to detect binding events on an electrode microarray using enzymes |
| CN1943560A (en) * | 2005-10-09 | 2007-04-11 | 北京师范大学 | Sandwich structure magnetic composite micro ball with functional shell layer, its preparing method and use |
| CN101168781A (en) * | 2007-10-17 | 2008-04-30 | 李越希 | In vitro activity measuring method for hepatitis C virus RNA depending RNA polymerase and application thereof |
| CN102087283A (en) * | 2009-12-08 | 2011-06-08 | 华中科技大学 | Chitosan-nano-gold enzyme immunosensor for detecting mycobacterium tuberculosis and application thereof |
| CN102072954A (en) * | 2010-10-29 | 2011-05-25 | 济南大学 | Research and application of electrochemiluminescent immunosensor for detecting tumor markers |
Non-Patent Citations (8)
| Title |
|---|
| Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor;Lijuan Bai等;《Biosensors and Bioelectronics》;20110427;4331－4336 * |
| Carbon Nanotube Amplification Strategies for Highly Sensitive Immunodetection of Cancer Biomarkers;Xin Yu等;《J Am Chem Soc》;20060830;11199－11205 * |
| Lijuan Bai等.Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor.《Biosensors and Bioelectronics》.2011,4331－4336. |
| Ruchika Malhotra等.Ultrasensitive Electrochemical Immunosensor for Oral Cancer Biomarker IL-6 Using Carbon Nanotube Forest Electrodes and Multilabel Amplification.《Anal. Chem.》.2010,3118－3123. |
| Ultrasensitive Electrochemical Immunosensor for Oral Cancer Biomarker IL-6 Using Carbon Nanotube Forest Electrodes and Multilabel Amplification;Ruchika Malhotra等;《Anal. Chem.》;20100415;3118－3123 * |
| Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification;Ying Zhuo等;《Biosensors and Bioelectronics》;20100925;2188－2193 * |
| Xin Yu等.Carbon Nanotube Amplification Strategies for Highly Sensitive Immunodetection of Cancer Biomarkers.《J Am Chem Soc》.2006,11199－11205. |
| Ying Zhuo等.Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification.《Biosensors and Bioelectronics》.2010,2188－2193. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102253194A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102253194B (en) | Secondary antibody probe for sandwich immune reaction by using amperometric immunosensor | |
| Zhou et al. | Emerging strategies to develop sensitive AuNP-based ICTS nanosensors | |
| Liu et al. | A sensitive electrochemiluminescence immunosensor based on luminophore capped Pd@ Au core-shell nanoparticles as signal tracers and ferrocenyl compounds as signal enhancers | |
| CN103278651B (en) | Kit for chemiluminescence immunity quantitative detection of MYO (myohaemoglobinnano) nano magnetic particle and preparation method of kit | |
| Wang et al. | Platinum porous nanoparticles hybrid with metal ions as probes for simultaneous detection of multiplex cancer biomarkers | |
| Cheng et al. | Highly sensitive luminol electrochemiluminescence immunosensor based on ZnO nanoparticles and glucose oxidase decorated graphene for cancer biomarker detection | |
| CN102262125B (en) | Electrochemical immune sensor for detecting diethylstilbestrol and preparation method and application of sensor | |
| Fu et al. | Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers | |
| WO2016127318A1 (en) | Cardiac troponin i ultra-sensitive detection reagent kit, and ultra-sensitive detection method therefor | |
| Peng et al. | A novel immunoassay based on the dissociation of immunocomplex and fluorescence quenching by gold nanoparticles | |
| Hassen et al. | Quantitation of influenza A virus in the presence of extraneous protein using electrochemical impedance spectroscopy | |
| CN108169303A (en) | Aptamer electrochemical sensor based on metal-organic framework material as signal probe | |
| Yang et al. | Hollow platinum decorated Fe3O4 nanoparticles as peroxidase mimetic couple with glucose oxidase for pseudobienzyme electrochemical immunosensor | |
| CN109521004A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) | |
| CN102207501A (en) | Biotinylation bovine serum albumin and streptavidin enzyme-labeled reaction plate and preparation method thereof | |
| CN103278623A (en) | Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) nano magnetic particle and preparation method of kit | |
| CN107796803A (en) | A kind of the magnetic microparticle chemiluminescence detection kit and preparation method of free β human chorionic gonadotrophins | |
| CN108931652A (en) | A kind of kit with Magnetism particulate immuno chemistry luminescence method detection myoglobin content | |
| CN106093396A (en) | A kind of preparation method and application of immunosensor based on Au GQD@PtPd | |
| Gan et al. | A novel signal-amplified immunoassay for the detection of C-reactive protein using HRP-doped magnetic nanoparticles as labels with the electrochemical quartz crystal microbalance as a detector | |
| Vairaperumal et al. | Optical nanobiosensor-based point-of-care testing for cardiovascular disease biomarkers | |
| Li et al. | A label-free electrochemical aptasensor based on Ti3C2Tx-Ag/Au nanoparticles as a signal amplification strategy for CRP detection | |
| Liu et al. | Zeolite-iron oxide nanocomposite from fly ash formed a ‘clubbell’structure: integration of cardiac biocapture macromolecules in serum on microelectrodes | |
| CN103969445B (en) | The preparation of heme-manganese dioxide composites and the method for detecting human IgG thereof | |
| CN108375612A (en) | A kind of method of composite nano materials Electrochemical Detection alpha-fetoprotein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |