CN109781976A - Fluorescence immune analysis method based on carbon quantum dot - Google Patents
Fluorescence immune analysis method based on carbon quantum dot Download PDFInfo
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- CN109781976A CN109781976A CN201811612977.3A CN201811612977A CN109781976A CN 109781976 A CN109781976 A CN 109781976A CN 201811612977 A CN201811612977 A CN 201811612977A CN 109781976 A CN109781976 A CN 109781976A
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Abstract
The present invention provides the fluorescence immune analysis method based on carbon quantum dot.The carbon quantum dot is using citric acid as carbon source, and thiocarbamide or urea are what nitrogen source was prepared.The carbon quantum dot is applied in enzyme-linked immune analytic method, establish a kind of highly sensitive fluoroimmunoassay new method based on carbon quantum dot, using efficient fluorescence inner filtering effect is generated between carbon quantum dot and the chromogenic substrate of enzymatic, horseradish peroxidase in ELISA/alkaline phosphatase substrate for enzymatic activity successfully can be reacted into the absorbance signal to be formed and be effectively converted into fluorescence signal.This method has many advantages, such as that easy to operate, at low cost, high sensitivity and stability are good, can be used for the quantitative detection of small haptens.In addition, this method has good scalability, it is expected to spread to other target detection systems.
Description
Technical field
The present invention relates to Antigen Detection Techniques fields, specifically, being related to the fluoroimmunoassay side based on carbon quantum dot
Method.
Background technique
Immunoassay is the quick analytical technology of one kind based on specific recognition between antigen and antibody and combination.Immune point
Analysis is applicable to macromolecular compound (such as protein, bacterium) and small molecule compound (such as pesticide, veterinary drug, mycotoxin)
Measurement.Due to immunoassay there is high specific, detection speed fast, low cost and be suitable for the screening of a large amount of sample sites etc. it is excellent
Point makes it be used widely in the analysis fields such as food safety detection, environmental monitoring.
Enzyme-linked immune analytic method is a kind of more common immunoassay method, operation letter low with instrument dependency degree
Just, low cost, high specificity, sensitivity are higher, mass detection can be achieved, easily commercialization the advantages that, have become in the market
One kind is widely used, mature detection and analytical technology, and has a large amount of relevant reagent kit product to launch.It is small in food
Molecular chemistry pollutant is because its molecular weight is smaller (≤6000Da), often only single antigenic determinat, thus generallys use competing
Enzyme-linked immunosorbent assay is striven to carry out quantitative detection to it.Traditional competitive enzyme-linked immune analytic approach generallys use horseradish peroxidating
Object enzyme or the colour developing of alkaline phosphatase substrate for enzymatic activity, and exported in the form of absorbance value as signal.However, substrate for enzymatic activity produces
Raw absorbance signal usually has lower signal-to-noise ratio, causes the sensitivity of ELISA lower.
In order to reach better detection sensitivity, it is a kind of effective that establishing, which has the fluorescence immune analysis method of high s/n ratio,
Strategy.Conventional fluorescent dye reagent has that fluorescent stability is poor, intolerant to photobleaching, the defects of Stokes shift is small, thus
Limit its application in fluorescence immune analysis method.It is the semiconductor-quantum-points such as traditional fluorescent nano material such as CdSe, glimmering
Light microballoon, upper conversion nano particle, rare-earth nano-fluorescent complex etc. usually have quantum yield high, and fluorescent stability is good, swash
The excellent optical characteristics such as hair spectrum is wider, Stokes shift is big, are commonly used for fluoroimmunoassay.However semiconductor-quantum-point
Harsh etc. traditional usual synthesis condition of fluorescent nano material, synthesis step is complicated, and containing heavy metal substance, there are certain lifes
Object toxic.And semiconductor-quantum-point etc. has to pass through " oil-water " phase inversion of complexity and surface modification just can apply to fluorescence
In immunoassay.The above factor limits the application of traditional fluorescent nano material, therefore in fluoroimmunoassay, light
Have excellent performance, convieniently synthesized, at low cost, stablizes, and the exploitation of the novel fluorescence probe of nonhazardous effect has important meaning
Justice.
Carbon quantum dot is a kind of novel carbon-based fluorescent nano material, with excellent optical property, good water solubility,
Without toxic element, environmental-friendly, synthesis material source is wide, at low cost, convieniently synthesized, good biocompatibility many advantages, such as.
These characteristics make carbon quantum dot bioanalysis and in terms of have broad application prospects.Carbon dots are immune at present
In analysis using less, the application potential in immunoassay still has to be developed.
Summary of the invention
The object of the present invention is to provide a kind of fluorescence immune analysis methods based on carbon quantum dot.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a kind of fluorescent carbon quantum dot, the carbon quantum dot is
Using citric acid as carbon source, thiocarbamide or urea are what nitrogen source was prepared.Specifically, the fluorescent carbon quantum dot presses following scheme I
Or II is prepared:
Scheme I: 3-5g citric acid and 5-7g thiocarbamide are added in 15-20mL water, and stirring dissolves compound, will be mixed
Liquid is placed in microwave reactor, heats 7-10min under 800-1000W power, until solution becomes dark brown solid from colourless, so
After be cooled to room temperature, be added 25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get；
Scheme II: 3-5g citric acid and 5-7g urea are added in 15mL water, and stirring dissolves compound, by mixed liquor
It is placed in microwave reactor, heats 7-10min under 800-1000W power, until solution becomes dark brown solid from colourless, then
Be cooled to room temperature, be added 25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get.
Preferably, the fluorescent carbon quantum dot is prepared by following scheme I ' or II ':
Scheme I ': 5g citric acid and 7g thiocarbamide are added in 15mL water, and stirring dissolves compound, and mixed liquor is placed in
It in microwave reactor, heats 7 minutes under 800W power, until solution becomes dark brown solid from colourless, then cools to room temperature,
Be added 25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get；
Scheme II ': 3g citric acid and 5g urea are added in 15mL water, and stirring dissolves compound, and mixed liquor is set
It in microwave reactor, is heated 7 minutes under 800W power, until solution becomes dark brown solid from colourless, is subsequently cooled to room
Temperature, be added 25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get.
In preceding method, the filtering is preferably using 0.22 μm of film filtering.
It is preferable to use the dialysis membranes of model 3500Da to dialyse in ultrapure water at least for 24 hours for the dialysis；And/or
The chromatographic column is preferably Sephadex G25 column.
Excitation wavelength according to the fluorescent carbon quantum dot (CDs-1) of scheme I preparation is 370nm-380nm, and launch wavelength is
520nm-530nm.370nm excitation, when 520nm emits, CDs-1 absolute quantum yield is 32.6%.
Excitation wavelength according to the fluorescent carbon quantum dot (CDs-2) of scheme II preparation is 400nm-410nm, and launch wavelength is
515nm-525nm.400nm excitation, when 515nm emits, CDs-2 absolute quantum yield is 27.1%.
Second aspect, the present invention provide following any application of the fluorescent carbon quantum dot:
1. preparing the application in bioprobe, biosensor and catalytic field；
2. the application in immunoassay.
The third aspect, the present invention provide bioprobe, biosensor and the inspection prepared by the fluorescent carbon quantum dot
Test agent, kit.
Fourth aspect, the present invention provide the fluorescence immune analysis method based on carbon quantum dot, comprising the following steps:
Each reacting hole of S1, manually antigen coat ELISA Plate；
S2, specific antibody and testing sample solution are added into reacting hole, after being incubated for a period of time, add into reacting hole
The secondary antibody for entering hole horseradish peroxidase or alkali phosphatase enzyme mark continues to be incubated for；
S3,3,3 ', 5,5 '-tetramethyl biphenyl of horseradish peroxidase substrate amine aqueous solution or alkaline phosphorus are added into reacting hole
Sour zymolyte 4-NPP solution carries out chromogenic reaction；
After S4, chromogenic reaction, the fluorescent carbon quantum dot of scheme I or II preparation, detection are added into reacting hole
Fluorescence intensity；
S5, the small haptens standard solution for preparing various concentration, are detected according to S2-S4, and it is glimmering to establish reflection
The standard curve of relationship between luminous intensity and small haptens concentration；
S6, the testing result according to testing sample solution, reference standard curve obtain testing sample solution small molecular half
The concentration of antigen.
Testing principle of the invention is as follows: horseradish peroxidase is in H2O2In the presence of, it can catalysis oxidation 3,3 ', 5,5 '-
Tetramethyl benzidine (TMB) generates tetramethyl benzidine oxypolymer (TMBox).TMBox is a kind of strong extinction material, it
Absorption spectrum have with the excitation spectrum of CDs-1 it is good overlapping, can be effectively sudden to generate apparent fluorescence inner filtering effect
Go out the fluorescence of CDs-1.Similar principle, alkaline phosphatase enzymatic 4-NPP (pNPP) hydrolysis generate to nitro
Phenol (pNP) is also a kind of strong extinction material, its absorption spectrum has good overlapping with the excitation spectrum of CDs-2, is generated obvious
Fluorescence inner filtering effect, so as to effectively quench the fluorescence of CDs-2.By monitoring the fluorescent quenching rate of CDs-1/CDs-2,
Horseradish peroxidase in ELISA/alkaline phosphatase substrate for enzymatic activity can successfully be reacted to the absorbance to be formed letter
Number be effectively converted into fluorescence signal, to increase detection window, improve detection signal-to-noise ratio, and it is significant improve it is enzyme-linked
The detection sensitivity of immunoassay.
In the specific embodiment of the present invention, the fluorescence immune analysis method includes the following steps (with Buddha's warrior attendant
For alkanamine fluoroimmunoassay):
1) (amantadine) artificial antigen is diluted to concentration with coating buffer is 3.3 × 10–5Mg/mL is added by 100 holes μ L/
Enter in 96 hole elisa Plates, after 4 DEG C of incubation 12h, with cleaning solution board-washing；
2) confining liquid is added by 150 holes μ L/ to be closed, 37 DEG C of incubation 1h, with cleaning solution board-washing；
3) by specific (amantadine) antibody of 50 holes μ L/ addition, (concentration is 2.5 × 10–5Mg/mL) and by 50 holes μ L/ add
Enter testing sample solution, 37 DEG C of incubation 0.5h, specific binding reaction occurs for Ag-Ab, then with cleaning solution board-washing；
4) secondary antibody (concentration 0.1mg/mL) of horseradish peroxidase or alkali phosphatase enzyme mark is added by 100 holes μ L/,
37 DEG C of incubation 0.5h form enzyme labeled immunoassay compound, with cleaning solution board-washing；
5) by 100 holes μ L/, by horseradish peroxidase 3,3 ', 5,5 '-tetramethyl biphenyl of substrate amine aqueous solution, (concentration is
2.5mM) or alkaline phosphatase substrate 4-NPP solution (concentration 5mM) is separately added into corresponding ELISA Plate hole
In, 37 DEG C of incubation 20min；
6) the carbon quantum dot solution of 20 μ g/mL is added by 20 holes μ L/, and detects fluorescent value with multi-function microplate reader；
Specifically, CDs-2 solution is added to the enzyme mark in step 5) after horseradish peroxidase enzyme catalytic chromogenic reaction
In plate hole (or in the ELISA Plate hole after step 5) alkaline phosphatase Catalytic color reaction is added in CDs-2 solution), and
Fluorescent value is detected with multi-function microplate reader；Excitation wavelength 370nm is set, the fluorescence spectrum of CDs-1 at 400-600nm is scanned, is read
Take fluorescent value at 520nm (fluorescence spectrum of or excitation wavelength 400nm, scanning 420-600nm place CDs-2, it is glimmering at reading 515nm
Light value)；
7) the small haptens standard solution for preparing various concentration gradient, according to 3) -6) it is detected, processing gained
Data draw standard curve；
8) according to the testing result of testing sample solution, reference standard curve obtains testing sample solution small molecular half
The concentration of antigen；
Preferably, the coating buffer are as follows: the carbonate buffer solution of 10mM, pH 9.6.
The cleaning solution are as follows: the phosphate buffer of 50mM, pH 7.4 containing 0.05%Tween-20.
The confining liquid are as follows: the phosphate buffer containing 2%BSA.
Small haptens of the present invention come from mycotoxin, pesticide, veterinary drug or environmental hormone.For example, described small point
Sub- haptens includes but is not limited to amantadine, aflatoxin B1 or Ribavirin.
The artificial antigen is the conjugate of the small haptens and bovine serum albumin(BSA).For example, amantadine-ox
Seralbumin conjugate, aflatoxin B1-bovine serum albumin(BSA) conjugate or Ribavirin-bovine serum albumin(BSA) coupling
Object.
The specific antibody is the monoclonal antibody for resisting the small haptens.For example, anti-amantadine source of mouse list
Clonal antibody, aspergillus flavus resisting toxin B1 monoclonal antibody or Ribavirin monoclonal antibody.
The quantitative detection of sample small molecular haptens can be realized using the above method.When the small haptens are gold
When rigid alkanamine, aflatoxin B1 or Ribavirin, corresponding linear detection range and minimum detection limit difference are as follows:
The linear detection range of amantadine: 0.035-1.1ng/mL, minimum detection limit: 0.02ng/mL.
The linear detection range of aflatoxin B1: 0.016-0.95ng/mL, minimum detection limit: 0.012ng/mL.
The linear detection range of Ribavirin: 0.18-0.74ng/mL, minimum detection limit: 0.015ng/mL.
In the present invention, the anti-amantadine source of mouse monoclonal antibody, aspergillus flavus resisting toxin B1 monoclonal antibody and benefit bar
Wei Lin monoclonal antibody is provided by China Agricultural University's national basic veterinary drug safety evaluation center, and preparation method can be found in:
Wang,Z.,Wen,K.,Zhang,X.,Li,X.,Wang,Z.,Shen,J.,Ding,S.(2018).New
hapten synthesis,antibody production,and indirect competitive enzyme-linked
immnunosorbent assay for amantadine in chicken muscle.Food Analytical
Methods,11(1),302-308.
Zhang,X.,Song,M.,Yu,X.,Wang,Z.,Ke,Y.,Jiang,H.,Wen,K.(2017)
.Development of a new broad-specific monoclonal antibody with uniform
affinity for aflatoxins and magnetic beads-based enzymatic immunoassay.Food
Control,79,309-316.
Wang,Z.,Yu,X.,Ma,L.,Liu,H.,Ding,S.,Wang,Z.,Wen,K.(2018).Preparation
of high affinity antibody for ribavirin with new haptens and residue analysis
in chicken muscle,eggs and duck muscle.Food Additives&Contaminants:Part A,35,
1247-1256.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
(1) the highly sensitive fluoroimmunoassay new method based on carbon quantum dot that the present invention provides a kind of, has behaviour
The advantages that making simple, at low cost, high sensitivity and good stability.
(2) carbon quantum dot can be applied to enzyme linked immunological directly as fluorescence probe without additional surface-functionalized modification
In analysis, operation is simplified.
It (three), can be successfully using efficient fluorescence inner filtering effect is generated between carbon quantum dot and the chromogenic substrate of enzymatic
Horseradish peroxidase in ELISA/alkaline phosphatase substrate for enzymatic activity is reacted the absorbance signal to be formed effectively to turn
Fluorescence signal is turned to, there is good scalability, be expected to spread to other target detection systems.
(4) carbon quantum dot fluorescence quantum yield height, stable luminescence, convieniently synthesized, non-toxic is used as detection probe can
It is stable, easy to operate, low in cost, environmentally friendly in control property.
(5) present invention is suitable for the quantitative detection of small haptens, such as mycotoxin, pesticide, veterinary drug, environmental hormone
Deng, sample treatment detection processing method according to national standards.By changing using identification antibody and corresponding artificial antigen,
Using carbon quantum dot as fluorescence probe, this method extends to the non-competing enzyme linked immunological point of the objects such as protein, nucleic acid
In analysis detection.
(6) present invention at least successfully solves following technical problem: 1. because of bottom of developing the color in existing enzyme-linked immune analytic method
The technical problem that object noise is relatively low and causes detection sensitivity lower；2. conventional substitutes enzyme as signal by carbon quantum dot
Probe, applied to small haptens detection competitive immunoassay method in, the coupling efficiency of carbon quantum dot and antibody is lower,
Coupling effect is difficult to be guaranteed；3., as fluorescence signal probe, being applied to small haptens when using carbon quantum dot and detecting
Competitive immunoassay method in when, generally require to carry out carbon quantum dot complicated surface-functionalized modification, modification effect is difficult
To be guaranteed.
Detailed description of the invention
Fig. 1 is the fluorescence enzyme-linked immune analytic method schematic illustration that amantadine is detected in the embodiment of the present invention 2.
Fig. 2 is the fluorescence spectra of the carbon quantum dot synthesized in the embodiment of the present invention 1.
Fig. 3 is the transmission electron microscope picture of the carbon quantum dot synthesized in the embodiment of the present invention 1.
Fig. 4 is respectively the inspection that amantadine, aflatoxin B1 and Ribavirin concentration are detected in 2-4 of the embodiment of the present invention
Survey canonical plotting.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, test reagent consumptive material used, such as without special
Illustrate, is conventional biochemical reagent.
Quantitative test involved in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
In following embodiment, phosphate buffer (PBS, 0.05M, pH7.4's) is formulated as follows: by NaCl 40g,
Na2HPO413.5g KH2PO41.0g, KCl 1.0g are dissolved in 1L ultrapure water.
Cleaning solution: 0.05M, PBS (pH7.4)+0.05% (w/w) Tween-20.
Confining liquid: 0.05M, PBS (pH7.4)+1% (w/w) BSA solution.
Coating buffer: carbonate buffer solution (CB, 0.01M, pH 9.6), by Na2CO31.59g and NaHCO32.94g plus super
Pure water is settled to 1L.
All small haptens standard items and conventional biochemical reagent etc. involved in following embodiment are purchased from
Sigma company.
The synthesis of 1 fluorescent carbon quantum dot of embodiment
5g citric acid and 7g thiocarbamide are added in 15mL water.Being stirred continuously dissolves compound, and mixed solution is placed in
In microwave reactor, 7min is heated under 800W power, until solution becomes dark brown solid from colourless, shows that reactant is carbonized,
There is carbon quantum dot generation.After being cooled to room temperature, 25mL water is added with lysate.Prepared carbon quantum dot is passed through 0.22 μm
Film filtering is dialysed in ultrapure water at least for 24 hours to remove unreacted small point with removing bulky grain using dialysis membrane (3500Da)
Son.It is separated and is purified using carbon quantum dot solution of the Sephadex G25 column chromatography to preparation, the maximum collected
Excitation wavelength is 370nm, and maximum emission wavelength is the carbon quantum dot solution of 520nm, is named as CDs-1.
3g citric acid and 5g urea are added in 15mL ultrapure water to form clear solution.Being stirred continuously keeps compound molten
Solution, mixed solution is placed in microwave reactor, heats 7min under 800W power, until solution is consolidated from the colourless dark-brown that becomes
Body shows that reactant is carbonized, there is carbon quantum dot generation.After being cooled to room temperature, 25mL water is added with lysate.It will be prepared
Carbon quantum dot removes bulky grain by 0.22 μm of film filtering, and is dialysed at least for 24 hours using dialysis membrane (3500Da) in ultrapure water
To remove unreacted small molecule.Using carbon quantum dot solution of the Sephadex G25 column chromatography to preparation carry out separation and it is pure
Change, the maximum excitation wavelength collected is 400nm, and maximum emission wavelength is the carbon quantum dot solution of 515nm, is named as CDs-
2。
The fluorescence spectra of the carbon quantum dot of synthesis is shown in that Fig. 2, transmission electron microscope picture are shown in Fig. 3.
The detection of 2 amantadine fluoroimmunoassay of embodiment
1) amantadine artificial antigen is diluted to concentration with coating buffer is 3.3 × 10–5Mg/mL is added by 100 holes μ L/
In 96 hole elisa Plates, after 4 DEG C of incubation 12h, with cleaning solution board-washing；
2) confining liquid is added by 150 holes μ L/ to be closed, 37 DEG C of incubation 1h, with cleaning solution board-washing；
3) by the anti-amantadine antibody of 50 holes μ L/ addition specificity, (concentration is 2.5 × 10–5Mg/mL) and by 50 holes μ L/ add
Enter testing sample solution, 37 DEG C of incubation 0.5h, specific binding reaction occurs for Ag-Ab, then with cleaning solution board-washing；
4) secondary antibody (concentration 0.1mg/mL) of horseradish peroxidase or alkali phosphatase enzyme mark is added by 100 holes μ L/,
37 DEG C of incubation 0.5h form enzyme labeled immunoassay compound, with cleaning solution board-washing；
5) by 100 holes μ L/, by horseradish peroxidase 3,3 ', 5,5 '-tetramethyl biphenyl of substrate amine aqueous solution, (concentration is
2.5mM) or alkaline phosphatase substrate 4-NPP solution (concentration 5mM) is separately added into corresponding ELISA Plate hole
In, 37 DEG C of incubation 20min；
6) conversion and amplification of signal: horseradish in step 5) is added in the carbon quantum dot CDs-1 solution of 20 hole μ L/, 20 μ g/mL
In ELISA Plate hole after Catalyzed Synthesis By Peroxidase chromogenic reaction (or CDs-2 solution is added step 5) alkaline phosphatase and urges
In ELISA Plate hole after change chromogenic reaction)；And fluorescent value is detected with PerkinElmer envision multi-function microplate reader,
Set excitation wavelength 370nm, the fluorescence spectrum of scanning 400-600nm place CDs-1, fluorescent value (or excitation wavelength at reading 520nm
400nm scans the fluorescence spectrum of CDs-2 at 420-600nm, reads fluorescent value at 515nm).
7) fluoroimmunoassay standard curve is established: the amantadine standard items of known concentration gradient are according to step 3) -6)
Method is detected, processing step 6) the data obtained, draw standard curve；Shown in the standard curve of acquisition such as Fig. 4 (a and b), with
Amantadine concentration increase, the decline of carbon quantum dot fluorescent quenching rate, intensity is gradually recovered.The party is calculated to obtain by standard curve
The amantadine linear detection range of method is 0.035-1.1ng/mL, detection line, that is, IC10For 0.02ng/mL.
The fluorescence enzyme-linked immune analytic method schematic illustration that the present invention detects amantadine is shown in Fig. 1.Detect amantadine
The examination criteria curve of concentration is shown in Fig. 4-a and Fig. 4-b.Wherein, Fig. 4-a is the amantadine fluorescence based on horseradish peroxidase
ELISA standard curve, Fig. 4-b are the amantadine fluorescence ELISA standard curve based on alkaline phosphatase.
Traditional ELISA is according to step 1) -5) method detected, after the completion of enzymatic reaction, every hole is added
The concentrated sulfuric acid solution (for the chromogenic substrate of horseradish peroxidase enzyme catalytic) of 50 μ L 2mol/L, with absorbance at 450nm wavelength
Measure each hole OD value；Alternatively, 2mol/L NaOH solution is added (for alkaline phosphatase enzymatic in every hole after the completion of enzymatic reaction
Chromogenic substrate), with each hole OD value of absorbance measurement at 405nm wavelength.The amantadine standard items of known concentration gradient are according to step
Rapid 1) -5) method is detected, processing step 5) the data obtained, draw standard curve；This method is calculated to obtain by standard curve
Linear detection range be 0.23-9.1ng/mL, detection line, that is, IC10For 0.2ng/mL.
The detection of 3 aflatoxin B1 fluoroimmunoassay of embodiment
1) aflatoxin B1 artificial antigen is diluted to concentration with coating buffer is 2.3 × 10–5Mg/mL, by 100 holes μ L/
It is added in 96 hole elisa Plates, after 4 DEG C of incubation 12h, with cleaning solution board-washing；
2) confining liquid is added by 150 holes μ L/ to be closed, 37 DEG C of incubation 1h, with cleaning solution board-washing；
3) by 50 holes μ L/ addition specificity aspergillus flavus resisting toxin B1 antibody, (concentration is 2.1 × 10–5Mg/mL) and by 50 μ L/
Testing sample solution, 37 DEG C of incubation 0.5h is added in hole, and specific binding reaction occurs for Ag-Ab, then with cleaning solution board-washing；
4) secondary antibody (concentration 0.1mg/mL) of horseradish peroxidase or alkali phosphatase enzyme mark is added by 100 holes μ L/,
37 DEG C of incubation 0.5h form enzyme labeled immunoassay compound, with cleaning solution board-washing；
5) by 100 holes μ L/, by horseradish peroxidase 3,3 ', 5,5 '-tetramethyl biphenyl of substrate amine aqueous solution, (concentration is
2.5mM) or alkaline phosphatase substrate 4-NPP solution (concentration 5mM) is separately added into corresponding ELISA Plate hole
In, 37 DEG C of incubation 20min；
6) conversion and amplification of signal: horseradish in step 5) is added in the carbon quantum dot CDs-1 solution of 20 hole μ L/, 20 μ g/mL
In ELISA Plate hole after Catalyzed Synthesis By Peroxidase chromogenic reaction (or CDs-2 solution is added step 5) alkaline phosphatase and urges
In ELISA Plate hole after change chromogenic reaction)；And fluorescent value is detected with PerkinElmer envision multi-function microplate reader,
Set excitation wavelength 370nm, the fluorescence spectrum of scanning 400-600nm place CDs-1, fluorescent value (or excitation wavelength at reading 520nm
400nm scans the fluorescence spectrum of CDs-2 at 420-600nm, reads fluorescent value at 515nm).
7) fluoroimmunoassay standard curve is established: the aflatoxin B1 standard items of known concentration gradient are according to step
3) -6) method is detected, processing step 6) the data obtained, draw standard curve；The standard curve of acquisition such as Fig. 4 (c and d) institute
Show, as aflatoxin B1 concentration increases, carbon quantum dot fluorescent quenching rate decline, intensity is gradually recovered.Pass through standard curve
Calculate this method linear detection range be 0.016-0.95ng/mL, detection line, that is, IC10For 0.012ng/mL.Wherein, Fig. 4-
C is the aflatoxin B1 fluorescence ELISA standard curve based on horseradish peroxidase, and Fig. 4-d is based on alkaline phosphorus
The aflatoxin B1 fluorescence ELISA standard curve of sour enzyme.
Traditional ELISA is according to step 1) -5) method detected, after the completion of enzymatic reaction, every hole is added
The concentrated sulfuric acid solution (for the chromogenic substrate of horseradish peroxidase enzyme catalytic) of 50 μ L 2mol/L, with absorbance at 450nm wavelength
Measure each hole OD value；Alternatively, 2mol/L NaOH solution is added (for alkaline phosphatase enzymatic in every hole after the completion of enzymatic reaction
Chromogenic substrate), with each hole OD value of absorbance measurement at 405nm wavelength.The amantadine standard items of known concentration gradient are according to step
Rapid 1) -5) method is detected, processing step 5) the data obtained, draw standard curve；This method is calculated to obtain by standard curve
Linear detection range be 0.14-3.75ng/mL, detection line, that is, IC10For 0.1ng/mL.
The detection of 4 Ribavirin fluoroimmunoassay of embodiment
1) Ribavirin artificial antigen is diluted to concentration with coating buffer is 3.0 × 10–5Mg/mL is added by 100 holes μ L/
In 96 hole elisa Plates, after 4 DEG C of incubation 12h, with cleaning solution board-washing；
2) confining liquid is added by 150 holes μ L/ to be closed, 37 DEG C of incubation 1h, with cleaning solution board-washing；
3) by the anti-Ribavirin antibody of 50 holes μ L/ addition specificity, (concentration is 1.5 × 10–5Mg/mL) and by 50 holes μ L/ add
Enter testing sample solution, 37 DEG C of incubation 0.5h, specific binding reaction occurs for Ag-Ab, then with cleaning solution board-washing；
4) secondary antibody (concentration 0.1mg/mL) of horseradish peroxidase or alkali phosphatase enzyme mark is added by 100 holes μ L/,
37 DEG C of incubation 0.5h form enzyme labeled immunoassay compound, with cleaning solution board-washing；
5) by 100 holes μ L/, by horseradish peroxidase 3,3 ', 5,5 '-tetramethyl biphenyl of substrate amine aqueous solution, (concentration is
2.5mM) or alkaline phosphatase substrate 4-NPP solution (concentration 5mM) is separately added into corresponding ELISA Plate hole
In, 37 DEG C of incubation 20min；
6) conversion and amplification of signal: horseradish in step (5) is added in the carbon quantum dot CDs-1 solution of 20 hole μ L/, 20 μ g/mL
In ELISA Plate hole after Catalyzed Synthesis By Peroxidase chromogenic reaction (or CDs-2 solution is added step (5) alkaline phosphatase and urges
In ELISA Plate hole after change chromogenic reaction)；And fluorescent value is detected with PerkinElmer envision multi-function microplate reader,
Set excitation wavelength 370nm, the fluorescence spectrum of scanning 400-600nm place CDs-1, fluorescent value (or excitation wavelength at reading 520nm
400nm scans the fluorescence spectrum of CDs-2 at 420-600nm, reads fluorescent value at 515nm).
7) fluoroimmunoassay standard curve is established: the ribavirin standard product of known concentration gradient are according to step 3) -6)
Method is detected, processing step 6) the data obtained, draw standard curve；Shown in the standard curve of acquisition such as Fig. 4 (e and f), with
Ribavirin concentration increase, the decline of carbon quantum dot fluorescent quenching rate, intensity is gradually recovered.The party is calculated to obtain by standard curve
The linear detection range of method is 0.18-0.74ng/mL, detection line, that is, IC10For 0.015ng/mL.Wherein, Fig. 4-e is based on horseradish
The Ribavirin fluorescence ELISA standard curve of peroxidase, Fig. 4-f are the Ribavirin based on alkaline phosphatase
Fluorescence ELISA standard curve.
Traditional ELISA is according to step 1) -5) method detected, after the completion of enzymatic reaction, every hole is added
The concentrated sulfuric acid solution (for the chromogenic substrate of horseradish peroxidase enzyme catalytic) of 50 μ L 2mol/L, with absorbance at 450nm wavelength
Measure each hole OD value；Alternatively, 2mol/L NaOH solution is added (for alkaline phosphatase enzymatic in every hole after the completion of enzymatic reaction
Chromogenic substrate), with each hole OD value of absorbance measurement at 405nm wavelength.The ribavirin standard product of known concentration gradient are according to step
Rapid 1) -5) method is detected, processing step 5) the data obtained, draw standard curve；This method is calculated to obtain by standard curve
Linear detection range be 0.23ng/mL-8.25ng/mL, detection line, that is, IC10For 0.15ng/mL.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. fluorescent carbon quantum dot, which is characterized in that the fluorescent carbon quantum dot is prepared by following scheme I or II:
Scheme I: 3-5g citric acid and 5-7g thiocarbamide are added in 15-20mL water, and stirring dissolves compound, and mixed liquor is set
In microwave reactor, 7-10min is heated under 800-1000W power, until solution becomes dark brown solid from colourless, it is then cold
But to room temperature, be added the dissolution of 25mL water, gained carbon quantum dot solution through filtering, dialysis and chromatography to get；
Scheme II: 3-5g citric acid and 5-7g urea are added in 15mL water, and stirring dissolves compound, and mixed liquor is placed in
In microwave reactor, 7-10min is heated under 800-1000W power, until solution becomes dark brown solid from colourless, is then cooled down
To room temperature, be added the dissolution of 25mL water, gained carbon quantum dot solution through filtering, dialysis and chromatography to get.
2. fluorescent carbon quantum dot according to claim 1, which is characterized in that the fluorescent carbon quantum dot presses following scheme I '
Or II ' is prepared:
Scheme I ': 5g citric acid and 7g thiocarbamide are added in 15mL water, and stirring dissolves compound, and mixed liquor is placed in microwave
It in reactor, heats 7 minutes under 800W power, until solution becomes dark brown solid from colourless, then cools to room temperature, be added
25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get；
Scheme II ': 3g citric acid and 5g urea are added in 15mL water, and stirring dissolves compound, mixed liquor are placed in micro-
It in wave reactor, is heated 7 minutes under 800W power, until solution becomes dark brown solid from colourless, then cools to room temperature, add
Enter 25mL water dissolution, gained carbon quantum dot solution through filtering, dialysis and chromatography to get.
3. fluorescent carbon quantum dot according to claim 1, which is characterized in that the filtering is using 0.22 μm of film filtering；With/
Or
The dialysis is dialysed at least for 24 hours using the dialysis membrane of model 3500Da in ultrapure water；And/or
The chromatographic column is Sephadex G25 column.
4. fluorescent carbon quantum dot according to claim 1-3, which is characterized in that the fluorescence carbon amounts of scheme I preparation
The excitation wavelength of son point is 370-380nm, launch wavelength 520-530nm, and preferably excitation wavelength is 370nm, and launch wavelength is
520nm；Scheme II preparation fluorescent carbon quantum dot excitation wavelength be 400-410nm, launch wavelength 515-525nm, preferably
Excitation wavelength is 400nm, launch wavelength 515nm.
5. following any application of any one of the claim 1-4 fluorescent carbon quantum dot:
1. preparing the application in bioprobe, biosensor and catalytic field；
2. the application in immunoassay.
6. the fluorescence immune analysis method based on carbon quantum dot, which comprises the following steps:
Each reacting hole of S1, manually antigen coat ELISA Plate；
S2, specific antibody and testing sample solution are added into reacting hole, after being incubated for a period of time, the adding hole into reacting hole
The secondary antibody of horseradish peroxidase or alkali phosphatase enzyme mark continues to be incubated for；
S3,3,3 ', 5,5 '-tetramethyl biphenyl of horseradish peroxidase substrate amine aqueous solution or alkaline phosphatase are added into reacting hole
Substrate 4-NPP solution carries out chromogenic reaction；
After S4, chromogenic reaction, any one of the claim 1-4 fluorescent carbon quantum dot is added into reacting hole, detects glimmering
Luminous intensity；
S5, the small haptens standard solution for preparing various concentration, are detected according to S2-S4, and it is strong to establish reflection fluorescence
The standard curve of relationship between degree and small haptens concentration；
S6, the testing result according to testing sample solution, reference standard curve obtain testing sample solution small molecular haptens
Concentration.
7. according to the method described in claim 6, characterized by comprising the following steps:
1) artificial antigen is diluted to concentration 3.3 × 10 with coating buffer–5Mg/mL is added in 96 hole elisa Plates, 4 by 100 holes μ L/
DEG C be incubated for 12h after, with cleaning solution board-washing；
2) confining liquid is added by 150 holes μ L/ to be closed, 37 DEG C of incubation 1h, with cleaning solution board-washing；
3) concentration 2.5 × 10 is added by 50 holes μ L/–5The specific antibody of mg/mL and by 50 holes μ L/ be added testing sample solution,
Specific binding reaction occurs for 37 DEG C of incubation 0.5h, Ag-Ab, then with cleaning solution board-washing；
4) horseradish peroxidase of concentration 0.1mg/mL or the secondary antibody of alkali phosphatase enzyme mark are added by 100 holes μ L/, 37 DEG C incubate
0.5h is educated, enzyme labeled immunoassay compound is formed, with cleaning solution board-washing；
5) by 100 holes μ L/ by horseradish peroxidase 3,3 ', 5,5 '-tetramethyl biphenyl of substrate amine aqueous solution of concentration 2.5mM or dense
The alkaline phosphatase substrate 4-NPP solution of degree 5mM is separately added into corresponding ELISA Plate hole, 37 DEG C of incubations
20min；
6) the carbon quantum dot solution of 20 μ g/mL is added by 20 holes μ L/, and detects fluorescent value with multi-function microplate reader；
7) the small haptens standard solution for preparing various concentration gradient, according to 3) -6) it is detected, handle institute's total
According to drafting standard curve；
8) according to the testing result of testing sample solution, reference standard curve obtains testing sample solution small molecular haptens
Concentration；
Wherein, the coating buffer are as follows: the carbonate buffer solution of 10mM, pH 9.6；
The cleaning solution are as follows: the phosphate buffer of 50mM, pH 7.4 containing 0.05%Tween-20；
The confining liquid are as follows: the phosphate buffer containing 2%BSA.
8. according to the method described in claim 6, it is characterized in that, the small haptens come from mycotoxin, pesticide, beast
Medicine or environmental hormone, preferably amantadine, aflatoxin B1 or Ribavirin.
9. according to the method described in claim 6, it is characterized in that, the artificial antigen is the small haptens and ox blood
The conjugate of pure albumen；And/or
The specific antibody is the monoclonal antibody for resisting the small haptens.
10. the method according to claim 6, which is characterized in that when the small haptens are adamantane
When amine, aflatoxin B1 or Ribavirin, corresponding linear detection range and minimum detection limit difference are as follows:
The linear detection range of amantadine: 0.035-1.1ng/mL, minimum detection limit: 0.02ng/mL；
The linear detection range of aflatoxin B1: 0.016-0.95ng/mL, minimum detection limit: 0.012ng/mL；
The linear detection range of Ribavirin: 0.18-0.74ng/mL, minimum detection limit: 0.015ng/mL.
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