CN102353768A - Quantum dot based immunofluorescence detection method for malachite green and special kit - Google Patents
Quantum dot based immunofluorescence detection method for malachite green and special kit Download PDFInfo
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Abstract
The invention discloses a quantum dot based immunofluorescence detection method for malachite green and a special kit. The immunofluorescence detection kit for detecting malachite green provided by the invention comprises a malachite green coating antigen and malachite green antibody labelled by a quantum dot. The method can realize quantitative determination on malachite green residual, has low detection limit, high sensitivity and good singularity, and is suitable for detections of a plurality of samples. Accordingly, the invention has wide application prospect in the field of malachite green detection.
Description
Technical field
The present invention relates to a kind of method and dedicated kit that detects malachite green based on the immunofluorescence of quantum dot.
Background technology
Malachite green (Malachite Green; MAL) have another name called Viride Nitens, green, the peacock green of salt matrix; Aniline green, Victoria green or China are green etc., belong to triphenylmethane dye, saprolegniasis, branchiomycosis and the ich etc. that once were widely used in preventing and treating all kinds of aquatic livestocks.But malachite green and metabolic product leucomalachite green thereof can produce high residue in the aquatic products body.Be prone to cause spinoffs such as carcinogenic, teratogenesis and mutagenesis after the people is edible.European Union, the U.S. and part East Asian countries have forbidden all that with the antifungal drug of malachite green as the fishing class China was also listed malachite green in " veterinary drug and the compound inventory thereof of food animal forbidding " in May, 2002.At present, the goldstandard that is used for malachite green vestigial thing detection by quantitative is chromatogram/mass spectroscopy, but its sample pretreatment process is loaded down with trivial details time-consuming, and testing cost is high.ELISA method based on competitiveness enzyme-linked immune response principle need not carried out complicated sample pre-treatment process, and detection time is short relatively, but its precision is not enough, can only be used for qualitatively screening, can't be used for quantitative conclusive evidence.The sample size that detects malachite green vestigial and pollution situation investigation thereof in the current outlet aquatic products is big, and task is heavy, and cost is high, and sense cycle is long.Therefore, be badly in need of stable, quick, the economic standard method of testing result.
(Quantum Dots, QDs) marker material is one type of new material that development in recent years is got up to quantum dot, comprises II-VI family and III-V family semiconductor nano.Because the luminous and absorption characteristic that quantum dot is outstanding; Make its have fluorescence lifetime length and width excitation spectrum, narrow emission spectrum, can be accurately tuning emission wavelength, very high photochemical stability, can carry out advantageous characteristic such as multi-color marking; Adopt its material that serves as a mark, can realize the ultramicron of target molecules is detected.
Requirements such as quantum yield height, fluorescence are strong because the quantum dot-labeled material that is applicable to immune diagnostic reagent must satisfy, good biocompatibility, highly stable and cost are low; And existing external its quantum yield of business-like quantum dot is many below 40%; Its size is less than normal; Need shine with laser optical apparatus and excite; Only can be applied in cellular immunofluorescence detection and aspects such as fluidic cell detection and sorting at present, so also not have the quantum dot immune detectable to come out on the international market.
Summary of the invention
An object of the present invention is to provide a kind of immunofluorescence detection agent box that is used to detect malachite green.
The immunofluorescence detection agent box that is used to detect malachite green provided by the present invention comprises malachite green coating antigen and quantum dot-labeled malachite green antibody.
In the mentioned reagent box, said malachite green antibody is that antibody titer is 10
6More than (be specially 10
6), the antibody affinity costant is 10
6～10
8M
-1Or 10
7～10
8M
-1Or 10
8M
-1The malachite green monoclonal antibody; Being specially antibody titer is 10
6, the antibody affinity costant is 10
8M
-1The malachite green monoclonal antibody, said malachite green monoclonal antibody steps the emerging Bioisystech Co., Ltd of clone available from Beijing, catalog number is 0010.
Said quantum dot is that surperficial carboxyl-content is 1 * 10
-3～9 * 10
-3Mmol/mg or 1 * 10
-3～6 * 10
-3Mmol/mg or 5 * 10
-3The quantum dot of the water-soluble CdSe of mmol/mg/ZnS nucleocapsid structure; The quantum yield of said quantum dot is 40～70% or 50～70% or 60%; The excitation wavelength of said quantum dot is 345nm, and emission wavelength is 620nm.
In above-mentioned arbitrary said kit, the particle diameter of said quantum dot is 10～20nm, and said particle diameter is specially 13～20nm, and said particle diameter especially is preferably 20nm; The deviation of its particle diameter (CV) is specially between 10～20% between 10～30%, is specially 15% again;
In above-mentioned arbitrary said kit, in the said quantum dot-labeled malachite green antibody, the amino on carboxyl on the said quantum dot and the said malachite green antibody forms peptide bond, and then said quantum dot is connected with said malachite green antibody.
In above-mentioned arbitrary said kit; Said malachite green coating antigen is the conjugate of malachite green and carrier protein, and wherein said malachite green and said carrier protein are connected through the peptide bond that the carboxyl of the introducing in the malachite green and amino in the carrier protein form; Said carrier protein is any in the gamma globulin of bovine serum albumin(BSA), human serum albumins, keyhole limpet hemocyanin, thyroglobulin, albumin rabbit serum, ovalbumin, fibrinogen and rabbit and chicken.
In above-mentioned arbitrary said kit, also comprise malachite green standard items, dilution, cleansing solution in the said kit, contain porose polystyrene board, encapsulate damping fluid and confining liquid;
Said malachite green standard items are that tetramethyl is for the diamido triphenylmethane;
Said dilution is the PBS damping fluid; Be specially the PBS damping fluid of 0.02M, pH7.4.
Said cleansing solution is the PBST damping fluid; Specifically according to following method preparation: the NaN3 that gets 0.2ml Tween20 and 0.1g is dissolved in the said dilution, and the dissolving back is settled to 1L with dilution.
The said damping fluid that encapsulates is a carbonate buffer solution; Be specially the carbonate buffer solution of 0.05M, pH9.6.
Said confining liquid is to contain the PBS damping fluid that is useful on the albumen that encapsulates; The said albumen that is used for encapsulating is any of BSA, ovalbumin and hemocyanin.Specifically according to following method preparation: 10g BSA and 0.2mlTween20 are dissolved in the above-mentioned dilution, and the dissolving back is settled to 1L with dilution.
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L in above-mentioned arbitrary said kit, said standard items are the standard items of following solution form: the solution that said tetramethyl is diluted to following each concentration for the diamido triphenylmethane with said dilution:.
In above-mentioned arbitrary said kit, said malachite green coating antigen is coated on said containing on the porose polystyrene board, and method for coating adds 100 μ l for to dilute the coating buffer that said malachite green coating antigen obtains 10 μ g/ml with the said damping fluid that encapsulates in every hole.
In above-mentioned arbitrary said kit, said quantum dot-labeled malachite green antibody is present in the kit with following solution form: the solution that the said quantum dot-labeled malachite green antibody of every 25ug is obtained with the said diluted of 5ml.
Another object of the present invention provides the method for malachite green in a kind of test sample.
The method of malachite green in the test sample provided by the present invention; Comprise the steps: testing sample to be detected with above-mentioned arbitrary described kit; Said testing sample is animal muscle tissue's sample; Be specially can practical seafood edible muscle, be specially the edible muscle of fish, shrimp, crab or soft-shelled turtle again.
Detection side's ratio juris of the present invention: adopt quantum dot as the fluorescence signal labeled molecule; With malachite green antigen direct coated in the micropore of polystyrene board; Add malachite green standard items or test sample; And quantum dot-labeled malachite green antibody; Make it form Ag-Ab binary electrochemiluminescent immunoassay compound; Excite and detect the fluorescence intensity of this immunofluorescence compound with fluorescence detector, through with measure the concentration that the typical curve contrast that forms obtains malachite green to be measured.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled malachite green antibody; The malachite green antigen that encapsulates combines malachite green antibody with malachite green in the test sample competitively, and the specificity through Ag-Ab combines to form Ag-Ab binary immune complex.
The formation of Ag-Ab binary electrochemiluminescent immunoassay compound is after adding quantum dot-labeled malachite green antibody and test sample simultaneously; Be coated on malachite green antigen and competitive ground of the malachite green in the test sample and malachite green antibodies in the polystyrene micropore, formation is fixed on the Ag-Ab binary immune complex in the microwell plate after wherein combining remaining malachite green antibody with test sample and being coated on the malachite green antigen generation specific bond in the microwell plate.
Malachite green immunofluorescence detection agent of the present invention is relevant with quantum dot-labeled immunofluorescence detection technique; Be to adopt quantum dot as the fluorescence signal marker material; Carry out class methods of immunofluorescence quantitative measurement, this technology has been integrated the research of association areas such as the chemosynthesis of fluorescence quantum nano material, finishing and labelling technique, indirect competition formula immunoassay technology.
Why the present invention can detect malachite green; Be to adopt a kind of method based on quantum dot-labeled immunofluorescence quantitative assay; Be about to malachite green antigen direct coated in the micropore of XPS; Measuring principle based on quantum dot-labeled indirect competition immunofluorescence assay; Adding malachite green standard items or test sample; And behind the quantum dot-labeled malachite green antibody; The fluorescence intensity that is attached to the Ag-Ab binary immune complex in the XPS micropore by detection realizes the detection to malachite green: be attached to the quantity difference of the quantum dot-labeled antibody of XPS micropore, the fluorescence intensity that is produced is also different.The content of the malachite green in the finite concentration scope in the height of fluorescence intensity level and the sample is inversely proportional to.Malachite green standard items through adding variable concentrations can be made into typical curve, can obtain the malachite green drug concentrations value of correspondence according to the fluorescence intensity level of this each test sample of typical curve inquiry.
Its concrete technical step comprises:
(1) preparation of fluorescence quantum point mark probe: the water soluble fluorescence quantum dot that adopt to be fit to, activate its surperficial carboxyl after, adopt the mode of chemical coupling that malachite green antibody orientation is connected to the quantum dot surface.
(2) envelope antigen: adopt malachite green antigen with the bovine serum albumin(BSA) coupling as envelope antigen, the method through physisorption with this antigen direct coated in the polystyrene board micropore.
(3) formation of Ag-Ab fluorescence immunoassay compound: in the above-mentioned polystyrene board micropore that encapsulates, add malachite green standard items or test sample; And quantum dot-labeled malachite green antibody; The malachite green antigen that is adsorbed in the hole combines with malachite green antibody with the malachite green in standard or the sample is emulative, through the specificity combination formation Ag-Ab binary electrochemiluminescent immunoassay compound of Ag-Ab.
(4) quantitative fluorescence detects: adopt fluorescence microplate reader to excite and detect the fluorescence intensity of above-mentioned formed Ag-Ab fluorescence immunoassay compound; Excitation wavelength: 345nm; Emission wavelength: 620nm; Fluorescence intensity through measuring serial corresponding standard items forms typical curve, through contrasting the concentration that obtains malachite green to be measured with the typical curve of measuring formation.
The formation of said Ag-Ab binary electrochemiluminescent immunoassay compound is: after adding quantum dot-labeled malachite green antibody; The malachite green antigen that encapsulates combines malachite green antibody with malachite green in standard or the sample competitively; Specificity through Ag-Ab combines to form Ag-Ab binary immune complex; The quantum dot of its marked can fluoresce after exciting; The excitation wavelength that the present invention uses is 345nm; Emission wavelength is 620nm, the Ag-Ab immune complex that obtains glowing.
The detection of said fluorescence intensity is the fluorescence intensity that excites and detect formed Ag-Ab binary electrochemiluminescent immunoassay compound with fluorescence microplate reader; Because the antibody that is adopted is fixed concentration; Usually the malachite green drug concentrations in the testing sample is high more; Many more by the medication amount of antibody capture; The antibody that combines with the malachite green antigen that encapsulates is few more, and the fluorescence intensity level that records is low more.
Because quantum dot carrying out will carrying out separation and purification with ultracentrifugation in the coupling with antibody, quantum dot that particle diameter is too little such as 8nm and 10nm's can't be centrifugal, can't purifying after the coupling, and result of use is relatively poor; Quantum dot that particle diameter is too big such as the ratio more than the 60nm are easier to assemble, and it is relatively poor to be used on the kit homogeneity.
The particle diameter of said quantum dot is 10～20nm, and said particle diameter is specially 13～20nm, and said particle diameter especially is preferably 20nm; The deviation of its particle diameter (CV) is preferably between 10～20% between 10～30%, and preferred 15%.
The fluorescence quantum yield of quantum dot and fluorescence intensity thereof have directly determined the height of detection sensitivity and accuracy thereof, and the fluorescence quantum yield of the quantum dot of classic method preparation all is lower than 40% usually, fluorescence intensity a little less than.
For improving its sensitivity and accuracy, the quantum yield of said quantum dot is 40～70%, and the fluorescence quantum yield of said quantum dot is specially 50～70%, and the fluorescent quantum product of said quantum dot especially is preferably 60%.
Survey for being used for the malachite green vestigial quality testing; The quantum dot surface need have the group that is easy to the malachite green antibody coupling; These groups can be carboxyl, amino groups; The group of optimizing is the surface functional group of band carboxyl; Usually adopt chemical method to connect antibody; Promptly, close reaction and accomplish coupling reaction with antibody generation carboxylic again with behind EDC and the NHS activation quantum dot.
Before with malachite green antibody and the condensate of quantum dot, also comprise the step that activates said quantum dot surface functional group with the formation of peptide bond covalent bond.The carboxyl-content difference on quantum dot surface can have influence on the sensitivity of detection, and for improving sensitivity, said functional group is specially carboxyl, and the content of said carboxyl is 1 * 10
-3～9 * 10
-3Mmol/mg, the content of said carboxyl is specially 1 * 10
-3～6 * 10
-3Mmol/mg, the content of said carboxyl especially is preferably 5 * 10
-3Mmol/mg.
In immune detection, the performance index of antibody are most important for the accuracy that detects, and usually, high specificity, the antibody that affinity is high can improve the accuracy of detection significantly.Discover that for improving sensitivity, said malachite green antibody affinity costant is 10
6～10
8M
-1Said malachite green antibody affinity costant is specially 10
7～10
8M
-1Said malachite green antibody affinity costant especially is preferably 10
8M
-1
Because malachite green is a small-molecule substance; Its molecular surface characteristic is unfavorable for combining with the direct of polystyrene micropore plate, itself and carrier protein need be carried out could reaching by means of the character of surface of carrier protein after the coupling good combination with polystyrene micropore plate.The seralbumin that various animals are arranged that can be used as carrier protein; Like bovine serum albumin(BSA) (Bovine Serum Albumin; BSA), human serum albumins (Human Serum Albumin; HSA); Also has keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin; KLH), thyroglobulin, albumin rabbit serum (RSA), ovalbumin (Ovalbumin, OVA), the gamma globulin of fibrinogen or rabbit and chicken.Discover; The BSA physicochemical property is stable; Lysine content is high; Free amino group is many; Bigger solubleness is all arranged under different pH and ionic strength, under the situation that contains organic solvent (like pyridine, DMF etc.), all can carry out coupling, and after coupling, still keep solvable state with haptens; Be splendid selection, so the present invention selects for use BSA as coupling protein as carrier protein.
The present invention passes through fluorescence quantum, malachite green antigen and malachite green antibody molecule The Characteristic Study; Through optimization to the preparation of various water soluble fluorescence quantum dot, coating and finishing condition; Water soluble fluorescence quantum dot and the specific antibody selecting to be fit to carry out directed covalent chemical coupling; Obtain functional fluorescence quantum point mark probe; And, reach quick and highly sensitive quantitative measurement to the malachite green vestigial medicine through optimizing the various conditions of competitive immunization reaction.Experiment showed, kit of the present invention highly sensitive, specificity good, accuracy is high.The inventive method can realize the detection by quantitative to the malachite green vestigial medicine, and detectability is low, detection sensitivity is high, specificity is good, also is applicable to the detection of several samples.Therefore, the present invention has broad application prospects at the detection range of malachite green.
Description of drawings
Fig. 1 water-soluble CdSe/ZnS fluorescence quantum Electronic Speculum (TEM) photo.
Fig. 2 detects the typical curve of malachite green with quantum dot-labeled indirect competition immunofluorescence technique.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The composition of embodiment 1, detection kit and preparation
Malachite green opens a day chemical industry Institute for Research and Technology available from the permanent unit in Beijing, and catalog number is 1245 BW3811.Its chemical name is that tetramethyl is for the diamido triphenylmethane.
1, malachite green coating antigen
The conjugate of malachite green and carrier protein BSA, malachite green and BSA are connected through the peptide bond that carboxyl in the malachite green intermediate and amino among the BSA form.
The intermediate of earlier synthetic malachite green is introduced carboxyl, again with albumen coupling.
Concrete steps are:
1) in there-necked flask, injects the 60ml absolute ethyl alcohol; Beyond the Great Wall with the plug of thermometer with the control temperature; Install condensing reflux pipe and nitrogen inlet; Slowly heat up; Prevent bumping; Treat that the stable boiling of alcohol (85 ℃) under bottle interior stirrer rotation begins slowly to open nitrogen valve, control speed air-flow can in bottle, be stablized bloats bubble; After 3 minutes; Treat that bottle interior air is replaced nitrogen protection atmosphere of formation by nitrogen fully, adds the anhydrous ZnCl2 of the about 18mmol of 24g and makees catalyzer, and it is dissolved fully; Add catalytic amount (about 5mg) solid strong acid ion exchange resin (Aladdin company; 1098585), add the about 6mmol 4-of 0.9g to carboxyl benzaldehyde, and make it abundant mixing; Add the about 19mmol N of 2.4ml; The N-dimethylaniline keeps above conditioned response 25h, obtains reactant liquor a;
2) after reaction finishes; Cooling reaction liquid a leaves standstill 12 hours (room temperature), and solution becomes bottle green by light green in the bottle, after 62.4ml cools off and adds 2ml 1mol/L hydrochloric acid and 30ml absolute methanol again among the afterreaction liquid a; Bottle green crystallization and colloid substance slowly occur in the bottle, obtain carboxylated malachite green;
3) get 82mg (about 0.2mmol) step 2) the carboxylated malachite green that obtains is dissolved in 8ml DMF (N; The N-dimethylaniline) makes it abundant dissolving in; Add 20.6mg (about 0.1mmol) DCC (N; The N-dicyclohexyl carbodiimide) activates 15min down in room temperature (25 ℃); Add NHS (N-maloyl imines) 28.6mg (about 0.2mmol) and react 1h down in room temperature (25 ℃); Change 4 ℃ of reaction 1.5h down afterwards over to, the malachite green that obtains activating;
4) get 100mg BSA and be dissolved in 8ml, the malachite green 0.1mol/L that obtains activating, pH in 8.5 the dobell's solution, obtain the BSA BAS, preserve for use down for 4 ℃;
5) 4 ℃ of reaction 3.5h in the malachite green of the activation that every 82mg step 3) the is obtained BSA BAS that slow adding 8ml step 4) obtains under ice bath;
6) product is in 0.02mol/L, and dialysis is 24 hours among the pH=7.5 PBS, and every 6h changes time dislysate, and products obtained therefrom freeze dryer freeze-drying is in-20 ℃ of preservations.
2, the malachite green coating antigen encapsulates
Employing encapsulates damping fluid the dilution of malachite green coating antigen is the coating buffer of concentration 10 μ g/ml, and every hole adds 100 μ l in 96 hole polystyrene micropore laths, places in 4 ℃ of refrigerators and spends the night.Discarded coating buffer in second day, with behind each plate hole of cleansing solution flushing 3 times, each hole adds 200 μ l confining liquids, handles 2h in 37 ℃ of sealings.Discard confining liquid afterwards, with behind each plate hole of cleansing solution flushing 3 times, carry out vacuum drain, with being positioned over-20 ℃ of preservations after the aluminium foil bag sealing.
3, quantum dot-labeled malachite green antibody:
Malachite green antibody is 10 for tiring
6, affinity costant is 10
8M
-1The malachite green monoclonal antibody, it steps the emerging Bioisystech Co., Ltd of clone available from Beijing, catalog number is 0010.
Quantum dot is available from Shenzhen's TELUS Science and Technology Ltd., and catalog number does
LumiQD
TM20.The sign of this quantum dot is following: particle diameter is that the CV of 20nm, particle diameter is 15%, and quantum yield is 60%, and surperficial carboxyl-content is 5 * 10
-3Mmol/mg, water-soluble, CdSe/ZnS nucleocapsid structure, excitation wavelength are 345nm, emission wavelength is 620nm; The red fluorescence quantum dot.The scintigram of quantum dot as shown in Figure 2.
In the said quantum dot-labeled malachite green antibody, the amino on carboxyl on the said quantum dot and the said malachite green antibody forms peptide bond, and then said quantum dot is connected with said malachite green antibody.
Quantum dot marking method is:
1) get the above-mentioned quantum dot of 2.5mg with the MES damping fluid of 0.1M (take by weighing 1.066g MES, 0.45g NaCl is dissolved in the 50ml pure water; Transfer pH to 4.7) washing and remove supernatant with the 20000rpm centrifugal enrichment after; Using 1ml concentration is that 4.7 MES damping fluid is resuspended as 0.1M, pH value, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15mg (final concentration is 10mM) the N-maloyl imines (NHS) that adds 0.96mg (final concentration is 5mM) is in wherein.Temperature of reaction is 37 ℃, the reaction half an hour after, the quantum dot after obtaining activating;
2) with the washing of the borate buffer solution of 50mM pH=8.5, get 0.15mg malachite green monoclonal antibody and 2.5mg activation afterwards the quantum dot borate buffer solution that is mixed into 0.8ml 50mM pH=8.5 (take by weighing 1.9g Na
2B
4O
7.10H
2O is dissolved in the 100ml pure water, transfers pH to 8.5) in abundant mixing.Down reaction 3.5 hours of room temperature (25 ℃) allows antibody and quantum dot form stable peptide bond covalent bond, obtains containing the reactant liquor of quantum dot after the coupling;
3) after reaction finishes; To step 2) to add final concentration in the reactant liquor that obtains be the BSA (Sigma-Aldrich of 5% (quality percentage composition); 85041C) the residual activity amino sites is sealed, be reflected at and carried out under 37 ℃ 0.5 hour, obtain containing the reactant liquor of sealing back quantum dot; After the completion, (take by weighing 2.3g Na with the 0.02M PBS damping fluid of pH=7.4
2HPO
4, 0.524g NaH
2PO
4.H
2O, 8.77g NaCL are dissolved in the 1L pure water, transfer pH to 7.4) washing, the 20000rpm centrifugal enrichment is removed supernatant, and resuspended back 4 ℃ of preservations are for use, obtain quantum dot-labeled malachite green antibody.
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L 4, malachite green standard items: the solution that malachite green is diluted to following each concentration with dilution:;
5, the PBS damping fluid of dilution: 0.02M, pH7.4;
Preparation: take by weighing 2.3g Na
2HPO
4, 0.524g NaH
2PO
4.H
2O and 8.77g NaCL are dissolved in the 1L pure water, transfer pH to 7.4.
6, cleansing solution: PBST damping fluid; Get the NaN of 0.2ml Tween20 and 0.1g
3Be dissolved in the above-mentioned PBS damping fluid, the dissolving back is settled to 1L with above-mentioned PBS damping fluid.
7, encapsulate damping fluid: the carbonate buffer solution of 0.05M, pH9.6.
8, confining liquid: 10g BSA and 0.2ml Tween20 are dissolved in the above-mentioned PBS damping fluid, and the dissolving back is settled to 1L with the PBS damping fluid.
The preparation method of embodiment 2, typical curve
In the malachite green micropore lath for preparing, add concentration and be 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5, the malachite green standard solution of 10ug/L; The 50ul/ hole; Quantum dot-labeled MAL antibody is diluted [being the 25ug labelled antibody: the 5ml dilution] with the PBS-T dilution at 1: 50; Add 50ul in each micropore; Room temperature vibration 1 hour detects its fluorescence intensity numerical value with fluorescence microplate reader after cleansing solution is washed 3 times.Fluorescence microplate reader is set to excitation wavelength 345nm, emission wavelength 620nm.
The mass concentration of the competition medicine of (B0 is 0 standard sample detected value, and B is for treating the test sample detected value) was confirmed the sensitivity of detection architecture when the detectability of competing detection was decided to be B0/B=1.2 according to Regression Equations.Testing result is as shown in table 1 below, and detection limit is decided to be 0.005ug/L.
The quantum dot kit detected value of table 1 malachite green variable concentrations sample
The mensuration of embodiment 3, cross reaction
Select concealed malachite green (through oxidation), crystal violet, recessive crystal violet (through oxidation), be made into series concentration respectively, detect with the quantum dot kit bar.Calculate the IC50 that respectively competes thing, calculate the cross reacting rate of these 5 kinds of medicines and malachite green quantum dot kit with following formula respectively.Computing formula is: cross reacting rate (%)=[IC50 (malachite green)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.The result shows that the malachite green quantum dot kit is complete intersection after oxidation to concealed malachite green, and crystal violet and the recessive crystal violet crossing-over rate after oxidation are reached 92%.
The cross reaction of table 2 malachite green quantum dot kit and other medicines
The mensuration of embodiment 4, accuracy
(1) sample extraction:
Fish scales, skin, gets muscle along ridge; Shrimp decaptitates, shell, gets edible muscle parts; Crab, soft-shelled turtle etc. are got edible part.Take by weighing sample 5g (being accurate to 0.001g); Place the homogenate cup; In cup, add 1mL oxammonium hydrochloride solution (0.25g/ml), 1mL p-toluenesulfonic acid solution (0.05mol/L) and 2mL ammonium acetate solution (0.1mol/L) and 10mL acetonitrile successively; Homogenate 2min; Add the 5g alkali alumina; With the oscillator 5min that vibrates, centrifugal 10 minutes of 4000g gets supernatant.Add the 10ml methylene chloride, 10ml water, vibration 3min, standing demix is collected lower floor's organic phase, and nitrogen dries up, and behind 1mL 0.02mol/L PBS dissolving residue, gets 50ul and is used for detecting.
(2) mensuration of the recovery
Detect 20 parts of negative peeled shrimp samples, wherein 5 parts of negative peeled shrimp samples add the MAL titer (0.1,0.5,1,2,5ug/L) of variable concentrations.Add each test of sample 5 times, and calculate recovery rate.
The determination of recovery rates result sees table 3, and the recovery that malachite green adds appearance is 83%～97.5%, average recovery rate 90.42%, and the coefficient of variation 7.61%～10.58%, average coefficient of variation 8.66%, accuracy is better.
Table 3 determination of recovery rates
Claims (8)
1. an immunofluorescence detection agent box that is used to detect malachite green comprises malachite green coating antigen and quantum dot-labeled malachite green antibody.
2. kit according to claim 1 is characterized in that:
Said malachite green antibody is that antibody titer is 10
6More than, the antibody affinity costant is 10
6～10
8M
-1The malachite green monoclonal antibody;
Said quantum dot is that surperficial carboxyl-content is 1 * 10
-3～9 * 10
-3The quantum dot of the water-soluble CdSe of mmol/mg/ZnS nucleocapsid structure; The quantum yield of said quantum dot is 40～70%.
3. kit according to claim 1 and 2 is characterized in that:
The particle diameter of said quantum dot is 10～20nm, and the deviation of its particle diameter is between 10～30%;
Said malachite green coating antigen is the conjugate of malachite green and carrier protein.
4. according to arbitrary described kit among the claim 1-3, it is characterized in that: also comprise malachite green standard items, dilution, cleansing solution in the said kit, contain porose polystyrene board, encapsulate damping fluid and confining liquid;
Said malachite green standard items are that tetramethyl is for the diamido triphenylmethane;
Said dilution is the PBS damping fluid;
Said cleansing solution is the PBST damping fluid;
The said damping fluid that encapsulates is a carbonate buffer solution;
Said confining liquid is to contain the PBS damping fluid that is useful on the albumen that encapsulates.
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L 5. according to arbitrary described kit among the claim 1-4, it is characterized in that: said standard items are the standard items of following solution form: the solution that said tetramethyl is diluted to following each concentration for the diamido triphenylmethane with said dilution:.
6. according to arbitrary described kit among the claim 1-5; It is characterized in that: said malachite green coating antigen is coated on said containing on the porose polystyrene board; Method for coating adds 100 μ l for to dilute the coating buffer that said malachite green coating antigen obtains 10 μ g/ml with the said damping fluid that encapsulates in every hole.
7. according to arbitrary described kit among the claim 1-6, it is characterized in that: said quantum dot-labeled malachite green antibody is present in the kit with following solution form: the solution that the said quantum dot-labeled malachite green antibody of every 25ug is obtained with the said diluted of 5ml.
8. the method for malachite green in the test sample comprises the steps: with arbitrary described kit among the claim 1-7 testing sample to be detected, and said testing sample is animal muscle tissue's sample, be specially can practical seafood edible muscle.
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| CN103713026A (en) * | 2014-01-08 | 2014-04-09 | 济南大学 | Preparation method and applications of aptamer electrochemical sensor for detecting malachite green (MG) |
| CN113759119A (en) * | 2021-09-16 | 2021-12-07 | 天津温阳生物技术有限公司 | Kit for fluorescence immunoassay rapid detection of malachite green carbon quantum dots in aquatic products and detection method thereof |
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| CN103713026A (en) * | 2014-01-08 | 2014-04-09 | 济南大学 | Preparation method and applications of aptamer electrochemical sensor for detecting malachite green (MG) |
| CN113759119A (en) * | 2021-09-16 | 2021-12-07 | 天津温阳生物技术有限公司 | Kit for fluorescence immunoassay rapid detection of malachite green carbon quantum dots in aquatic products and detection method thereof |
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