CN102253214B - Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit - Google Patents
Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit Download PDFInfo
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Abstract
The invention discloses a quantum dot-based method for detecting ciprofloxacin by immunofluorescence and a special kit. The immunofluorescence detection kit for detecting the ciprofloxacin comprises a ciprofloxacin peridium and a ciprofloxacin antibody marked by a quantum dot. By the method, the quantitative detection of ciprofloxacin residual medicines can be realized, the detection limit is low, the detection sensitivity is high, the specificity is high, and the method is suitable for detecting various samples. Therefore, the method has a bright application prospect in the field of the detection of the ciprofloxacin.
Description
Technical field
The present invention relates to a kind of method and dedicated kit of the detecting ciprofloxacin by immunofluorescence based on quantum dot.
Background technology
Quinolones (QNS) medicine is the very important broad-spectrum antibiotic of a class that develops rapidly over nearly 20 years, can anti-bacteria the DNA helicase, has a broad antifungal spectrum, efficient, low toxicity, tissue penetration are strong.Become the animal doctor face examine with aquaculture in one of most important anti-infectives, be used for the treatment of in a large number, prevention and growth promotion pay close attention to because its drug resistance and potential carcinogenicity cause widely.In tissue, the sign residue of Ciprofloxacin is Ciprofloxacin and Ciprofloxacin, wherein the highest with the residue concentration in liver organization and the renal tissue, secondly be the skin histology that muscle and fat adhere to, its metabolic product Ciprofloxacin (CIP) of Ciprofloxacin still has biologically active.At present, being used for the quantitative goldstandard that detects of residue of ciprofloxacin thing is the Chromatography/Mass Spectrometry method, but its sample pretreatment process is loaded down with trivial details time-consuming, and testing cost is high.Need not carry out complicated sample pretreatment process based on the ELISA method of competitiveness enzyme-linked immune response principle, detection time is relatively short, but its precision is inadequate, can only be used for qualitatively screening, can't be used for quantitatively conclusive evidence.The sample size that detects residue of ciprofloxacin and Survey of contaminating status thereof in the agricultural product such as current outlet aquatic products is large, 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 dot (Quantum Dots, QDs) marker material is the class new material that development in recent years is got up, and comprises II-VI family and III-V family semiconductor nano.Because the luminous and absorption characteristic that quantum dot is outstanding, but make its emission wavelength with fluorescence lifetime length and width excitation spectrum, narrow emission spectrum precision tuning, very high photochemical stability, can carry out the advantageous characteristic such as multi-color marking, adopt its material that serves as a mark, can realize the ultramicron of target molecules is detected.
The 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 to shine with laser optical apparatus and excite, only can be applied at present cellular immunofluorescence detection and the aspects such as fluidic cell detection and sorting, so also not have quantum dot immune to detect the reagent appearance on the international market.
Summary of the invention
An object of the present invention is to provide a kind of immunofluorescence detection agent box for detection of Ciprofloxacin.
Immunofluorescence detection agent box for detection of Ciprofloxacin provided by the present invention comprises Ciprofloxacin coating antigen and quantum dot-labeled Ciprofloxacin antibody.
In the mentioned reagent box, described Ciprofloxacin 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
-1Monoclonal antibody against ciprofloxacin; Being specially antibody titer is 10
6, the antibody affinity costant is 10
8M
-1Monoclonal antibody against ciprofloxacin, described monoclonal antibody against ciprofloxacin is available from Beijing Sheng Dilong bio tech ltd, catalog number is ENX-088.
Described 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 core shell structure; The quantum yield of described quantum dot is 40～70% or 50～70% or 60%; The excitation wavelength of described quantum dot is 345nm, and emission wavelength is 620nm.
In above-mentioned arbitrary described kit, the particle diameter of described quantum dot is 10～20nm, and described particle diameter is specially 13～20nm, and described 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 described kit, in the described quantum dot-labeled Ciprofloxacin antibody, the amino on the carboxyl on the described quantum dot and the described Ciprofloxacin antibody forms peptide bond, and then described quantum dot is connected with described Ciprofloxacin antibody.
In above-mentioned arbitrary described kit, described Ciprofloxacin coating antigen is the conjugate of Ciprofloxacin and carrier protein, and wherein said Ciprofloxacin and described carrier protein are connected by the peptide bond that the carboxyl in the Ciprofloxacin and amino in the carrier protein form; Described 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 described kit, also comprise Ciprofloxacin standard items, dilution, cleansing solution in the described kit, contain porose polystyrene board, be coated with damping fluid and confining liquid;
Described Ciprofloxacin standard items are 1-cyclopropyl-6-fluoro-Isosorbide-5-Nitrae-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid;
Described dilution is the PBS damping fluid; Be specially the PBS damping fluid of 0.02M, pH7.4.
Described cleansing solution is the PBST damping fluid; Specifically be prepared as follows: the NaN3 that gets 0.2ml Tween20 and 0.1g is dissolved in the described dilution, is settled to 1L with dilution after the dissolving.
Described coated damping fluid is carbonate buffer solution; Be specially the carbonate buffer solution of 0.05M, pH9.6.
Described confining liquid is to contain the PBS damping fluid that is useful on coated albumen; Described albumen for being coated with is any of BSA, ovalbumin and hemocyanin.Specifically be prepared as follows: 10g BSA and 0.2mlTween20 are dissolved in the above-mentioned dilution, are settled to 1L with dilution after the dissolving.
In above-mentioned arbitrary described kit, 0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L described standard items are the standard items of following solution form: with described dilution described 1-cyclopropyl-6-fluoro-Isosorbide-5-Nitrae-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid is diluted to the solution of following each concentration:.
In above-mentioned arbitrary described kit, described Ciprofloxacin coating antigen is coated on described containing on the porose polystyrene board, and method for coating adds 100 μ l for to dilute the coating buffer that described Ciprofloxacin coating antigen obtains 10 μ g/ml with described coated damping fluid in every hole.
In above-mentioned arbitrary described kit, described quantum dot-labeled Ciprofloxacin antibody is present in the kit with following solution form: every 25ug is stated the solution that quantum dot-labeled Ciprofloxacin antibody obtains with the described diluted of 5ml.
Another object of the present invention provides the method for Ciprofloxacin in a kind of test sample.
The method of Ciprofloxacin in the test sample provided by the present invention comprises the steps: with above-mentioned arbitrary described kit testing sample to be detected, and described testing sample is animal muscle tissue's sample, animal urine or honey.Described testing sample is specially pig urine.
Detection side's ratio juris of the present invention: adopt quantum dot as the fluorescence signal labeled molecule, with Ciprofloxacin antigen direct coated in the micropore of polystyrene board, add Ciprofloxacin standard items or test sample, and quantum dot-labeled Ciprofloxacin antibody, make it form Ag-Ab binary electrochemiluminescent immunoassay compound, excite and detect the fluorescence intensity of this immunofluorescence compound with fluorescence detector, by with the concentration of measuring the typical curve contrast that forms and obtain Ciprofloxacin to be measured.
Ag-Ab binary electrochemiluminescent immunoassay complex formation is after adding quantum dot-labeled Ciprofloxacin antibody, the coated Ciprofloxacin of Ciprofloxacin antigen with test sample in is combined Ciprofloxacin antibody competitively, by the specific binding formation Ag-Ab binary immune complex of Ag-Ab.
Ag-Ab binary electrochemiluminescent immunoassay complex formation is after adding simultaneously quantum dot-labeled Ciprofloxacin antibody and test sample, the Ciprofloxacin of Ciprofloxacin antigen in test sample that is coated in the polystyrene micropore is combined with Ciprofloxacin antibody competitively, wherein be combined with test sample remaining Ciprofloxacin antibody be coated on Ciprofloxacin antigen generation specific bond in the microwell plate after form the Ag-Ab binary immune complex that is fixed in the microwell plate.
Ciprofloxacin immunofluorescence detection agent of the present invention is relevant with quantum dot-labeled immunofluorescence detection technique, to adopt quantum dot as the fluorescence signal marker material, carry out class methods of immunofluorescence quantitative measurement, this Technology Integration the research of the 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 Ciprofloxacin, be to adopt a kind of method based on quantum dot-labeled immunofluorescence quantitative measurement, be about to Ciprofloxacin antigen direct coated in the micropore of polystyrene board, measuring principle based on quantum dot-labeled indirect competition immunofluorescence assay, adding Ciprofloxacin standard items or test sample, and behind the quantum dot-labeled Ciprofloxacin antibody, the fluorescence intensity that is attached to the Ag-Ab binary immune complex in the polystyrene board micropore by detection realizes the detection to Ciprofloxacin: the quantity of quantum dot-labeled antibody that is attached to the polystyrene board micropore is different, and the fluorescence intensity that produces is also different.The content of the Ciprofloxacin in the finite concentration scope in the height of fluorescence intensity level and the sample is inversely proportional to.Can be made into typical curve by the Ciprofloxacin standard items that add variable concentrations, the fluorescence intensity level of inquiring about each test sample according to this typical curve can obtain the concentration value of corresponding Ciprofloxacin medicine.
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 Ciprofloxacin antibody orientation is connected to the quantum dot surface.
(2) envelope antigen: adopt Ciprofloxacin antigen with the bovine serum albumin(BSA) coupling as envelope antigen, the method by physisorption with this antigen direct coated in the polystyrene board micropore.
(3) Ag-Ab fluorescence immunoassay complex formation: in above-mentioned coated good polystyrene board micropore, add Ciprofloxacin standard items or test sample, and quantum dot-labeled Ciprofloxacin antibody, the Ciprofloxacin antigen that is adsorbed in the hole combines with Ciprofloxacin antibody with the Ciprofloxacin in standard or the sample is emulative, by the specific binding 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; Form typical curve by the fluorescence intensity of measuring serial corresponding standard items, by contrasting the concentration that obtains Ciprofloxacin to be measured with the typical curve of measuring formation.
Described Ag-Ab binary electrochemiluminescent immunoassay complex formation is: after adding quantum dot-labeled Ciprofloxacin antibody, the coated Ciprofloxacin of Ciprofloxacin antigen in standard or sample is combined Ciprofloxacin antibody competitively, specific binding by Ag-Ab forms Ag-Ab binary immune complex, quantum dot of mark can fluoresce after exciting on it, 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 described 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 adopts is fixed concentration, usually the concentration of the Ciprofloxacin medicine in the testing sample is higher, medication amount by antibody capture is more, the antibody of being combined with coated Ciprofloxacin antigen is fewer, and the fluorescence intensity level that records is lower.
Because quantum dot carrying out will carrying out separation and purification with ultracentrifugation in the coupling with antibody, the 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; The quantum dot that particle diameter is too large 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 described quantum dot is 10～20nm, and described particle diameter is specially 13～20nm, and described particle diameter especially is preferably 20nm; The deviation of its particle diameter (CV) between 10～30%, preferably between 10～20%, 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 described quantum dot is 40～70%, and the fluorescence quantum yield of described quantum dot is specially 50～70%, and the fluorescent quantum product of described quantum dot especially is preferably 60%.
Survey for being used for the residue of ciprofloxacin quality testing, quantum dot surface needs with the group that is easy to the Ciprofloxacin antibody coupling, these groups can be carboxyl, amino groups, the group of optimizing is the surface functional group with carboxyl, usually adopt chemical method to connect antibody, namely with behind EDC and the NHS activation quantum dot, close reaction and finish coupling reaction with antibody generation carboxylic again.
Before the condensate with the formation of peptide bond covalent bond with Ciprofloxacin antibody and quantum dot, also comprise the step that activates described quantum dot surface functional group.The carboxyl-content difference on quantum dot surface can have influence on the sensitivity of detection, and for improving sensitivity, described functional group is specially carboxyl, and the content of described carboxyl is 1 * 10
-3～9 * 10
-3Mmol/mg, the content of described carboxyl is specially 1 * 10
-3～6 * 10
-3Mmol/mg, the content of described 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.Research finds that for improving sensitivity, described Ciprofloxacin antibody affinity costant is 10
6～10
8M
-1Described Ciprofloxacin antibody affinity costant is specially 10
7～10
8M
-1Described Ciprofloxacin antibody affinity costant especially is preferably 10
8M
-1
Because Ciprofloxacin is small-molecule substance, its molecular surface characteristic is unfavorable for the direct combination with polystyrene micropore plate, itself and carrier protein need to be carried out could reaching by means of the character of surface of carrier protein after the coupling good combination with polystyrene micropore plate.Can be used as the seralbumin that various animals are arranged of carrier protein, such as bovine serum albumin(BSA) (Bovine Serum Albumin, BSA), human serum albumins (Human Serum Albumin, HSA), also has keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin, KLH), the gamma globulin of thyroglobulin, albumin rabbit serum (RSA), ovalbumin (Ovalbumin, OVA), fibrinogen or rabbit and chicken.Research is found, the BSA physicochemical property is stable, lysine content is high, free amino group is many, larger solubleness is all arranged under different pH and ionic strength, in the situation that contains organic solvent (such as pyridine, DMF etc.), all can carry out coupling with haptens, and after coupling, still keep solvable state, the splendid selection as carrier protein, so the present invention selects BSA as coupling protein.
The present invention is by the research to fluorescence quantum, Ciprofloxacin antigen and Ciprofloxacin antibody molecule characteristic, by the 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 by optimizing the various conditions of competitive immunization reaction, reach the quick and highly sensitive quantitative measurement to the residue of ciprofloxacin medicine.Experiment showed, kit of the present invention highly sensitive, specificity good, accuracy is high.The inventive method can realize the quantitative detection to the residue of ciprofloxacin 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 field of Ciprofloxacin.
Description of drawings
Fig. 1 water-soluble CdSe/ZnS fluorescence quantum Electronic Speculum (TEM) photo.
Fig. 2 is with the typical curve of quantum dot-labeled indirect competition immuno-fluorescence assay Ciprofloxacin.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The composition of embodiment 1, detection kit and preparation
Ciprofloxacin opens a day Chemical Engineering Technology research institute available from the permanent unit in Beijing, and catalog number is 30233CDCT-C11668500.Its chemical name is 1-cyclopropyl-6-fluoro-Isosorbide-5-Nitrae-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid.Ciprofloxacin is QNS, and molecule band carboxyl itself can be directly and albumen coupling.
1, Ciprofloxacin coating antigen
The conjugate of Ciprofloxacin and carrier protein BSA, Ciprofloxacin and BSA are connected by the peptide bond that the carboxyl in the Ciprofloxacin and amino among the BSA form.
The preparation method of Ciprofloxacin coating antigen is as follows:
1) get 33.1mg Ciprofloxacin, 3ml DMF (DMF) and 3ml dioxane and be mixed to get mixed liquor a, the temperature of mixing is 25 ℃, and the time of mixing is 30min, and the mode of mixing is concussion;
2) the mixed liquor a that the step 1 of 6ml) obtains, 26.2 μ l (final concentration is 0.1mmol) the positive amine of tributyl is at 4 ℃ of lower 15min that stir, obtain mixed liquor b, in mixed liquor b, add 1.5 μ l isobutyl chlorocarbonates (about 0.1mmol) at 25 ℃ of lower stir-activating 1h; The ciprofloxacin solution that obtains activating;
3) 100mg BSA is dissolved in 10ml 0.1mol/L, and pH obtains the BSA BAS in 8.5 the dobell's solution, 4 ℃ lower preserve stand-by;
4) ciprofloxacin solution that 6ml is activated slowly dropwise adds in the 10mlBSA BAS by separating funnel under ice bath (4 ℃), and stirring reaction 12h obtains mixed liquor c;
5) be 0.02mol/L with gained mixed liquor c in concentration, pH is 4 ℃ of dialysis 48h in 7.4 the PBS solution, and every 6h changes a dislysate, removes unreacted little molecule.Products obtained therefrom freeze dryer freeze-drying in-20 ℃ of preservations, obtains Ciprofloxacin antigen (CIP-BSA).
2, the Ciprofloxacin coating antigen is coated
Adopt coated damping fluid that the dilution of Ciprofloxacin coating antigen is the coating buffer of concentration 10 μ g/ml, every hole adds l00 μ l in 96 hole polystyrene micropore laths, places in 4 ℃ of refrigerators and spends the night.Second day discards coating buffer, wash each plate hole 3 times with cleansing solution after, each hole adds 200 μ l confining liquids, processes 2h in 37 ℃ of sealings.After discarding afterwards confining liquid, washing each plate hole 3 times with cleansing solution, carry out vacuum drain, with being positioned over-20 ℃ of preservations after the aluminium foil bag sealing.
3, quantum dot-labeled Ciprofloxacin antibody:
Ciprofloxacin antibody is 10 for tiring
6, affinity costant is 10
8M
-1Monoclonal antibody against ciprofloxacin, it is available from Beijing Sheng Dilong bio tech ltd, catalog number is ENX-088.
Quantum dot is available from Shenzhen's TELUS Science and Technology Ltd., and catalog number is
LumiQD
TM20.The sign of this quantum dot is as follows: 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 described quantum dot-labeled Ciprofloxacin antibody, the amino on the carboxyl on the described quantum dot and the described Ciprofloxacin antibody forms peptide bond, and then described quantum dot is connected with described Ciprofloxacin 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, be that 0.1M, pH value are that 4.7 MES damping fluid is resuspended with 1ml concentration, add the 1-ethyl of 0.96mg (final concentration is 5mM)-(3-dimethylaminopropyl) carbodiimide (EDC) and 1.15mg (final concentration is 10mM) N-maloyl imines (NHS) in wherein.Temperature of reaction is 37 ℃, the reaction half an hour after, the quantum dot after obtaining activating;
2) with the borate buffer solution washing of 50mM pH=8.5, get the borate buffer solution that quantum dot after 0.15mg monoclonal antibody against ciprofloxacin and the 2.5mg activation is mixed into 0.8ml 50mM pH=8.5 and (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.The lower reaction of room temperature (25 ℃) 3.5 hours 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, reaction was carried out under 37 ℃ 0.5 hour, obtained containing the reactant liquor of the rear quantum dot of sealing; After finishing, (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 rear 4 ℃ of preservations are stand-by, obtain quantum dot-labeled Ciprofloxacin antibody.
0,0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L 4, Ciprofloxacin standard items: the solution that Ciprofloxacin 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, be settled to 1L with above-mentioned PBS damping fluid after the dissolving.
7, the carbonate buffer solution of coated damping fluid: 0.05M, pH9.6.
8, confining liquid: 10g BSA and 0.2ml Tween20 are dissolved in the above-mentioned PBS damping fluid, are settled to 1L with the PBS damping fluid after the dissolving.
The preparation method of embodiment 2, typical curve
In the Ciprofloxacin micropore lath for preparing, add concentration and be 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5, the Ciprofloxacin standard solution of 10ug/L, the 50ul/ hole, quantum dot-labeled CIP 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 determined the sensitivity of detection system 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 0.001ug/L.
The quantum dot kit detected value of table 1 Ciprofloxacin variable concentrations sample
Ciprofloxacin Concentration (ug/L)
The mensuration of embodiment 3, cross reaction
(a day Chemical Engineering Technology research institute is opened by the permanent unit in Beijing to select Enrofloxacin, 30235CDCT-C13170000), Danofloxacin (Sigma-aldrich, 33700-100MG-R), (a day Chemical Engineering Technology research institute is opened to Norfloxacin by the permanent unit in Beijing, 30234CDCT-C15648000), (a day Chemical Engineering Technology research institute is opened to Enoxacin by the permanent unit in Beijing, 29641NIC-130453), (a day Chemical Engineering Technology research institute is opened to Ofloxacin by the permanent unit in Beijing, 30237CDCT-C15717000) with Pei Nuosha star (Sigma-aldrich, 33899-100MG-R), be made into respectively series concentration, detect with quantum dot kit.Calculate the IC50 that respectively competes thing, calculate respectively the cross reacting rate of these 5 kinds of medicines and Ciprofloxacin quantum dot kit with following formula.Computing formula is: cross reacting rate (%)=[IC50 (Ciprofloxacin)/IC50 (medicine to be measured)] * 100.
Mensuration and result of calculation are as shown in table 2.The result shows that the Ciprofloxacin quantum dot kit is complete intersection to Ciprofloxacin, and is less to all the other several crossing-over rates.
The cross reaction of table 2 Ciprofloxacin quantum dot kit and other medicines
The mensuration of embodiment 4, accuracy
(1) sample extraction:
1, musculature sample: take by weighing musculature sample 8g (being accurate to 0.01g), add acetonitrile-NaOH solution (84ml acetonitrile and 16ml 0.1M NaOH solution mix) 10ml, abundant mixing 10min, the centrifugal 10min of 4000g gets supernatant 4ml, add 0.02M PBS 4ml, add methylene chloride 8ml, fully mixed the centrifugal 10min of 4000g 10 minutes, remove supernatant, take off a layer organic phase 4ml, nitrogen dries up, with 1ml 0.01M NaOH dissolution residual substance, add 1ml 0.02M PBS mixing, add the 1ml normal hexane, mix 2min, the centrifugal 5min of 4000g, discard upper organic phase and center section liquid, take off layer liquid 50ul for detection of.
2, urine sample: the urine of clarification can be directly used in detection, if cloudy urine needs first centrifugal (4000g) 10min, gets supernatant and detects.
3, honey: the sample to without crystallization stirs it; To the sample of crystallization is arranged, under encapsulation situations, place the water-bath that is no more than 60 ℃ warm, vibration stirs evenly after sample all melts, and is cooled to room temperature.Take by weighing 2g sample (being accurate to 0.01g), add 2ml 0.02M PBS and methylene chloride 8ml, mechanical shaking extraction 10min, the centrifugal 10min of 4000g discards supernatant liquid, and lower floor's organic phase is dried up with nitrogen.With 500ul 0.01M NaOH dissolution residual substance, add 500ul 0.02M PBS mixing, get 50ul for detection of.
(2) mensuration of the recovery
Detect 30 parts of negative pig urine samples, wherein 5 portions of negative CIP titers that add variable concentrations (0.01,0.1,0.5,1,5ug/L).Add 5 urine samples of each test of sample, and calculate recovery rate.
Determination of recovery rates the results are shown in Table 3, and the recovery that Ciprofloxacin adds sample is 88%～98%, average recovery rate 93.66%, and the coefficient of variation 6.12%～9.09%, average coefficient of variation 8.02%, accuracy is better.
Table 3 determination of recovery rates
Claims (4)
1. the immunofluorescence detection agent box for detection of Ciprofloxacin comprises Ciprofloxacin coating antigen and quantum dot-labeled Ciprofloxacin antibody;
Described Ciprofloxacin antibody is that antibody titer is 10
6More than, the antibody affinity costant is 10
6～10
8M
-1Monoclonal antibody against ciprofloxacin;
Described 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 core shell structure; The quantum yield of described quantum dot is 40～70%;
The particle diameter of described quantum dot is 10～20nm, and the deviation of its particle diameter is between 10～30%;
Described Ciprofloxacin coating antigen is the conjugate of Ciprofloxacin and carrier protein;
Also comprise Ciprofloxacin standard items, dilution, cleansing solution in the described kit, contain porose polystyrene board, be coated with damping fluid and confining liquid;
Described Ciprofloxacin standard items are 1-cyclopropyl-6-fluoro-Isosorbide-5-Nitrae-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid;
Described dilution is the PBS damping fluid;
Described cleansing solution is the PBST damping fluid;
Described coated damping fluid is carbonate buffer solution;
Described confining liquid is to contain the PBS damping fluid that is useful on coated albumen;
0.001,0.005,0.01,0.05,0.1,0.5,1,5,10ug/L described standard items are the standard items of following solution form: with described dilution described 1-cyclopropyl-6-fluoro-Isosorbide-5-Nitrae-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid is diluted to the solution of following each concentration:.
2. kit according to claim 1, it is characterized in that: described Ciprofloxacin coating antigen is coated on described containing on the porose polystyrene board, method for coating adds 100 μ l for to dilute the coating buffer that described Ciprofloxacin coating antigen obtains 10 μ g/ml with described coated damping fluid in every hole.
3. kit according to claim 2, it is characterized in that: described quantum dot-labeled Ciprofloxacin antibody is present in the kit with following solution form: the solution that the described quantum dot-labeled Ciprofloxacin antibody of every 25ug is obtained with the described diluted of 5ml.
4. the method for Ciprofloxacin in the test sample comprises the steps: with arbitrary described kit among the claim 1-3 testing sample to be detected, and described testing sample is animal muscle tissue's sample, animal urine or honey.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110187929 CN102253214B (en) | 2011-07-06 | 2011-07-06 | Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit |
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| CN105628929A (en) * | 2014-11-25 | 2016-06-01 | 北京维德维康生物技术有限公司 | Quantum-dot immunofluorescence kit for simultaneously detecting gentamycin and quinolone medicines |
| CN106568949B (en) * | 2016-11-02 | 2018-07-27 | 南昌大学 | A kind of small haptens detection method based on direct competitive fluoroimmunoassay |
| CN107490565B (en) * | 2017-06-27 | 2019-12-03 | 昆明理工大学 | A kind of method of nitrogen-doped carbon quantum dot fluorescence enhanced sensitivity detection Ciprofloxacin |
| CN107722968B (en) * | 2017-11-10 | 2019-07-16 | 青岛大学 | A kind of preparation method of the Ciprofloxacin ratio fluorescent probe based on nano-complex |
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| CN101308145A (en) * | 2008-06-17 | 2008-11-19 | 江南大学 | Method for quantum dot mark indirect competition fluoroimmunoassay detection for aeroseb-d |
| CN101762706A (en) * | 2010-01-05 | 2010-06-30 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
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| CN101762706A (en) * | 2010-01-05 | 2010-06-30 | 中国农业大学 | Method of detecting residue of small-molecule substance harmful to human body and a special kit |
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