CN107656062A - A kind of method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol - Google Patents
A kind of method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol Download PDFInfo
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Abstract
The invention discloses a kind of method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol, it comprises the following steps：Prepare carboxylic polystyrene nanoparticle, prepare nanometer fluorescent microspheres；Wherein, in the nanometer fluorescent microspheres, the mol ratio of rare earth element ion, beta diketone body class chelate and Fluorescence Increasing synergist is 2: 2: 3, and the rare earth element ion is Nd3+And Sm3+, prepare nanoparticle time-resolved fluorescence chloramphenicol probe, preparative chromatography test strips, utilize above-mentioned probe and chromatograph test strip detection chloramphenicol.The detection method detection sensitivity is high, and detection range is wide, reliable and stable, possesses good commercial applications prospect.
Description
Technical field
The invention belongs to detection technique field, and in particular to one kind utilizes nanoparticle time-resolved fluorescence probe in detecting chlorine
The method of mycin.
Background technology
Time-resolved fluoroimmunoassay (Time resolved fluoroisnmunoassay, TRFIA) is last century 80
Age propose a kind of more new detection means, TRFIA be by the use of rare earth ion as tracer-labelling protein, polypeptide,
Hormone, antibody, nucleic acid probe or biologically active cell, with its chelating agent, enhancing liquid (some is not needed) in question response body
System is (such as:Antigen-antibody immune response, biotin-labeled pentylamine reaction, nucleic acid probe hybridization reaction, target cell and effector cell kill
Traumatic response etc.) react after, with time-resolved fluorescence instrument determine end product in fluorescence intensity, according to fluorescence intensity and phase
To fluorescence intensity ratio, the concentration of analyte in reaction system is speculated, reach the purpose of quantitative analysis.TRFIA and chemiluminescence,
Electrochemical luminescence immunoassay technology and referred to as three big super sensitivity detection technologies, in food inspection, clinical medicine detection, biology
Scientific research detection and environment measuring etc. have relatively broad application.
Because the fluorescence intensity of rare earth element complex is all relatively low, it is therefore desirable to improve detection using Fluorescence Increasing technology
Sensitivity.At present, according to the difference of Signal Enhanced Technology, TRFIA can be divided into three types, and dissociation strengthens technology again
(DELFIA), Cyber Fluor systems and the TRFIA (Nano-TRFIA) based on nanoparticle.Wherein, Nano-TRFIA is one
The brand-new time-resolved fluorescence detection means of kind, it combines long-life and the signal amplification of nanoparticle of rare earth element fluorescence
Effect, by rare earth element and its complex co-doped in nanometer and microballoon, after surface active, by antibody labeling in mark
Remember thing surface, form compound, the compound is used for immune detection, sensitivity can be greatly enhanced, and obtain more broad
The range of linearity.
Chloramphenicol is the antibiotic as caused by Venezuela Streptothrix, belongs to biocidal property broad-spectrum antibiotic.Chloramphenicol is to control
Cure the wound cold, paratyphoid drug of first choice, be one of specific medicament for treating anaerobic infection, chloramphenicol is also used for sensitive microbial
The treatment of caused various infectious diseases.Method that is quick, easy, reliable, detecting chloramphenicol exactly is urgently obtained at present.
The content of the invention
The technical problem to be solved in the present invention aiming at it is existing, lack it is quick, easy, reliable, detect chlorine exactly
A kind of method deficiency of mycin, there is provided method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol.Described side
Method can quickly, content that is reliable, detecting chloramphenicol exactly, and high sensitivity, reproducible, stability is high, when saving
Between.This method can be widely applied for being commercialized the detection to chloramphenicol.
Purpose for detecting chloramphenicol, the present inventor to the structure of nanoparticle time-resolved fluorescence probe,
Especially to the kind of the preparing raw material rare earth elements ion of its part nanometer fluorescent microspheres --- samarium element and neodymium member
Element carries out special selection so that the fluorescence intensity of the nanometer fluorescent microspheres prepared is greatly enhanced, and make rare earth element ion,
The mol ratio of beta-diketon body class chelate and Fluorescence Increasing synergist is 2: 2: 3, by above-mentioned improvement, improves to utilize to contain and is somebody's turn to do
Sensitivity, accuracy and the reliability of the nanoparticle time-resolved fluorescence probe in detecting chloromycetin content of nanometer fluorescent microspheres.
The technical scheme is that：A kind of method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol,
It comprises the following steps：
(1) carboxylic polystyrene nanoparticle is prepared；
(2) the carboxylic polystyrene nanoparticle prepared using step (1), prepares nanometer fluorescent microspheres；The nanometer
In fluorescent microsphere, the mol ratio of rare earth element ion, beta-diketon body class chelate and Fluorescence Increasing synergist is 2: 2: 3, described
Rare earth element ion is Nd3+And Sm3+；
(3) nanometer fluorescent microspheres and chloramphenicol monoclonal antibody prepared using step (2), prepare the nanoparticle time point
Distinguish fluorescence-chloramphenicol probe；The nanometer fluorescent microspheres and rabbit igg monoclonal antibody prepared using step (2) prepare nanoparticle
Time-resolved fluorescence-rabbit igg probe；
(4) the nanoparticle time-resolved fluorescence-chloramphenicol probe and nanoparticle time resolution that prepared by step of freeze drying (3)
Fluorescence-rabbit igg probe, obtain the chloramphenicol monoclonal antibody and and lyophilized nano fluorescent of lyophilized nanometer fluorescent microspheres mark
The rabbit igg monoclonal antibody of microballoon mark；
(5) detection line T lines are prepared using chloramphenicol, prepares nature controlling line C line using goat-anti rabbit secondary antibody, spraying, assembling obtain
Chromatograph test strip；
(6) the lyophilized nanometer fluorescent microspheres for preparing step (4) mark chloramphenicol monoclonal antibody and and lyophilized
The rabbit igg monoclonal antibody of nanometer fluorescent microspheres mark is added in the hole of ELISA Plate；
(7) testing sample is taken to add in the hole of the ELISA Plate of step (6)；
(8) chromatograph test strip that into the hole of step (7) prepared by inserting step (5), sample pad one end is immersed in the hole
In liquid 0.5cm, immerse 5min after insert Fluorescent reader in reading, obtain testing result.
In the present invention, it is preferred that step (1) the carboxylic polystyrene nanoparticle, by the side comprised the following steps
Method is made：
10mM styrene monomers and 0.95mM acrylic monomers are dissolved in 10mL dodecyl sodium sulfates containing 0.45mM
In deionized water, round-bottomed flask is added, is stirred with magnetic stir bar；
The air in round-bottomed flask is eliminated with high pure nitrogen, heated sealed is to 70 DEG C.Add 0.15mM potassium peroxydisulfates
0.5mL, sealing oxygen barrier stirring reaction are down to room temperature after 8 hours；
Reaction solution is filtered with whatman 2v filter paper, it is saturating to deionized water with molecular cut off 25,000Da bag filter
Analysis 5 days, liquid in bag filter is collected, add 4 DEG C of preservations after 0.05% Sodium azide, the percentage is mass percent.
In the present invention, it is preferred that a diameter of 45 ± 8nm of prepared carboxylic polystyrene nanoparticle, surface electricity
Lotus is 200-240 μ eq/g.
In step (2) of the present invention, the beta-diketon body class chelate is the conventional beta-diketon body class chelate in this area, compared with
It is β-naphthoyltrifluoroacetone goodly.The Fluorescence Increasing synergist is the conventional Fluorescence Increasing synergist in this area, preferably
For trioctyl phosphine oxide and/or phenanthroline.
More preferably, step (2) described nanometer fluorescent microspheres are made by the method comprised the following steps：
The carboxylic polystyrene nanoparticle for taking step (1) to prepare, add 10mL deionized waters and acetone mixture, institute
The volume ratio for stating deionized water and the acetone is 1:1, obtain reaction solution A；
The density for making the polystyrene microsphere of carboxylated in reaction solution A is 1 × 1014Individual/mL, stirs, and sequentially adds
μ L of 0.1M neodymium trichlorides 100, the μ L of 0.1M samarium trichlorides 100, μ L of 0.1M β-naphthoyltrifluoroacetone 200, the oxidation of 0.1M trioctylphosphines
The μ L of 150 μ L and 0.1M phenanthroline of phosphine 150, it is heated to 60 DEG C of constant temperature stirring lucifuges and reacts 10 hours, it is small to be then down to room temperature reaction 2
When, obtain reaction solution B；
Organic solvent in solution is removed by reaction solution B by being evaporated under reduced pressure mode, deionized water is dialysed 5 days, gone
Except remaining remaining small-molecule substance, liquid in bag filter is collected, adds 0.05% 4 DEG C of preservations of Sodium azide, the percentage is
Mass percent.
In the present invention, it is preferred that step (3) nanoparticle time-resolved fluorescence-chloramphenicol probe is by including following
The method of step is made：
Nanometer fluorescent microspheres prepared by step (2), are dissolved in 10mL 0.01M pH8.0 borate buffer, make fluorescence
Microsphere Density is about 1.0 × 1012Individual/mL.400W is ultrasonically treated 30 seconds, is then slowly added into the 15mg/mL μ of carbodiimide 200
L, obtain reaction solution I；
Reaction solution I is at the uniform velocity stirred at ambient temperature after incubating 15 minutes, 150000g is centrifuged 10 minutes, collects precipitation.
Precipitation is washed repeatedly with 0.01M pH 8.0 borate buffer.Repeated centrifugation step 2 time, produce the fluorescent microsphere of activation；
The fluorescent microsphere of activation is redissolved in 5mL 0.01M pH8.0 borate buffer, adds 250 μ g chloramphenicol lists
Clonal antibody, 4 DEG C of stirring reactions 12 hours, obtains reaction solution II；
The 12000g of reaction solution II is centrifuged 10 minutes, collects precipitation.The precipitation is redissolved in containing 1.5% trehalose and 2%
In bovine serum albumin(BSA), 0.01M pH7.4 phosphate buffer, the percentage is quality percent by volume.
In the present invention, it is preferred that the preparation method of nanoparticle time-resolved fluorescence-rabbit igg probe is received with described
The preparation method of meter Wei Qiu time-resolved fluorescences-chloramphenicol probe is identical, and chloramphenicol monoclonal antibody only is replaced with into rabbit igg list
Clonal antibody.
In the present invention, it is preferred that the chloramphenicol monoclonal antibody of step (4) the lyophilized nanometer fluorescent microspheres mark and
The rabbit igg monoclonal antibody marked with lyophilized nanometer fluorescent microspheres is made by the method comprised the following steps：
Nanoparticle time-resolved fluorescence-chloramphenicol probe and nanoparticle time-resolved fluorescence prepared by step (3)-
Rabbit igg probe dilutes 20 times and 30 times successively respectively with lyophilized dilution, obtains diluent A and dilution B, the lyophilized dilution
For containing 6% sucrose, 4% bovine serum albumin(BSA), 1% mannitol 0.05M pH 7.4 PBPS buffer solutions, the percentage
For mass percent；
The diluent A and dilution B are pressed 1:Vacuum freezedrying after 1 volume ratio mixes.
In the present invention, it is preferred that step (5) described chromatograph test strip is made by the method comprised the following steps：
By chloramphenicol use containing 1.5% trehalose, 2% bovine serum albumin(BSA), 0.05% Tween-20 0.01M pH7.4
Phosphoric acid buffer is dissolved to final concentration 0.1mg/mL, must spray film liquid A, is sprayed on Membrane jetter by film liquid A is sprayed away from nitrocellulose filter
Detection line T lines are formed at left end 2mm, the percentage is quality percent by volume；
Goat-anti rabbit secondary antibody is used to the 0.01M pH7.4 for containing 1.5% trehalose, 2% bovine serum albumin(BSA), 0.05% polysorbas20
Phosphoric acid buffer be dissolved to final concentration 1.0mg/mL, film liquid B must be sprayed, with Membrane jetter by spray film liquid B be sprayed on away from nitrocellulose
Nature controlling line C line is formed at film right-hand member 4mm, nature controlling line is separated by 5mm with detection line, and the percentage is quality percent by volume；
The nitrocellulose filter sprayed is placed in 25 DEG C of vacuum drying ovens and dried；
Overlapped successively on hardboard paper and paste nitrocellulose filter, glass fibre element pads, draws the nitric acid for there are detection line T lines
Cellulose membrane, nitrocellulose filter, filter paper, sample pad and the blotting paper for having nature controlling line C line are drawn, then cut into the wide examinations of 4mm
Paper slip.
It on the basis of common sense in the field is met, above-mentioned each optimum condition, can be combined, it is each preferably real to produce the present invention
Example.
Agents useful for same and raw material of the present invention are commercially available.
The positive effect of the present invention is：Detection method detection sensitivity provided by the invention is high, its quantitative limit energy
Enough it is minimal to 7ng/mL；The detection range of the detection method is wide, and its quantitative linearity scope can reach 7-650ng/mL；The detection
Method detection is reliable and stable, and its sample TIANZHU XINGNAO Capsul is up to 95-103%.In addition, the detection method is easy to operation, only
Need just can obtain accurate, reliable measurement result within 5 minutes, possess good commercial applications prospect.
Embodiment
The present invention is further illustrated below by the mode of embodiment, but is not therefore limited the present invention to described
Among scope of embodiments.The experimental method of unreceipted actual conditions in the following example, conventionally and condition, or according to
Catalogue selects.
The implication of normal temperature described in embodiment is the implication of the conventional normal temperature in this area, can be carried out described in embodiment
Reaction.Preferably 15~30 DEG C.
The preparation of the carboxylic polystyrene nanoparticle of embodiment 1
10mM styrene monomers (being purchased from BASF Aktiengesellschaft) and 0.95mM acrylic monomers (are purchased from German BASF
Company) it is dissolved in the deionized water of 10mL dodecyl sodium sulfates containing 0.45mM, round-bottomed flask is added, uses magnetic stir bar
Stir.Then the air in round-bottomed flask is eliminated with high pure nitrogen, heated sealed is to 70 DEG C.Add 0.15mM persulfuric acid
Potassium 0.5mL, sealing oxygen barrier stirring reaction are down to room temperature, then (are purchased from reaction solution with whatman 2v filter paper after 8 hours
Whatman companies, 8 μm of aperture) filtering, finally with bag filter (be purchased from Spectrum companies of the U.S., molecular cut off 25,
000Da) deionized water is dialysed 5 days, collects liquid in bag filter, after adding 0.05% (w/w) Sodium azide, carboxylated is produced and gathers
Styrene nanoparticle, it is placed on 4 DEG C of preservations.
Pass through measure, a diameter of 45 ± 8nm of prepared carboxylic polystyrene nanoparticle, surface charge 200-
240μeq/g。
The preparation of the nanometer fluorescent microspheres of embodiment 2
(1) the carboxylic polystyrene nanoparticle prepared in Example 1,10mL deionized waters and acetone mixing are added
(wherein, deionized water and the volume ratio of acetone are 1 to liquid:1) reaction solution A, is obtained.
(2) density of polystyrene microsphere is about 1 × 10 in the reaction solution A obtained by step (1)14Individual/mL, stirs,
Sequentially add μ L of 0.1M neodymium trichlorides 100, the μ L of 0.1M samarium trichlorides 100, the μ L of 0.1M β-naphthoyltrifluoroacetone 200,0.1M tri-
The μ L of 150 μ L and 0.1M phenanthroline of octyl group phosphine oxide 150.It is heated to 60 DEG C of constant temperature stirring lucifuges to react 10 hours, is then down to room
Temperature reaction 2 hours, obtains reaction solution B.
(3) the reaction solution B obtained by step (2) is removed into organic solvent therein by being evaporated under reduced pressure mode.Spend again from
Sub- water is dialysed 5 days, removes other wherein remaining small-molecule substances, is collected bag filter and (is purchased from Spectrum companies of the U.S., retains
Molecular weight 10,000Da) interior liquid, after adding 0.05% (w/w) Sodium azide, 4 DEG C of preservations are placed on, it is micro- to produce nano fluorescent
Ball.Neodymium ion chelate and terbium ion chelate have been wrapped up in the nanometer fluorescent microspheres.
By testing and calculating (method disclosed with reference to GB/T 21883-2008), receiving obtained by average each step (3)
The quantity of the neodymium ion chelate wrapped up in rice fluorescent microsphere is 300,000.
The preparation of the chloramphenicol monoclonal antibody of the nanometer fluorescent microspheres of embodiment 3 mark
(1) nanometer fluorescent microspheres prepared in Example 2, are dissolved in 10mL 0.01M pH8.0 borate buffer,
It is about 1.0 × 10 to make fluorescent microsphere density12Individual/mL.400W is ultrasonically treated 30 seconds, and the carbon two for being then slowly added into 15mg/mL is sub-
The μ L of amine (EDC) 200, obtain reaction solution I.
(2) reaction solution I obtained by step (1) is at the uniform velocity stirred at ambient temperature after incubating 15 minutes, 150000g centrifugations
10 minutes, collect precipitation.Precipitation is washed repeatedly with 0.01M pH8.0 borate buffer.Centrifugation step described in repeat step (2)
Rapid 2 times, produce the fluorescent microsphere of activation.
(3) fluorescent microsphere by the activation obtained by step (2) is redissolved in 5mL 0.01M pH8.0 borate buffer,
Add 250 μ g chloramphenicol monoclonal antibodies (being purchased from Melamine companies), 4 DEG C of stirring reactions 12 hours, obtain reaction solution II.
(4) 12000g of reaction solution II obtained by step (3) is centrifuged 10 minutes, collects precipitation.By the precipitation redissolve in containing
In 1.5% (m/v) trehalose and 2% (m/v) bovine serum albumin(BSA), 0.01M pH7.4 phosphate buffer, it is glimmering to produce nanometer
The chloramphenicol monoclonal antibody of light microballoon mark, is placed in 4 DEG C and saves backup.
The preparation of the rabbit igg monoclonal antibody of the nanometer fluorescent microspheres of embodiment 4 mark
(1) nanometer fluorescent microspheres prepared in Example 2, are dissolved in 10mL 0.01M pH8.0 borate buffer,
It is about 1.0 × 10 to make fluorescent microsphere density12Individual/mL.400w is ultrasonically treated 30 seconds, and the carbon two for being then slowly added into 15mg/mL is sub-
The μ L of amine 200, obtain reaction solution I.
(2) reaction solution I obtained by step (1) is at the uniform velocity stirred at ambient temperature after incubating 15 minutes, 150000g centrifugations
10 minutes, collect precipitation.Precipitation is washed repeatedly with 0.01M pH8.0 borate buffer.Centrifugation step described in repeat step (2)
Rapid 2 times, produce the fluorescent microsphere of activation.
(3) fluorescent microsphere by the activation obtained by step (2) is redissolved in 5mL 0.01M pH8.0 borate buffer,
Add 600 μ g rabbit iggs monoclonal antibodies (giving bio tech ltd purchased from Shang Haiji), 4 DEG C of stirring reactions 12 hours, obtain instead
Answer liquid III.
(4) 12000g of reaction solution III obtained by step (3) is centrifuged 10 minutes, collects precipitation.By the precipitation redissolve in containing
In 1.5% (m/v) trehalose and 2% (m/v) bovine serum albumin(BSA), 0.01M pH7.4 phosphate buffer, it is glimmering to produce nanometer
The rabbit igg monoclonal antibody of light microballoon mark, is placed in 4 DEG C and saves backup.
The monoclonal antibody of the nanometer fluorescent microspheres of embodiment 5 mark freezes
(1) prepared in the chloramphenicol monoclonal antibody and embodiment 4 for marking nanometer fluorescent microspheres prepared by embodiment 3
The rabbit igg monoclonal antibody of nanometer fluorescent microspheres mark (can be referred to as " nanoparticle time-resolved fluorescence-chloramphenicol spy again
Pin " and " nanoparticle time-resolved fluorescence-rabbit igg probe ") with lyophilized dilution (containing 6% sucrose, 4% bovine serum albumin(BSA),
The 0.05M pH 7.4 of 1% mannitol PBPS buffer solutions, the percentage are mass percent) respectively successively dilute 20 times and
30 times, obtain diluent A and dilution B.
(2) diluent A and dilution B prepared step (1) presses 1:1 (v/v) is well mixed, is dispensed with 100 μ L per hole
In 96 hole detachable micropore plates, (lyophilized technique is shown in Table 1) is dried using vacuum freezedrying mode, produces lyophilized receive
The chloramphenicol monoclonal antibody of rice fluorescent microsphere mark and the rabbit igg monoclonal antibody with lyophilized nanometer fluorescent microspheres mark,
It can be sealed with silica gel plug.
The lyophilized technique of table 1
| Temperature | Regulating time | Retention time | Pressure |
| -55℃ | 30min | 240min | 100KPa |
| -35℃ | 30min | 180min | 0.15mbar |
| -15℃ | 30min | 480min | 0.15mbar |
| -5℃ | 30min | 120min | 0.11mbar |
| 5℃ | 30min | 120min | 0.11mbar |
| 25℃ | 30min | 240min | 0.15mbar |
| 25℃ | 5min | 60min | 0mbar |
The preparation of the chromatograph test strip of embodiment 6
1) preparation of nitrocellulose filter C/T lines
(1) by chloramphenicol (being purchased from Jilin Province Huagang pharmaceutical Co. Ltd) with containing 1.5% (m/v) trehalose, 2% (m/v)
Bovine serum albumin(BSA), the 0.01M pH7.4 phosphoric acid buffer of 0.05% (v/v) Tween-20 are dissolved to final concentration 0.1mg/mL,
Film liquid A must be sprayed.Film liquid A will be sprayed with Membrane jetter (being purchased from Beijing Jin Shitai bioengineering Science and Technology Ltd.) to be sprayed on away from nitric acid fibre
Tie up and form detection line T lines at plain film left end 2mm.
(2) by goat-anti rabbit secondary antibody (being purchased from Elabscience companies) with containing 1.5% (m/v) trehalose, 2% (m/v) ox blood
Pure albumen, the 0.01M pH7.4 phosphoric acid buffer of 0.05% (m/v) polysorbas20 are dissolved to final concentration 1.0mg/mL, obtain spray film
Liquid B, it will spray film liquid B with Membrane jetter and be sprayed on away from nature controlling line C line is formed at nitrocellulose filter right-hand member 4mm, nature controlling line is with detecting
Line is separated by 5mm.
(3) nitrocellulose filter sprayed is placed in 25 DEG C of vacuum drying ovens and dried, in drying at room temperature environment
Save backup.
2) assembling of test strips
Overlap and paste successively on hardboard paper：Nitrocellulose filter, glass fibre element pad, draw the nitre for there are detection line T lines
Acid cellulose film, draw nitrocellulose filter, filter paper, sample pad and the blotting paper for having nature controlling line C line.Cut into after assembling
Test strips wide 4mm, produce chromatograph test strip.The chromatograph test strip is put into dry plastics keg and be sealed, guaranteed the quality
Phase was up to more than 1 year.
The detection of the chloramphenicol of embodiment 7
A, detecting step
(1) the lyophilized nanometer fluorescent microspheres for preparing embodiment 5 mark chloramphenicol monoclonal antibody and and lyophilized
The rabbit igg monoclonal antibody of nanometer fluorescent microspheres mark is added to 96 hole detachable micropore ELISA Plates (being purchased from CORNING companies)
Hole in.
(2) 100 μ L testing samples are taken to add in the hole of 96 hole detachable micropore ELISA Plates of step (1) again.
(3) chromatograph test strip prepared by embodiment 6 is inserted into the hole of step (2), sample pad one end is immersed in step (2)
Hole liquid in 0.5cm, insert in Portable fluorescence readout instrument (being purchased from Nanjing micrometering bio tech ltd) after 5min
That is readable, by built-in standard curve can quantitative testing result.
B, accuracy is tested
(1) it is mould to chlorine is detected through high performance liquid chromatography GC-MS (being purchased from Nanjing Ke Jie Analytical Instrument Co., Ltd)
Cellulose content is to add chloramphenicol in 0 deionized water, its concentration is respectively reached 0ng/mL, 10ng/mL, 20ng/mL, 40ng/
ML, 80ng/mL, 160ng/mL, 320ng/mL and 640ng/mL, detected by step A detection method.
(2) step (1) is repeated into experiment 15 times.
As a result show：Chromatograph test strip is limited to 5ng/mL to quantifying for the chloramphenicol in sample, and quantitative linearity scope is in 5-
In 1000ng/mL, sample TIANZHU XINGNAO Capsul fully meets the needs of quantitative detection within 100%~120%.Its is sensitive
Degree is higher than the colloidal gold immuno-chromatography test paper strip prepared with chloramphenicol monoclonal antibody more than 15 times.
So as to obtain nanometer fluorescent microspheres.
Comparative example 1
A, the comparison of different nanometer fluorescent microspheres fluorescence intensities
The density of polystyrene microsphere is about 1 × 10 in reaction solution A14Individual/mL, stirs, and sequentially adds：
1., μ L of 0.1M neodymium trichlorides 100, the μ L of 0.1M samarium trichlorides 100, μ L of 0.1M β-naphthoyltrifluoroacetone 200,0.1M
The μ L of 300 μ L and 0.1M phenanthroline of trioctyl phosphine oxide 100；
2., μ L of 0.1M neodymium trichlorides 100, the μ L of 0.1M samarium trichlorides 100, μ L of 0.1M β-naphthoyltrifluoroacetone 200,0.1M
The μ L of 150 μ L and 0.1M phenanthroline of trioctyl phosphine oxide 150；
It is heated to 60 DEG C of constant temperature stirring lucifuges to react 10 hours, is then down to room temperature reaction 2 hours, obtains reaction solution B.
Remaining all steps and condition are completely the same with embodiment 2.So as to obtain successively nanometer fluorescent microspheres 2., nanometer
Fluorescent microsphere 3. with nanometer fluorescent microspheres 4..
Nanometer fluorescent microspheres made from embodiment 2,8 and above-mentioned nanometer fluorescent microspheres are carried out to the comparison of fluorescence intensity, knot
Fruit is shown in Table 2.
The detection of the fluorescence intensity of table 2
Wherein, fluorescence intensity is equivalent to list caused by after the definition of fluorescence intensity excites for a nanoparticle in table 2
The multiple of the produced fluorescence intensity of individual free neodymium ion chelate.
B, accuracy is tested
(1) nanometer fluorescent microspheres for preparing step A 1. with nanometer fluorescent microspheres 2. according to embodiment 3~5 the step of, according to
The chloramphenicol monoclonal antibody and and lyophilized nanometer fluorescent microspheres mark of secondary acquisition nanometer fluorescent microspheres mark lyophilized accordingly
The rabbit igg monoclonal antibody of note.
(2) the chloramphenicol monoclonal antibody that marks the nanometer fluorescent microspheres lyophilized accordingly obtained by step (1) and and
The step of rabbit igg monoclonal antibody of lyophilized nanometer fluorescent microspheres mark is according to embodiment 6,8, it is accurate that testing sample is carried out
Property test.
(3) antibody freezed corresponding to each nanometer fluorescent microspheres in step (2) is carried out repeating experiment 15 times.As a result such as
Shown in table 3.
The accuracy test result of table 3
| The nanometer fluorescent microspheres used | Quantitative limit (ng/mL) | Quantitative linearity scope (ng/mL) | Sample TIANZHU XINGNAO Capsul |
| Nanometer fluorescent microspheres are 1. | 9 | 9-600 | 90%~100% |
| Nanometer fluorescent microspheres are 2. | 10 | 10-580 | 87%~101% |
| Made from embodiment 2 | 7 | 7-650 | 95%~103% |
The result explanation of table 3, when containing neodymium, samarium simultaneously in the preparing raw material of nanometer fluorescent microspheres in itself, its final inspection
The quantitative limit for surveying chloramphenicol is minimum --- detection sensitivity highest；Quantitative linearity scope is maximum --- and detection range is most wide；Sample
TIANZHU XINGNAO Capsul highest --- detection is reliable and stable.
The scope of the present invention is not limited by the specific embodiments described, and the embodiment is only used as illustrating the present invention respectively
The single example of individual aspect, the method and component of functional equivalent are also included in the scope of the invention.In fact, except as described herein
Outside content, those skilled in the art can easily grasp a variety of improvement to the present invention with reference to described above.The improvement
Also fall within the scope of the appended claims.Every bibliography mentioned above is all included in herein as reference in full.
Claims (7)
- A kind of 1. method using nanoparticle time-resolved fluorescence probe in detecting chloramphenicol, it is characterised in that it includes following The step of：(1) carboxylic polystyrene nanoparticle is prepared；(2) the carboxylic polystyrene nanoparticle prepared using step (1), prepares nanometer fluorescent microspheres；The nano fluorescent In microballoon, the mol ratio of rare earth element ion, beta-diketon body class chelate and Fluorescence Increasing synergist is 2: 2: 3, the rare earth Element ion is Nd3+And Sm3+；(3) nanometer fluorescent microspheres and chloramphenicol monoclonal antibody prepared using step (2), it is glimmering to prepare nanoparticle time resolution Light-chloramphenicol probe；The nanometer fluorescent microspheres and rabbit igg monoclonal antibody prepared using step (2) prepare the nanoparticle time Resolved fluorometric-rabbit igg probe；(4) the nanoparticle time-resolved fluorescence-chloramphenicol probe and nanoparticle time resolution that prepared by step of freeze drying (3) are glimmering Light-rabbit igg probe, obtain lyophilized nanometer fluorescent microspheres mark chloramphenicol monoclonal antibody and and lyophilized nano fluorescent it is micro- The rabbit igg monoclonal antibody of ball mark；(5) detection line T lines are prepared using chloramphenicol, prepares nature controlling line C line using goat-anti rabbit secondary antibody, spraying, assembling are chromatographed Test strips；(6) the chloramphenicol monoclonal antibody and and lyophilized nanometer that the lyophilized nanometer fluorescent microspheres for preparing step (4) mark The rabbit igg monoclonal antibody of fluorescent microsphere mark is added in the hole of ELISA Plate；(7) testing sample is taken to add in the hole of the ELISA Plate of step (6)；(8) chromatograph test strip that into the hole of step (7) prepared by inserting step (5), sample pad one end is immersed in the hole Liquid 0.5cm, reading in Fluorescent reader is inserted after immersing 5min, obtains testing result.
- 2. the method as described in claim 1, it is characterised in that step (1) the carboxylic polystyrene nanoparticle, by wrapping The method for including following steps is made：By 10mM styrene monomers and 0.95mM acrylic monomers be dissolved in 10mL dodecyl sodium sulfates containing 0.45mM go from In sub- water, round-bottomed flask is added, is stirred with magnetic stir bar；The air in round-bottomed flask is eliminated with high pure nitrogen, heated sealed adds 0.15mM potassium peroxydisulfate 0.5mL to 70 DEG C, Oxygen barrier stirring reaction is sealed after 8 hours, is down to room temperature；Reaction solution is filtered with whatman 2v filter paper, with molecular cut off 25,000Da bag filter is to deionized water dialysis 5 My god, liquid in bag filter is collected, adds 4 DEG C of preservations after 0.05% Sodium azide, the percentage is mass percent.
- 3. the method as described in claim 1, it is characterised in that in step (2), the beta-diketon body class chelate is β-naphthalene first Acyl trifluoroacetone；And/or the Fluorescence Increasing synergist is trioctyl phosphine oxide and/or phenanthroline.
- 4. method as claimed in claim 3, it is characterised in that step (2) described nanometer fluorescent microspheres are by comprising the following steps Method be made：The carboxylic polystyrene nanoparticle for taking step (1) to prepare, 10mL deionized waters and acetone mixture are added, it is described to go The volume ratio of ionized water and the acetone is 1:1, obtain reaction solution A；The density for making the polystyrene microsphere of carboxylated in reaction solution A is 1 × 1014Individual/mL, stirs, and sequentially adds 0.1M μ L of neodymium trichloride 100, the μ L of 0.1M samarium trichlorides 100, μ L of 0.1M β-naphthoyltrifluoroacetone 200,0.1M trioctyl phosphine oxides 150 The μ L of μ L and 0.1M phenanthroline 150, it is heated to 60 DEG C of constant temperature stirring lucifuges and reacts 10 hours, be then down to room temperature reaction 2 hours, obtain Reaction solution B；Organic solvent in solution is removed by reaction solution B by being evaporated under reduced pressure mode, deionized water is dialysed 5 days, removes it Small-molecule substance more than surplus, liquid in bag filter is collected, add 0.05% 4 DEG C of preservations of Sodium azide, the percentage is quality Percentage.
- 5. the method as described in claim 1, it is characterised in that step (3) nanoparticle time-resolved fluorescence-chloramphenicol Probe is made by the method comprised the following steps：Nanometer fluorescent microspheres prepared by step (2), are dissolved in 10mL 0.01M pH8.0 borate buffer, make fluorescent microsphere Density is about 1.0 × 1012Individual/mL, 400W are ultrasonically treated 30 seconds, are then slowly added into the 15mg/mL μ L of carbodiimide 200, are obtained Reaction solution I；Reaction solution I is at the uniform velocity stirred at ambient temperature after incubating 15 minutes, 150000g is centrifuged 10 minutes, collects precipitation, use 0.01M pH 8.0 borate buffer washs precipitation repeatedly, repeated centrifugation step 2 time, produces the fluorescent microsphere of activation；The fluorescent microsphere of activation is redissolved in 5mL 0.01M pH8.0 borate buffer, adds 250 μ g chloramphenicol monoclonals Antibody, 4 DEG C of stirring reactions 12 hours, obtains reaction solution II；The 12000g of reaction solution II is centrifuged 10 minutes, precipitation is collected, the precipitation is redissolved in containing 1.5% trehalose and 2% ox blood In pure albumen, 0.01M pH7.4 phosphate buffer, the percentage is quality percent by volume.
- 6. the method as described in claim 1, it is characterised in that the chlorine of step (4) the lyophilized nanometer fluorescent microspheres mark Mycin monoclonal antibody and and lyophilized nanometer fluorescent microspheres mark method of the rabbit igg monoclonal antibody by comprising the following steps It is made：Nanoparticle time-resolved fluorescence-chloramphenicol probe prepared by step (3) and nanoparticle time-resolved fluorescence-rabbit IgG probes dilute 20 times and 30 times successively respectively with lyophilized dilution, obtain diluent A and dilution B, the lyophilized dilution is Containing 6% sucrose, 4% bovine serum albumin(BSA), 1% mannitol 0.05M pH 7.4 PBPS buffer solutions, the percentage is Mass percent；The diluent A and dilution B are pressed 1:Vacuum freezedrying after 1 volume ratio mixes.
- 7. the method as described in claim 1, it is characterised in that step (5) chromatograph test strip is by comprising the following steps Method is made：The 0.01MpH7.4 phosphoric acid containing 1.5% trehalose, 2% bovine serum albumin(BSA), 0.05% Tween-20 is used to delay in chloramphenicol Punching is dissolved to final concentration 0.1mg/mL, must spray film liquid A, is sprayed on Membrane jetter by film liquid A is sprayed away from nitrocellulose filter left end 2mm Place forms detection line T lines, and the percentage is quality percent by volume；By goat-anti rabbit secondary antibody use containing 1.5% trehalose, 2% bovine serum albumin(BSA), 0.05% polysorbas20 0.01M pH7.4 phosphorus Acid buffering is dissolved to final concentration 1.0mg/mL, must spray film liquid B, and will spray film liquid B with Membrane jetter is sprayed on away from the nitrocellulose filter right side Nature controlling line C line is formed at the 4mm of end, nature controlling line is separated by 5mm with detection line, and the percentage is quality percent by volume；The nitrocellulose filter sprayed is placed in 25 DEG C of vacuum drying ovens and dried；Overlapped successively on hardboard paper and paste nitrocellulose filter, glass fibre element pads, draws the cellulose nitrate for there are detection line T lines Plain film, nitrocellulose filter, filter paper, sample pad and the blotting paper for having nature controlling line C line are drawn, then cut into the wide test strips of 4mm.
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