CN110187117A - The detection kit and its application of beta-Lactam antibiotic - Google Patents
The detection kit and its application of beta-Lactam antibiotic Download PDFInfo
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- CN110187117A CN110187117A CN201910525200.1A CN201910525200A CN110187117A CN 110187117 A CN110187117 A CN 110187117A CN 201910525200 A CN201910525200 A CN 201910525200A CN 110187117 A CN110187117 A CN 110187117A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses the detection kit of beta-Lactam antibiotic and its applications.The present invention provides beta-Lactam antibiotic detection kit comprising: 1) antibody or its solution of the anti-beta-Lactam antibiotic of fluorescent marker；2) particles with superparamagnetism label beta-Lactam antibiotic coupling protein antigen or its solution；The beta-Lactam antibiotic coupling protein antigen is the artificial antigen formed after beta-Lactam antibiotic and carrier protein couplet；3) micro-fluidic chip detection card；The detection sensitivity height of the kit, high specificity, can quickly, easily detect beta-Lactam antibiotic.The application of the kit will be helpful to the detection of the main residues such as harmful beta-lactam antibiotic in raw material milk supply and its dairy produce.
Description
Technical field
The present invention relates to field of detection of food safety, and in particular, to the detection kit of beta-Lactam antibiotic and its
Using the side of detection kit and detection beta-Lactam antibiotic more particularly, to beta-Lactam antibiotic in milk
Method.
Background technique
Milk has become a kind of Major Nutrient drink of Chinese, and it is general that China's milk safety problem has become a whole people
All over the social concern of concern.Beta-lactam antibiotic is widely used in animal husbandry as veterinary drug, passes through in milk cow production
It is commonly used to prevent and treat the diseases such as bovine mastitis, pneumonia, bacterial diarrhea and bacterial arthritis.When treating lactation period infected cattle
The beta-lactam antibiotic used can remain from mammary gland is moved on in milk cow and enter in milk, as precautionary measures, in milk cow
The antibiotic added in feed will also result in the residual of antibiotic in milk.Long-term consumption has the milk of antibiotic residue, energy
The problems such as enough causing allergy and increasing the drug resistance of human body, so countries in the world forbid the dairy produce containing antibiotic in city
It circulates on.The main body management mode of China milk industry is still that dispersion cultivates at present, concentrated processing after being purchased by milking station, but I
The milking station infrastructure construction of state is overall more to fall behind, and detectability is to be improved.Milking station construction is extremely heavy in milk supply development
The content wanted.It is reported that the time of venomous injurant quality detection is longer (needing -3 hours 2 hours)；Testing cost is also higher, milking station
Complete detection can not be carried out from door to door, and can only carry out Preliminary detection to basic physical and chemical index.If certain family milk goes wrong,
Also it only waits until just detected after delivering Dairy Enterprise together after problem milk is mixed with qualified milk.Many is often led in this way
The dairy farmer that there is no problem is involved and is sustained a loss.In addition, milking station is general lack of professional technician, former milk quality it is preliminary
It checks on not enough, is badly in need of carrying out relatively complete professional technique training to milking station staff and instrument operator.Therefore, it builds
Vertical raw material milk and quality of dairy products safety detection standards system and whole-course quality control system, and it is interior to the biggish β-of health hazard
Measure that amide antibiotics residue project establishes corresponding rapid detection method and standard is very important.
Summary of the invention
For current dairy produce enterprise because lacking quick, cheap, accurate and efficient antibiotics leftover detection technology due to nothing
Method obtains the technology missing link of safety " nonreactive " milk supply, and beta-lactam antibiotic is super quick fast in present invention preparation detection milk
Fast detection kit, specific technical solution are as follows:
A purpose of the invention is to provide a kind of detection kit of beta-Lactam antibiotic.
Detection kit provided by the invention comprising:
1) antibody or its solution of the anti-beta-Lactam antibiotic of fluorescent marker；
2) particles with superparamagnetism label beta-Lactam antibiotic coupling protein antigen or its solution；The beta-lactam is anti-
Raw element coupling protein antigen is the artificial antigen formed after beta-Lactam antibiotic and carrier protein couplet；
3) micro-fluidic chip detection card；
For micro-fluidic chip detection card as shown in Figure 1, A is sectional view, B is front elevation, by bottom plate 1 and is fixed on bottom plate
Micro-fluidic chip 2 form；Micro-fluidic chip is equipped with sense channel 9；Sense channel is by foot passage 3 and is located at its both ends
Loading channel 45 compositions are connected to imbibition channel, the opening in the Loading channel and imbibition channel is respectively well 6 and inhales
Fluid apertures 7, the foot passage are fixed with filter core 8 (in the embodiment of the present invention, filter core is located among foot passage), filter core
It is equipped with multiple through-holes for the sample adding liquid that circulates.
Other than both ends are respectively as the opening of well and imbibing hole, rest part seals the entire sense channel
It closes.The filter core is securable to the foot passage middle position.It is seamless between the filter core and the foot passage to be connected,
Sample adding liquid is only capable of circulating by the through-hole on the filter core.
The diameter of particles with superparamagnetism is greater than the through-hole aperture on filter core；And the diameter of particles with superparamagnetism is less than locating for it
Foot passage internal diameter.The through-hole aperture of the filter core is 0.5 μm -5 μm, it is preferable that is 1-2 μm.
In an embodiment of the present invention, Loading channel and imbibition channel are perpendicular to foot passage, and well and imbibing hole
Horizontal position be higher by the foot passage；It can also be with other angle design, as long as allowing the sample of addition from Loading channel
Well flows into, and flows through filter core, and can be sucked out from the imbibing hole in imbibition channel.
For the sense channel, it can be designed to that the internal diameter in the Loading channel and the imbibition channel is greater than the bottom
The internal diameter in portion channel can preferably cooperate the head of sample injector when using in this way.
In a specific embodiment of the invention, in the sense channel, the Loading channel and the imbibition channel
Perpendicular to the foot passage (the i.e. described foot passage be parallel to ground, the Loading channel and the imbibition channel perpendicular to
Ground) so that the horizontal position of the well and the imbibing hole is higher by the foot passage.The sense channel it is interior
Diameter is 1-100 μm；Wherein, the internal diameter of the foot passage is specially 20 μm, the Loading channel and the imbibition channel it is interior
Diameter is specially 100 μm (diameter of the well and the imbibing hole is 100 μm).
Wherein, in the micro-fluidic chip detection card, the horizontal position of the well and the imbibing hole is higher by described
The foot passage top edge 1-3mm, such as 1-2mm of sense channel.
In mentioned reagent box, the fluorescence is fluorescein isothiocynate, phycoerythrin, rare-earth fluorescent microballoon or fluorescence volume
Sub- point；
Further, the fluorescence quantum is CdSe/ZnS fluorescence quantum；
Or, in the antibody of the anti-beta-Lactam antibiotic of the fluorescent marker, the antibody of the anti-beta-Lactam antibiotic
For penicillin binding protein.
In mentioned reagent box, the particles with superparamagnetism is Fe3O4Magnetic nano-particle is coated SiO2And surface modification
The microballoon of carboxyl-functional group.
In mentioned reagent box, the particles with superparamagnetism diameter is 100nm-100um；
Or, further, the particles with superparamagnetism diameter is 1-10um；
Or, further, the particles with superparamagnetism diameter is 4-10um.
In mentioned reagent box, the albumen is BSA, OVA or KLH.
In mentioned reagent box, the kit further includes the sample treatment liquid for dilute sample；
Further, the sample treatment liquid is the 0.02M PBS solution containing 0.05%Tween20.
In mentioned reagent box, the through-hole aperture on the filter core is less than the diameter of the particles with superparamagnetism；
The diameter of the particles with superparamagnetism is less than the internal diameter of foot passage locating for the filter core.
Or, the diameter of the particles with superparamagnetism is less than the sense channel on micro-fluidic chip detection card most
Small internal diameter.
Another object of the present invention is to provide the preparation method of mentioned reagent box.
Preparation method provided by the invention includes the following steps: to prepare the anti-beta-lactam through fluorescent marker respectively
The beta-Lactam antibiotic coupling protein antigen and the micro-fluidic core of the antibody of antibiotic, particles with superparamagnetism label
Piece detection card；
The method for preparing the antibody of the anti-beta-Lactam antibiotic through fluorescent marker is as mentioned before；
Prepare the method such as institute above of the beta-Lactam antibiotic coupling protein antigen through particles with superparamagnetism label
It states；
The method for preparing the micro-fluidic chip detection card is as mentioned before.
Micro-fluidic chip detection card in above-mentioned detection kit is also the scope of protection of the invention；
Or following any application is also the scope of protection of the invention:
Application of the above-mentioned kit in detection beta-Lactam antibiotic；
Or application of the mentioned reagent box in preparation detection beta-Lactam antibiotic product；
Or whether mentioned reagent box contains the application in beta-Lactam antibiotic in detection sample to be tested；
Or whether mentioned reagent box contains the application in beta-Lactam antibiotic product in preparation detection sample to be tested；
Or application of the mentioned reagent box in detection sample to be tested in beta-Lactam antibiotic content；
Or application of the mentioned reagent box in preparation detection sample to be tested in beta-Lactam antibiotic content product.
It is a still further object of the present invention to provide following methods.
Method provided by the invention is following either method:
(A) a kind of method for detecting beta-Lactam antibiotic content in sample to be tested, comprising:
(A1) it draws standard curve: the standard solution of the beta-Lactam antibiotic of series of concentrations is prepared, if by resulting
The standard solution of dry beta-Lactam antibiotic of the part containing various concentration is respectively and at the sample in above-mentioned kit
Liquid mixing is managed, then the antibody of the anti-beta-Lactam antibiotic of the fluorescent marker being separately added into the kit and described
The beta-Lactam antibiotic coupling protein antigen of particles with superparamagnetism label in kit, reaction obtain reaction solution；
The reaction solution is added in the well of the detection card of the micro-fluidic chip in the kit, then described in
Sample treatment liquid is rinsed；Detect the glimmering of the particles with superparamagnetism for being located at the filter core front end in the micro-fluidic chip detection card
Luminous intensity；Then standard curve is drawn according to the concentration value of the standard items of the fluorescence intensity level and beta-Lactam antibiotic measured,
Obtain calibration curve equation；
Wherein, the concentration value of the standard items of the basis measures fluorescence intensity level and beta-Lactam antibiotic draws standard
Curve can according to the LOG value of the concentration values of the standard items of fluorescence intensity level and beta-Lactam antibiotic that measures make standard song
Line chart；
(A2) sample to be tested detects: the sample to be tested is mixed with the sample treatment liquid in the kit, then
Described in being added in the antibody and the kit of the anti-beta-Lactam antibiotic through fluorescent marker in the kit
The beta-Lactam antibiotic coupling protein antigen marked through particles with superparamagnetism, reaction, obtains reaction solution；By the reaction solution
It is added in the well of the micro-fluidic chip detection card in the kit, then rushed with the sample treatment liquid
It washes；Detect the fluorescence intensity for being located at the particles with superparamagnetism of the filter core front end in the micro-fluidic chip detection card；Then root
The content of beta-Lactam antibiotic in the sample to be tested is calculated according to calibration curve equation obtained by (A1)；
(B) it is a kind of detection or auxiliary detection sample to be tested in whether the method containing beta-Lactam antibiotic, comprising:
(B1) sample to be tested is mixed with the sample treatment liquid in the kit, is added in the kit
The anti-beta-Lactam antibiotic through fluorescent marker antibody and the kit in it is described through particles with superparamagnetism mark
The beta-Lactam antibiotic coupling protein antigen of note, reaction, obtains reaction solution；The reaction solution is added to examination described previously
The micro-fluidic chip in agent box detects in the well of card, then is rinsed with the sample treatment liquid；Then institute is detected
State the fluorescence intensity for being located at the microballoon of the filter core front end in micro-fluidic chip detection card；
(B2) according to testing result, according to determining in the sample to be tested whether contain beta-Lactam antibiotic as follows: if
(B1) fluorescence intensity measured is not less than standard fluorescence intensity, then does not contain in the sample to be tested or candidate is without containing β-
Beta-lactam antibiotics；If the fluorescence intensity that (B1) is measured is lower than the standard fluorescence intensity, or detects unstressed configuration through (B1),
Then contain in the sample to be tested or candidate contains beta-Lactam antibiotic；
The standard fluorescence intensity is measured according to the method included the following steps: by sample to be tested described in (B1)
The sample without beta-Lactam antibiotic is replaced with, remaining is operated with (B1).
Further, in (A1) and (B1), the condition of the reaction can be room temperature reaction 10min.
Further, the sample to be tested can be milk.
Micro-fluidic chip hereinbefore detects card, and channel internal diameter is designed as 20 μm, and the filter core aperture in channel is 1-2 μ
M (aperture is uneven, between 1-2um)；The horizontal position of well and imbibing hole is higher by the middle section top of sense channel
Edge 1-2mm.
Above-mentioned beta-Lactam antibiotic is ampicillin, ampicillin sodium salt, Amoxicillin, benzyl penicillin, mould
Plain V, oxacillin, Cloxacillin, Cefquinome, Ceftiofur, cefapirin or Cefacetrile.
The experiment proves that the present invention prepares a kind of detection kit for detecting beta-Lactam antibiotic, the reagent
The detection sensitivity height of box, high specificity, can quickly, easily detect beta-Lactam antibiotic.The application of the kit will have
The detection for helping in raw material milk supply and its dairy produce the main residues such as harmful beta-lactam antibiotic, solve currently due to
It abuses antibiotic and is difficult to ensure the technical bottleneck problem of China's milk supply quality and safety, thus to promote the strong of China milk industry
Kang Fazhan, Chinese is improved to the confidence of domestic dairy produce, the health offer technical support of the guarantee common people and product branch
It holds.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of micro-fluidic chip detection card in the detection kit of beta-Lactam antibiotic；A is to cut open
Face figure, B are front elevation；1 is bottom plate；2 be micro-fluidic chip；3 be foot passage；4 be Loading channel；5 be imbibition channel；6 are
Well；7 be imbibing hole；8 be filter core；9 be sense channel.
Fig. 2 is the detected value of the detection kit of beta-Lactam antibiotic and the curve synoptic diagram of concentration.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment 1, the detection kit of beta-Lactam antibiotic
One, the preparation of the penicillin binding protein of fluorescent marker
The penicillin binding protein of fluorescent marker is the sulfydryl of penicillin binding protein (anti-beta-Lactam antibiotic antibody)
What the amino covalence key coupling with fluorophor was realized, the specific method is as follows:
1, mercapto is introduced in penicillin binding protein
3- (the 2- pyridine of 10uM is added in 5mg penicillin binding protein (Zhen Kang Science and Technology Ltd., Shenzhen, Ag021)
Dimercapto) propionic acid N-hydroxy-succinamide ester (SPDP) solution (SPDP and methanol composition), it reacts at room temperature 60 minutes, uses hole
The super filter tube that diameter is 10kDa purifies, and collects filtrate, then the dithiothreitol (DTT) solution (dithiothreitol (DTT) of 1uM is added into filtrate
Formed with water), it reacts 30 minutes and is purified with the super filter tube that aperture is 10kDa, collect filtrate again, as contain free mercaptan
Penicillin binding protein solution.
2, the amino functional CdSe/ZnS quantum dot (activating quantum dot amino with sulfo-SMCC) activated
By the amino functional CdSe/ZnS fluorescence quantum (Zhen Kang Science and Technology Ltd., Shenzhen, CQ003) of 10mg with
The sulfo-SMCC solution (sulfo-SMCC and DMSO solvent composition) of 0.01mM mixes, and incubates 30 min at room temperature and ultrafiltration is de-
Salt collects filtrate, as the amino functional CdSe/ZnS quantum dot solution of desalination.
3, the penicillin binding protein of fluorescent marker
By the amino function of the above-mentioned 1 penicillin binding protein solution containing free mercaptan prepared and the desalination of above-mentioned 2 preparation
CdSe/ZnS quantum dot solution can be changed to mix and incubate 3h at room temperature.The n- second for being 1% with final concentration mass percent concentration
30min is closed and incubated to base maleimide amine aqueous solution, is finally purified with 300kDa super filter tube (with containing 0.1% (quality volume hundred
Point content g: ml) pH value of BSA be 7.4 concentration is the washing of 0.02M PBS buffer solution), it collects ultrafiltration product and is simultaneously stored up at 4 DEG C
Deposit, obtain fluorescent marker penicillin binding protein solution (solvent be containing 0.1% (quality volumn concentration g:ml) BSA's
PH value is that 7.4 concentration are 0.02M PBS buffer solution；The concentration of the penicillin binding protein of fluorescent marker is 1mg/mL).
Two, the preparation of particles with superparamagnetism label beta-Lactam antibiotic coupling BSA antigen
It is that beta-Lactam antibiotic is coupled BSA antigen that particles with superparamagnetism, which marks beta-Lactam antibiotic coupling BSA antigen,
Aminoterminal and the c-terminus covalent coupling of particles with superparamagnetism obtain.
Above-mentioned particles with superparamagnetism is Fe3O4Magnetic nano-particle is coated SiO2And surface modification carboxyl-functional group
Microballoon, diameter 4-10um.
1, the superparamagnetism Fe of surface carboxyl groups modification and coated Si O23O4The preparation of particle
Take 100-300 nanometers of 100mg partial size of magnetic Fe3O4Nano particle, with HCl processing, the washing of 0.1M.Then will
In its ethanol water for being scattered in 100ml 20%.0.5mL concentrated ammonia liquor (concentration 25%) is added under ultrasound condition.Afterwards plus
Enter 1mL tetraethyl orthosilicate, reacts 24 hours.It is scattered in after washing in 100ml ethyl alcohol, 0.5mL concentrated ammonia liquor is added, is then added
1mL OTMS is reacted 24 hours, is scattered in chloroform after washing, and 1g PMA-ODE is then added, 25% ammonium hydroxide is added after dry
Dissolution obtains surface carboxyl functionalized and coated Si O2 Superparamagnetic Fe_3O_4 particle, and diameter is 4-10um.
2, the preparation of particles with superparamagnetism label beta-Lactam antibiotic coupling BSA antigen
With it is above-mentioned 1 preparation average diameter be 4-10um, surface carboxyl functionalized and coated Si O2 superparamagnetism
Fe3O4 particle, ampicillin are coupled BSA antigen, and it is even to prepare particles with superparamagnetism label ampicillin by the following method
Join BSA antigen:
(1) surface carboxyl functionalized and coated Si O2 the superparamagnetism Fe of above-mentioned 1 preparation of 10mg is taken3O4Particle is used
It after MES buffer (0.1M, pH4.7) washing and Magneto separate, is resuspended with 1ml MES buffer (0.1M, pH4.7), 1- second is added
Base-(3- dimethylaminopropyl) carbodiimide (EDC) extremely final concentration of 5mM, NHS (N- hydroxysuccinimide) is added extremely
Final concentration of 10mM, room temperature are protected from light, the superparamagnetism microballoon of carboxyl modified after reaction half an hour is activated.
(2) the superparamagnetism grain of carboxyl modified after washing the activation that (1) obtains with the borate buffer solution of 50mM pH8.5
Son；
Take 0.2mg ampicillin coupling BSA antigen (Zhen Kang Science and Technology Ltd., Shenzhen, Ag021) and 10mg above-mentioned
The particles with superparamagnetism of carboxyl modified is mixed into the borate buffer solution of 50mM pH8.5 and mixes well after activation after washing.
Room temperature is protected from light lower reaction 2 hours, allows the antigen and particles with superparamagnetism to form stable covalent peptide bonds and combines, obtains superparamagnetic
Property particle and ampicillin coupling BSA antigen conjugate.After reaction, (quality percentage contains for addition final concentration of 1%
Amount) ethanolamine solutions to particles with superparamagnetism and ampicillin coupling BSA antigen conjugate on residual activity carboxyl position
Point is closed, and room temperature is protected from light 0.5 hour.After the completion, with containing 0.1% (quality volumn concentration g:ml) BSA
0.02M PBS buffer solution；Washing, it is molten that resuspension obtains 10mg/ml particles with superparamagnetism label ampicillin coupling BSA antigen
Liquid (it is 0.02M PBS buffer solution that solvent, which is 7.4 concentration for the pH value containing 0.1% (quality volumn concentration g:ml) BSA), 4
It DEG C saves stand-by.
Three, micro-fluidic chip detection card
Micro-fluidic chip detection card is as shown in Figure 1, micro-fluidic chip detection card is by bottom plate 1 and is fixed on micro- on bottom plate
Fluidic chip 2 forms；Micro-fluidic chip 2 is equipped with sense channel 9；Sense channel 9 by foot passage 3 and be located at its two
The Loading channel 4 at end is connected to composition with imbibition channel 5, and the opening in Loading channel 4 and imbibition channel 5 is respectively well 6 and inhales
Fluid apertures 7, the centre of foot passage 3 are fixed with filter core 8, filter core 8 be equipped with it is multiple for the sample adding liquids that circulate through-hole (filter core and
Seamless between foot passage to be connected, sample adding liquid is only capable of circulating by the through-hole on filter core).In order to preferably cooperate sample injector
Head, sense channel 9 are designed to that the internal diameter in Loading channel 4 and imbibition channel 5 is greater than internal diameter (the i.e. sense channel 9 of foot passage 3
For the thick intermediate thin structure in both ends).In sense channel 9, Loading channel 4 and imbibition channel 5 are perpendicular to 3 (i.e. bottom of foot passage
Portion channel 3 is parallel to ground, and Loading channel 4 and imbibition channel 5 are perpendicular to ground) so that the level of well 6 and imbibing hole 7
Position is higher by the top edge of foot passage 3.The internal diameter of foot passage 3 is specially 20 μm, Loading channel 4 and imbibition channel 5 it is interior
Diameter is specially 100 μm (diameter of well 6 and imbibing hole 7 is 100 μm).The aperture of through-hole on filter core 8 is 1-2 μm of (hole
Diameter is uneven, between 1-2 μm)；The horizontal position of well 6 and imbibing hole 7 is higher by the top edge 1-2mm of foot passage 3.
The diameter of particles with superparamagnetism is greater than the through-hole aperture on filter core；And the diameter of particles with superparamagnetism is less than filter core institute
Locate the internal diameter of foot passage.
Four, the preparation of beta-Lactam antibiotic kit is detected
Detection beta-Lactam antibiotic kit contains:
1) the penicillin binding protein solution (concentration 1mg/mL) of the fluorescent marker of an above-mentioned preparation；It specifically will be real
The penicillin binding protein solution for applying the fluorescent marker that example 1 is prepared is used containing 0.1% (quality volumn concentration g:ml)
The pH value of BSA is that 7.4 concentration are that 100 times of 0.02M PBS buffer solution dilution (concentration after 100 times of dilution is 10ug/mL) divides afterwards
Dress.
2) it is above-mentioned two preparation particles with superparamagnetism label beta-Lactam antibiotics be coupled BSA antigenic solution: solvent be containing
The 0.02M PBS buffer solution of 0.1% (quality volumn concentration g:ml) BSA；Particles with superparamagnetism labelled antigen concentration 100,000
A/mL, in terms of particles with superparamagnetism number in every mL solution；The particles with superparamagnetism marks beta-that specifically above-mentioned two are prepared
Beta-lactam antibiotics coupling BSA antigen is used tricks rolling counters forward, and with the pH containing 0.1% (quality volumn concentration g:ml) BSA
Value is that 7.4 concentration are that particle is formulated as 100,000/mL of concentration and dispensed by 0.02M PBS buffer solution.
3) the micro-fluidic chip detection card of above-mentioned three preparation is packaged to be detection beta-Lactam antibiotic kit.
4) sample treatment liquid.
It is that 0.02M PBS is molten that sample treatment liquid, which is 7.4 concentration for the pH value containing 0.05% (volumn concentration) Tween20,
Liquid.
Five, the foundation of the detection method of beta-Lactam antibiotic kit is detected
1, reaction principle
Penicillin binding protein being capable of specific bond beta-Lactam antibiotic；If there is no beta-lactam anti-in sample to be tested
Raw element, then the beta-Lactam antibiotic albumen of fluorescence quantum point mark penicillin binding protein and magnetic nano-particle label is even
Associated antigen combines；Magnetic nano-particle shows very hyperfluorescence intensity.It is to be measured if there is beta-Lactam antibiotic in sample to be tested
The beta-Lactam antibiotic protein-coupled antigen competitive binding that beta-Lactam antibiotic and magnetic nano-particle mark in sample is glimmering
Light quanta point marks penicillin binding protein；Fluorescence intensity on magnetic nano-particle weakens.
2, detection method
Particles with superparamagnetism that above-mentioned two prepare label beta-Lactam antibiotic coupling BSA antigen is used tricks rolling counters forward,
And configure 100,000/mL of concentration for particle and dispense, obtain the particles with superparamagnetism marks beta-of concentration 100,000/mL particle
Beta-lactam antibiotics are coupled BSA antigen.
It is dispensed after the penicillin binding protein for the fluorescent marker that above-mentioned one prepares is diluted 100 times.
Application method is as follows:
20uL sample to be tested is taken, is added 20uL sample treatment liquid (the 0.02M PBS solution containing 0.05%Tween20), so
Be separately added into afterwards 20uL concentration 100,000/mL particle particles with superparamagnetism label beta-Lactam antibiotic coupling BSA antigen and
The penicillin binding protein of fluorescent marker after 100 times of dilution, incubation at room temperature reaction (25 DEG C) 10 minutes after mixing, obtain anti-
Answer liquid.It takes 10uL reaction solution to be added in the well of micro-fluidic chip detection card, and is rinsed with 20uL sample treatment liquid to complete
Portion is finished.
The detection of above-mentioned micro-fluidic chip is snapped into fluorescence detector, detection parameters are 365nm excitation wavelength, 610nm transmitting
Wavelength.
Magnetic bead is trapped at filter core, is flushed away less than the sample in filter core aperture and unbonded quantum dot, before detecting filter core
Hold magnetic bead fluorescent value.
Based on competition law principle, according to fluorescent measurement, can determine that anti-with the presence or absence of beta-lactam in the sample to be tested
Life is plain and there are the beta-Lactam antibiotic of how many content, and the specific method is as follows:
(A) a kind of method for detecting beta-Lactam antibiotic content in sample to be tested, comprising:
(A1) it draws standard curve: the standard solution of the beta-Lactam antibiotic of series of concentrations is prepared, if by resulting
The standard solution of dry beta-Lactam antibiotic of the part containing various concentration respectively with the sample in kit described previously
Treatment fluid mixing, then the antibody of the anti-beta-Lactam antibiotic of the fluorescent marker being separately added into kit described previously
With the beta-Lactam antibiotic coupling protein antigen of the particles with superparamagnetism label in kit described previously, reaction is obtained
To reaction solution；The reaction solution is added to the sample-adding of the detection card of the micro-fluidic chip in kit described previously
In hole (magnetic bead is trapped at filter core, is flushed away less than the sample in filter core aperture and unbonded quantum dot), then with the sample
Treatment fluid rinses；Detect the fluorescence intensity for being located at the magnetic bead of the filter core front end in the micro-fluidic chip detection card；Then root
Standard curve is drawn according to the concentration value of the standard items of the fluorescence intensity level and beta-Lactam antibiotic that measure, obtains standard curve
Equation；
Wherein, the concentration value of the standard items of the basis measures fluorescence intensity level and beta-Lactam antibiotic draws standard
Curve can according to the LOG value of the concentration values of the standard items of fluorescence intensity level and beta-Lactam antibiotic that measures make standard song
Line chart.
(A2) sample to be tested detects: the sample to be tested and the sample treatment liquid in kit described previously are mixed
It closes, adds the antibody of the anti-beta-Lactam antibiotic through fluorescent marker in kit described previously and described previously
The beta-Lactam antibiotic coupling protein antigen marked through particles with superparamagnetism in kit, reaction are reacted
Liquid；The reaction solution is added to (magnetic in the well of the detection card of the micro-fluidic chip in kit described previously
Pearl is trapped at filter core, is flushed away less than the sample in filter core aperture and unbonded quantum dot), then with the sample treatment liquid
It rinses；Detect the fluorescence intensity for being located at the magnetic bead of the filter core front end in the micro-fluidic chip detection card；Then according to (A1)
The content of beta-Lactam antibiotic in the sample to be tested is calculated in gained calibration curve equation.
(B) it is a kind of detection or auxiliary detection sample to be tested in whether the method containing beta-Lactam antibiotic, comprising:
(B1) sample to be tested is mixed with the sample treatment liquid in kit described previously, is added described previously
It is described through super in the antibody and kit described previously of the anti-beta-Lactam antibiotic through fluorescent marker in kit
The beta-Lactam antibiotic coupling protein antigen of paramagnetic particles label, reaction obtain reaction solution；The reaction solution is added
(magnetic bead is trapped at filter core, small in the well of micro-fluidic chip detection card into kit described previously
It is flushed away in the sample in filter core aperture and unbonded quantum dot), then rinsed with the sample treatment liquid；Then it detects described micro-
It is located at the fluorescence intensity of the microballoon of the filter core front end in fluidic chip detection card.
(B2) according to testing result, according to determining in the sample to be tested whether contain beta-Lactam antibiotic as follows: if
(B1) fluorescence intensity measured is not less than standard fluorescence intensity, then does not contain in the sample to be tested or candidate is without containing β-
Beta-lactam antibiotics；If the fluorescence intensity that (B1) is measured is lower than the standard fluorescence intensity, or detects unstressed configuration through (B1),
Then contain in the sample to be tested or candidate contains beta-Lactam antibiotic；The standard fluorescence intensity is according to including such as
What the method for lower step measured: sample to be tested described in (B1) being replaced with into the sample without beta-Lactam antibiotic, remaining behaviour
Make with (B1).
Embodiment 2, the Performance Evaluation for detecting beta-Lactam antibiotic kit
The detection beta-Lactam antibiotic kit that embodiment 1 obtains is assessed, the specific method is as follows:
1, detection sensitivity
With beta-Lactam antibiotic ampicillin standard items (Beijing Tan Mo quality inspection Science and Technology Ltd. 69-53-4) work
The sensitivity of the detection beta-Lactam antibiotic kit of embodiment 1 is measured for sample to be tested.
Beta-Lactam antibiotic ampicillin standard items are configured to the 0.02M PBS buffer solution that pH is 7.4 simultaneously
Series of concentrations (0,0.01,0.05,0.1,0.5,1,2,5,10ng/mL) is used as sample to be tested.
Sample to be tested is detected according to five 2 method of embodiment 1.
Make canonical plotting according to the LOG value of detected value and concentration value, canonical plotting is as shown in Figure 2.
It will test beta-Lactam antibiotic concentration when sensitivity is set to 50% inhibiting rate, will test and be limited to 90% inhibition
Beta-Lactam antibiotic concentration when rate, the results showed that, beta-Lactam antibiotic fluorescence detection test detects beta-lactam antibiosis
The sensitivity of plain ampicillin is 0.23ng/mL, and detection is limited to 0.03ng/mL.
The above results show that detection beta-Lactam antibiotic may be implemented.
2, accuracy and accuracy detection
The ampicillin of various concentration is added respectively to milk sample as sample to be tested.
Sample to be tested is detected according to five 2 method of embodiment 1.
Each sample to be tested is tested 10 times, and calculates the rate of recovery.
Measurement result is shown in Table 1, and ampicillin TIANZHU XINGNAO Capsul is 98.2%-104.3%, and accuracy is preferable；Variation lines
Number 3.9%-9.7%, average coefficient of variation is less than 15%.
Table 1 is that accuracy and accuracy detect
3, cross reaction detects
The obtained beta-Lactam antibiotic kit of embodiment 1 respectively intersects beta-lactam antibiotic
Reaction assay, and calculate cross reacting rate (table 2).
Cross reacting rate (%)=[IC50(ampicillin)/IC50(beta-Lactam antibiotic to be measured)] × 100.
2 cross reaction of table detection
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective,
The scope of the present invention is defined by the claims and their equivalents.
Claims (10)
1. a kind of detection kit of beta-Lactam antibiotic comprising:
1) antibody or its solution of the anti-beta-Lactam antibiotic of fluorescent marker；
2) particles with superparamagnetism label beta-Lactam antibiotic coupling protein antigen or its solution；The beta-Lactam antibiotic is even
Connection proteantigen is the artificial antigen formed after beta-Lactam antibiotic and carrier protein couplet；
3) micro-fluidic chip detection card；
The micro-fluidic chip detection card includes bottom plate and the micro-fluidic chip being fixed on bottom plate；It is set on the micro-fluidic chip
There is sense channel；The sense channel is connected to group with imbibition channel with the Loading channel for being located at its both ends by foot passage
At the opening in the Loading channel and the imbibition channel is respectively well and imbibing hole, and the foot passage is fixed with filter
Core, the filter core are equipped with multiple through-holes for the sample adding liquid that circulates.
2. detection kit according to claim 1, it is characterised in that:
The fluorescence is fluorescein isothiocynate, phycoerythrin, rare-earth fluorescent microballoon or fluorescence quantum；
Or, the antibody of the anti-beta-Lactam antibiotic is blueness in the antibody of the anti-beta-Lactam antibiotic of the fluorescent marker
Mycin binding protein.
3. detection kit according to claim 1 or 2, it is characterised in that:
The particles with superparamagnetism is Fe3O4Magnetic nano-particle is coated SiO2And surface modification carboxyl-functional group is micro-
Ball.
4. detection kit according to claim 1 to 3, it is characterised in that:
The particles with superparamagnetism diameter is 100nm-100um；
Or, the particles with superparamagnetism diameter is 1-10um；
Or, the particles with superparamagnetism diameter is 4-10um.
5. detection kit according to any one of claims 1-4, it is characterised in that:
The albumen is BSA, OVA or KLH.
6. any detection kit in -5 according to claim 1, it is characterised in that:
The kit further includes the sample treatment liquid for dilute sample.
7. any kit in -6 according to claim 1, it is characterised in that:
Through-hole aperture on the filter core is less than the diameter of the particles with superparamagnetism；
The diameter of the particles with superparamagnetism is less than the minimum diameter of the sense channel on micro-fluidic chip detection card；
Or, the diameter of the particles with superparamagnetism is less than the internal diameter of foot passage locating for the filter core.
8. the preparation method of any kit in claim 1-7 includes the following steps: to prepare respectively described through fluorescence mark
Note anti-beta-Lactam antibiotic antibody, the particles with superparamagnetism label beta-Lactam antibiotic coupling protein antigen and
The micro-fluidic chip detection card；
Prepare the method for the antibody of the anti-beta-Lactam antibiotic through fluorescent marker as recited in claim 7；
In the method such as claim 7 for preparing the beta-Lactam antibiotic coupling protein antigen through particles with superparamagnetism label
It is described；
Prepare the method for the micro-fluidic chip detection card as recited in claim 7.
9. the micro-fluidic chip in detection kit as claimed in any one of claims 1 to 5 detects card.
10. following any application:
Application of any kit in detection beta-Lactam antibiotic in claim 1-7；
Or application of any kit in preparation detection beta-Lactam antibiotic product in claim 1-7；
Or in claim 1-7 any kit in detection sample to be tested whether containing in beta-Lactam antibiotic
Using；
Or whether any kit contains beta-Lactam antibiotic in preparation detection sample to be tested in claim 1-7
Application in product；
Or any kit answering in beta-Lactam antibiotic content in detection sample to be tested in claim 1-7
With；
Or any kit beta-Lactam antibiotic content product in preparation detection sample to be tested in claim 1-7
In application.
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Application publication date: 20190830 |