CN106568949A - Direct competitive fluoroimmunoassay-based small molecule hapten detection method - Google Patents
Direct competitive fluoroimmunoassay-based small molecule hapten detection method Download PDFInfo
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Abstract
The invention provides a direct competitive fluoroimmunoassay-based small molecule hapten detection method. According to the method, fluorescent microspheres embedded with quantum dots are adopted instead of a conventional enzyme carrier to be subjected to small molecule Hapten conjugation. A coupled product is adopted as a competitive antigen to be subjected to direct competitive ELISA. According to the technical scheme of the invention, a large number of quantum dots are embedded inside a polymer carrier, so that the luminous intensity is higher. The detection sensitivity is effectively improved. In addition, the quantum dots are encapsulated in the microspheres, so that the quantum dots are less influenced by the external environment. In this way, the fluorescent quenching phenomenon and the microsphere coagulation phenomenon are avoided to a certain extent. At the same time, quantum dot microspheres are relatively large in particle size, so that quantum dot microspheres are simpler in separation and purification compared with quantum points. The microsphere separation in a conventional solution can be realized through the low-speed centrifugation process (wherein the speed is smaller than 10000 rpm). Moreover, the over-high affinity between competitive antigens and coated antibodies is reduced to a certain extent due to the relatively large particle sizes of the quantum dot microspheres. Therefore, the detection sensitivity is improved.
Description
Technical field
The present invention relates to Antigen Detection Techniques field, further to the improvement of direct competive ELISA method, and in particular to one
Plant based on the small haptens detection method of direct competitive fluorescence immunoassay.
Background technology
Immunoassay is the trace analysis between antigen and antibody based on specific recognition and reversibility association reaction
Method.Immunoassay is applicable not only to the detection of macromolecular compound (such as protein, nucleic acid, antibacterial), is also applied for small molecule
Compound (such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additive and physiologically active chemical substance etc.)
Determine.Because immunoassay has, high specificity, sensitivity are high, simple, quick, expense is low and are suitable to live batch samples sieves
The advantages of selecting, it obtains extensively should in the analysis fields such as environmental monitoring, food safety detection, clinical diagnosises and bioanalysiss
With.
Enzyme linked immunosorbent assay requires low, easy to carry, operation as one of the form of immunoassay with technical conditions
Easily and economically, effect duration length, sensitivity height, high specificity, be capable of achieving mass detection, often occur in the form of test kit and
The advantages of easy commercialization, have become and be most widely used and develop biological detection and analytical technology the most ripe.Now,
Conventional enzyme linked immunosorbent assay mainly includes two big class：One class is Double antibody sandwich-ELISA, and such method is
It is widely used in detecting the macromole antigen containing multiple antigenic determinants, such as albumen, microorganism and cell.Relative to tool
There is the macromole antigen of multiple antigenic determinants, micromolecular compound is often only single because of its molecular weight (being less than 6000)
Antigenic determinant, it is impossible to detected using Double antibody sandwich-ELISA.Competitive enzyme-linked immune absorption method is detection
One of small haptens most common method, mainly there is two kinds of detection patterns：Competitive ELISA absorption method and indirectly
Competitive enzyme-linked immune absorption method.Wherein, competitive ELISA absorption method is because of its simple and quick inspection in small haptens
Important role is played in survey.However, traditional competitive ELISA absorption method has two obvious shortcomings：First,
Cause sensitivity relatively low as signal output using horseradish peroxidase or the colour developing of alkali phosphatase catalytic chemistry substrate；2nd,
Enzyme-labelled antigen or the competition antigen antigen that constitutes competition of a relatively high with the affinity of antibody are difficult to be competed by target analytes, so as to
Cause sensitivity low.Therefore, the sensitivity for improving detection signal or the affinity for reducing competition antigen antagonist can be effective
Improve the sensitivity of traditional competitive ELISA absorption method in ground.
In traditional competitive ELISA absorption method, compete antigen preparation be by by small haptens with carry
Body protein (such as horseradish peroxidase, alkali phosphatase) is coupled.Because the size of carrier protein is less, constitute competition antigen
It is higher with the affinity of corresponding antibodies, it is impossible to be competed by object.Relative to carrier protein, nano-particle has bigger chi
Very little and weight, at that same temperature, its Brownian movement is slower.Can be with using nano-particle as the carrier of small haptens
Synthesize the lower competition antigen of affinity, so as to be easier to be competed by target analytes, and then it is sensitive to obtain higher detection
Degree.Additionally, substituting albumen as the carrier of competition antigen using nano material, traditional chemical synthesis or biological is effectively evaded
The limitation of synthetic antigen analog, such as complex operation are loaded down with trivial details, waste time and energy and occasionality is big.It is excellent based on above-mentioned technology
Gesture, the antigen vectors of nano-particle are of great interest, but in terms of the selection of carrier, have both been contemplated that molecular level
Coupling effect, while should also have the good characteristics of luminescence.In recent years, quantum dot excited with its width, narrow transmitting, stoke
This displacement is big and the excellent optical characteristics such as resistance to photobleaching are widely applied in immune analysis, has researcher to attempt profit
Replace conventional carrier HRP, ALP with quantum dot, but the combination effect of discovery quantum dot and antigen is undesirable in practical study, phase
The luminescent properties answered are difficult to ensure that, simultaneously because quantum dot nature is unstable therefore easily affected by environment and fluorescence occurs
It is quenched, further, since on the one hand the particle diameter of quantum dot is still relatively small, therefore there is a problem of isolating and purifying inconvenience, it is another
Aspect can still result in the too high phenomenon of affinity between competition antigen and antibody.
The content of the invention
It is contemplated that for the technological deficiency of prior art, there is provided a kind of based on the little of direct competitive fluorescence immunoassay
Molecule hapten detection method, with the direct competive ELISA method for solving prior art because chromogenic substrate luminescent properties are not good
Cause the technical problem that detection sensitivity is relatively low.
The invention solves the problems that another technical problem be prior art direct competive ELISA method in because competitive antigen with
The affinity of antibody is too high and causes detection sensitivity relatively low.
The invention solves the problems that another technical problem be to by quantum dot substitute zymophore direct competive ELISA method
In, the luminescent properties of quantum dot are difficult to be guaranteed.
The invention solves the problems that another technical problem be to by quantum dot substitute zymophore direct competive ELISA method
In, the chemical stability of quantum dot is relatively low.
The invention solves the problems that another technical problem be when using quantum dot microsphere as antigenic mark carrier to small molecule
When hapten performs enzyme linked immunosorbent assay detection, the biologic activity for being labeled antigen declines.
The invention solves the problems that another technical problem be when using quantum dot microsphere as antigenic mark carrier to small molecule
When hapten performs enzyme linked immunosorbent assay detection, antigen is lived in antigen-quantum dot microsphere complex during long-term holding
Property decline.
To realize above technical purpose, the present invention is employed the following technical solutions：
A kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method belongs to direct competitive
ELISA method, wherein the carrier being coupled with small haptens is the fluorescent microsphere of the quantum dot for being marked with carboxyl modified.
Preferably, the method is comprised the following steps：
1) coated antibody in ELISA Plate；
2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared；
3) by step 2) products therefrom and small haptens are coupled, that is, obtain competing antigen；
4) solution to be measured is added to step 1 with the competition antigen) ELISA Plate of antibody is coated with, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
Preferably, step 2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is to be prepared by the following method
's：With chloroform as solvent, the quantum dot concentration for preparing oleic acid moieties modification is 15～25mg/mL, PMMA concentration is 25～35mg/
ML, PMAO concentration is the mixed solution of 15～25mg/mL, keeps 25～35min, will then take sodium dodecyl sulfate aqueous solution
It is 3～4mg/mL to mix the quantum dot concentration modified to wherein oleic acid moieties with the mixed solution, and dechlorination is gone after mix homogeneously
Imitative, solid-liquid separation takes solid phase, that is, the fluorescent microsphere of the quantum dot for obtaining being marked with carboxyl modified.It is further preferred on this basis
's：The mix homogeneously can be realized under the conditions of ultrasonic vibration；The solid-liquid separation can be by the way that realization is centrifuged；
Solid-liquid separation takes can be with milli-Q water after solid phase, and washing times can be three times, and the product after washing can be in ultra-pure water
In in 4 DEG C preservation.
Preferably, the quantum dot is CdSe/ZnS quantum dots.
Preferably, the excitation wavelength of the quantum dot be 450nm, launch wavelength be 620nm.
Preferably, the removal of chloroform is realized using revolving.
Preferably, step 3) specifically include following operation：Carboxyl modified is marked with using bovine serum albumin coating
The fluorescent microsphere of quantum dot, product and small haptens are coupled, that is, obtain competing antigen.
Preferably, the utilization bovine serum albumin coating is marked with the fluorescent microsphere of the quantum dot of carboxyl modified, bag
Include following steps：The fluorescent microsphere and 1- ethyls-(3- diformazans of the quantum dot of carboxyl modified will be marked with phosphate buffer
Base aminopropyl) phosphinylidyne diimine, bovine serum albumin mixing, the quality volume fraction of bovine serum albumin is into solution
0.8%～1.2%, 25～35min is reacted in 35～39 DEG C, then add 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two
Imines 3～5 times, adds react 25～35min after 35～39 DEG C every time, adds and is fully completed rear solid-liquid separation and takes solid phase, washes
It is dissolved in sodium bicarbonate solution after washing.In optimal technical scheme more than, in order to obtain high repeatability, bovine serum albumin
Saturation flags are employed in vain.It is further preferred on this basis：The number of times added can be 4 times；The phosphate buffer
Initial concentration can be 0.04～0.06mol/L, its pH can be 5.8～6.2；Repeat the 1- ethyls-(3- bis- for adding every time
Dimethylaminopropyl) amount of phosphinylidyne diimine can be with equal；Taking can be with milli-Q water after solid phase, and washing times can be three
Secondary (washing step is used to remove unnecessary BSA)；The pH of the sodium bicarbonate solution can be 8.2～8.6；It is dissolved in carbonic acid
Can be in 4 DEG C of preservations after in hydrogen sodium solution.
Preferably, step 3) in solid-liquid separation be that 8～12min is centrifuged with the rotating speed of 12000～15000rpm.
Preferably, the coupling being marked between the fluorescent microsphere of the quantum dot of carboxyl modified and small haptens is profit
With active ester method realization.
Preferably, the fluorescent microsphere of the coated quantum dots for being marked with carboxyl modified of Jing BSA and small haptens idol
The coupling of connection is realized using active ester method.
It is the competitive antigen for obtaining different affinitys in above technical scheme, can be anti-by control small molecule half
It is former with the coated quantum dot microsphere of bovine serum albumin on bovine serum albumin mol ratio realizing, concrete consumption and operation
Condition can carry out adaptation Sexual behavior mode according to the general technology general knowledge of active ester method；Certainly, small haptens and bovine serum albumin
Concrete labelling mole can be than being respectively 5 between bovine serum albumin on white coated quantum dot microsphere:1、1:1、1:5、1:
10、1:20, preferably 1:10.
Preferably, the condition of the antigen antibody reaction is to react 25～35min at 35～39 DEG C.
Preferably, step 4) in the addition of competitive antigen and testing sample be 40～60 μ L/ holes；More optimizedly
50 μ L/ holes.
Preferably, Jing after antigen antibody reaction, first with the 0.01M phosphate buffer detersive enzymes containing 0.05% polysorbas20
Target three times, then ELISA Plate 1 time, then fluorescence intensity are washed with the phosphate buffer of 0.01M.
In above technical scheme, detect that the fluorescence intensity for obtaining is i.e. haptenic for reacting testing sample small molecular
Content, in practical operation using concentration known and distribution gradient small haptens standard solution, by above method
(percentage fluorescence rate (%)=F/F0 × 100%, wherein F0 is strong for the fluorescence of first standard (0 standard) to draw percentage fluorescence rate
Angle value, F is the meansigma methodss of the fluorescence intensity level of standard substance or sample) --- the standard curve of small haptens concentration, then profit
Corresponding percentage fluorescence rate is calculated with the fluorescence intensity of testing sample, little point of testing sample is then calculated from standard curve
Sub- hapten content.Concrete operation method can carry out adaptation Sexual behavior mode according to the general technology general knowledge of the art.Above-mentioned ladder
The titer of the small haptens of degree distribution, can respectively select 1.5,1,0.75,0.4,0.2,0.1,0.05,0pg/mL.
In above technical scheme, each reagent can reused using before prior to more than equilibrium at room temperature 30min.It is described
PMAO is maleic anhydride/1- vaccenic acid alternate copolymers, and the PMMA is polymethyl methacrylate, and the two can be from market
Buy.The quantum dot of the oleic acid moieties modification can be prepared according to the ordinary skill in the art.
The present invention suitable for small haptens detection by quantitative, such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated
Food additive and the chemical substance with physiologically active etc., are especially suitable for the trace detection of target analytes.Sample
Process according to conventional treatment method.
Had the advantages that using technical solution of the present invention：
1st, the present invention is embedded single fluorescence quantum into polymer microballoon by microemulsion method, has been prepared luminous
The higher fluorescence quantum microsphere of intensity, the more corresponding quantum dot of its luminous intensity improves 2800 times；Further, since quantum dot
The inside of microsphere is wrapped in, is affected little by external environment (solvent, heat, electricity, magnetic etc.), property is more steady under the protection of shell structure
It is fixed, avoid to a certain extent fluorescence be quenched and microsphere coagulation.Meanwhile, the particle diameter of quantum dot microsphere is about tens and arrives
Hundreds of nanometer, isolate and purify it is easy compared with quantum dot, the slow-speed of revolution (<10000rpm) centrifugation can be achieved with the microsphere point in conventional soln
From.
2nd, the inventive method prepares high luminous quantum dot fluorescence microsphere by using oil-soluble quantum dot is used for replacement amount
Son point is directly used in conventional immunological fluorescent labeling, greatly increases the intensity of fluorescence signal output, is conducive to improving detection
Sensitivity.
3rd, the inventive method substitutes little albumen particle as small molecule half by using the bigger quantum dot microsphere of particle diameter
The coupling carrier of antigen, it is possible to obtain the competition antigen of different affinitys, and affinity excursion is wider, is favorably improved straight
Connect the detection sensitivity of competition immune analysis.
4th, the technology of the present invention substitutes traditional enzymatic chemical colour reaction signal by using high luminous quantum dot microsphere, subtracts
Enzymatic step is lacked, therefore has operated more simply, detection time is shorter.
The invention provides a kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method
It is anti-with small molecule half conventional zymophore to be substituted using the fluorescent microsphere (Quantum dot beads, QBs) for being embedded with quantum dot
Original is coupled, and direct competive ELISA is performed as competition antigen using coupled product.In the technical scheme, fluorescent microsphere is by high poly-
Thing carrier has embedded substantial amounts of quantum dot, thus with higher luminous intensity, can effectively improve the sensitivity of detection.This
Outward, because quantum dot is wrapped in the inside of microsphere, affected little by external environment (solvent, heat, electricity, magnetic etc.), in the guarantor of shell structure
The lower property of shield is more stable, avoid to a certain extent fluorescence be quenched and microsphere coagulation.Meanwhile, the grain of quantum dot microsphere
Footpath is about tens to hundreds of nanometer, isolate and purify it is easy compared with quantum dot, the slow-speed of revolution (<10000rpm) centrifugation can be achieved with conventional molten
Microsphere in liquid is separated；It is additionally, since fluorescent microsphere and there is larger particle diameter, therefore can reduces to a certain extent competing antigen
The too high affinity between coated antibody, so as to lift the sensitivity of detection.
Description of the drawings
Fig. 1 is the principle schematic of the inventive method；
Fig. 2 is the standard curve of Aflatoxins M1 direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1；
Fig. 3 is the standard curve of parathion direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1；
Fig. 4 is the standard curve of enrofloxacin direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1；
Fig. 5 is the standard curve of 19- nortestosterones direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1；
Fig. 6 is the standard curve of tripolycyanamide direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1；
Fig. 7 is that the standard of 1,25- dihydroxyvitamin Ds direct competitive fluorescence immunoassay credit analysis in the embodiment of the present invention 1 is bent
Line.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
Will not be described in detail to belonging to known structure or function in following examples.
Approximating language used in following examples can be used for quantitative expression, show in the feelings for not changing basic function
Quantity can be allowed under condition to have certain variation.Therefore, with " about ", that the numerical value corrected of the language such as " left and right " is not limited to this is accurate
Numerical value itself.In certain embodiments, " about " expression allows scope of the numerical value that it is corrected positive and negative 10 (10%)
Interior change, such as, what " about 100 " represented can be any numerical value between 90 to 110.Additionally, " the about first numerical value is arrived
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
May be relevant with the precision of measuring instrument.
In addition to being defined, technology used and scientific terminology have and art technology people of the present invention in following examples
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent；The experiment
Method, if no special instructions, is conventional method；Quantitative test in following examples, is respectively provided with three repetitions and tests, as a result
Average；% in following examples, if no special instructions, is weight/mass percentage composition.
In tests below, the collocation method of phosphate buffer (PBS, 0.05M, pH 7.4) is as follows：NaCl 40g,
Na2HPO413.5g, KH2PO41.0g, KCl 1.0g is dissolved in 1L ultra-pure waters.With 0.1M NaOH pH value is adjusted to 8.0～9.0.
Mouse IgG class monoclonal antibody involved in test：Aspergillus flavus resisting toxin M1 monoclonal antibodies, anti-parathion
Monoclonal antibody, anti-enrofloxacin monoclonal antibody, anti-19- nortestosterones monoclonal antibody, anti-melamine monoclonal antibody
And anti-1,25- dihydroxyvitamin D monoclonal antibodies, provided by Wuxi Zhongde Bore Bioisystech Co., Ltd, this experiment
Involved all small haptens are bought from Sigma companies.
Embodiment 1
1st, the preparation of the quantum dot fluorescence microsphere of carboxyl modified
Quantum dot after the modification of 10mg oleic acid moieties (excitation wavelength of the quantum dot is 450nm, and launch wavelength is 620nm)
In being dissolved in the chloroform of 0.5mL, the polymethyl methacrylate (PMMA) and 10mg maleic anhydrides of 15mg/carbon of 1- 18 is added
Alkene alternate copolymer (PMAO), the mixed liquor is re-dissolved in the sodium dodecyl sulfate aqueous solution of 2.5mL most after half an hour
Final concentration about 3.3mg/mL, the mixed liquor is mixed under ultrasound condition, and this non-pole of chloroform is removed with the method for revolving after mixing
Property organic solvent after obtain the quantum dot fluorescence microsphere of water solublity carboxyl modified by way of centrifugation by quantum dot fluorescence microsphere
Separate, the milli-Q water three times of the quantum dot fluorescence microsphere after separation.Quantum dot fluorescence microsphere after washing is again molten
Solution 4 DEG C of preservations in ultra-pure water.
2nd, the preparation of the coated quantum dot microsphere of bovine serum albumin
The quantum dot fluorescence microsphere of water solublity carboxyl modified is dissolved in into pH 6.0, in 0.05mol/L phosphate buffers,
After adding appropriate 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine, the Ox blood serum that quality volume fraction is 1% is added
Albumin, 37 DEG C of reaction half an hour, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two that equivalent is continuously added after half an hour is sub-
Amine, repetition is added four times, and the coated quantum dot fluorescence microsphere of the bovine serum albumin saturation is centrifuged 10min under 14000rpm,
Remove the serum albumin for using three removals of milli-Q water unnecessary after supernatant, the quantum dot fluorescence microsphere after centrifugation is redissolved in pH
In 8.4 sodium bicarbonate solution, 4 DEG C of preservations.
3rd, Detection results checking test
This section performs this with the prepared coated quantum dot microsphere of bovine serum albumin of the 1st, 2 sections as experimental raw
Bright method, to verify the method to small-molecule substances such as mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additive
Detection results.
When the inventive method is used to detect small haptens content, implement sample pre-treatments by following steps, then
Detected with detection method, analysis result.
1) sample pre-treatments：The small haptens standard substance bought are diluted to into corresponding Concentraton gradient, concentration according to
Required actually detected limit determines；It is standby that the antigen sample for having diluted is put into 4 DEG C of refrigerators.Goal analysis in different samples to be checked
The purification process of thing is completed referring in particular to GB.
2) detection mycotoxin, pesticide, veterinary drug, environmental hormone, violated food additive are carried out with detection method
And the content of chemical substances with physiologically active.
3) analysis result.
With the standard substance 1.5,1,0.75,0.4,0.2,0.1,0.05,0pg/mL of 8 variable concentrations of above-mentioned preparation.
Excitation wavelength is 450nm, and launch wavelength is fluorescence intensity at 620nm.
The percentage fluorescence rate of the calculating of percentage fluorescence rate, standard substance or sample is equal to the fluorescence intensity level of standard substance or sample
Meansigma methodss remove in first standard (0 standard), i.e. percentage fluorescence rate (%)=F/F0× 100%, wherein F0For first standard
The fluorescence intensity level of (0 standard), F is the meansigma methodss (diplopore) of the fluorescence intensity level of standard substance or sample.
With percentage fluorescence rate as vertical coordinate, standard curve is drawn by abscissa of small haptens concentration (g/mL), asked
Linear equation.When actual sample detection is carried out, by the fluorescent value (F/F of sample0× 100%) value substitute into standard curve in,
The concentration of corresponding sample is read from standard curve, being multiplied by its corresponding extension rate, to be sample small molecular haptenic
Actual concentrations.
3.1 pairs of aflatoxin Ms1Test experience
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add the aspergillus flavus resisting toxin M of 0.1 μ g/mL1It is single
Per hole, 37 DEG C are incubated 120min, the PBS washings ELISA Plate of the 0.01M containing 0.05% polysorbas20 three times to the μ L of clonal antibody 100
Washed once with the PBS of 0.01M afterwards, add the μ L of bovine serum albumin 340 of 10mg/mL per hole, 37 DEG C of incubation 120min have
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of the 0.01M of 0.05% polysorbas20, add variable concentrations
Aflatoxin M1, it is added in ELISA Plate per the μ L of hole 50, add 50 μ L aflatoxin Ms1It is coupled bovine serum albumin coated
Quantum dot microsphere (competition antigen), after 37 DEG C of incubation 40min, is washed with the 0.01M phosphate buffers containing 0.05% polysorbas20
ELISA Plate washs afterwards ELISA Plate 1 time for three times with the phosphate buffer of 0.01M, is surveyed with SpectraMax i3x multi-function microplate readers
Determine ELISA Plate fluorescence intensity.Solution to be checked is added in the ELISA Plate for being coated with antibody per the μ L of hole 50, while adding 50 μ L competing
Strive antigen, 37 DEG C of incubations determine the fluorescence intensity per hole after 40 minutes, by calculating meansigma methodss after be updated to standard curve and obtain
Aflatoxin M in measuring samples1Concentration.Specific experiment result is as follows：Linear standard curve be y=-25.92ln (x)+
17.158, R2=0.9984, see accompanying drawing 2.The half-inhibition concentration IC of the method is calculated by standard curve50(i.e. F/F0×
100%=50%) it is 0.28pg/mL.
Above method is not limited to aflatoxin M1Detection, can be also used for the detection of other mycotoxins, Huang Qu
Syphilis element B1/B2/G1/G2, ochratoxin, vomitoxin, 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, fumonisins, T2 toxin etc..
3.2 the test experience to parathion
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add the anti-parathion monoclonal anti of 0.1 μ g/mL
The μ L of body 100 are used per hole after the PBS washings ELISA Plate of 37 DEG C of incubation 120min, the 0.01M containing 0.05% polysorbas20 three times
The PBS of 0.01M washed once, and add the μ L of bovine serum albumin 340 of 10mg/mL per hole, and 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate three times after washed once with the PBS of 0.01M, add variable concentrations to sulfur
Phosphorus, is added in ELISA Plate per the μ L of hole 50, adds 50 μ L parathion to be coupled bovine serum albumin coated quantum dot microsphere (competition
Antigen), after 37 DEG C of incubation 40min, washed with the 0.01M phosphate buffers for containing 0.05% polysorbas20 and used after ELISA Plate three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, determines ELISA Plate fluorescence strong with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added in the ELISA Plate for being coated with antibody per the μ L of hole 50, while adding 50 μ L competition antigens, 37 DEG C of incubations
The fluorescence intensity per hole is determined after 40 minutes, standard curve is updated to after calculating meansigma methodss and is obtained parathion in measuring samples
Concentration.Specific experiment result is as follows：Linear standard curve is y=-24.94ln (x)+15.374, R2=0.9965, see accompanying drawing 3.
The half-inhibition concentration IC of the method is calculated by standard curve50(i.e. F/F0× 100%=50%) it is 0.25pg/mL.
Above method is not limited to the detection of parathion, can be also used for the detection of other kinds of pesticide, such as other
Organophosphors or organochlorine pesticide, carbamate chemicals for agriculture etc..
The test experience of 3.3 pairs of enrofloxacins
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add the anti-enrofloxacin monoclonal of 0.1 μ g/mL
The μ L of antibody 100 are used per hole after the PBS washings ELISA Plate of 37 DEG C of incubation 120min, the 0.01M containing 0.05% polysorbas20 three times
The PBS of 0.01M washed once, and add the μ L of bovine serum albumin 340 of 10mg/mL per hole, and 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate three times after washed once with the PBS of 0.01M, add the En Nuosha of variable concentrations
Star, is added in ELISA Plate per the μ L of hole 50, adds 50 μ L enrofloxacins to be coupled the coated quantum dot microsphere of bovine serum albumin (competing
Strive antigen), after 37 DEG C of incubation 40min, washed with the 0.01M phosphate buffers for containing 0.05% polysorbas20 and used after ELISA Plate three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, determines ELISA Plate fluorescence strong with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added in the ELISA Plate for being coated with antibody per the μ L of hole 50, while adding 50 μ L competition antigens, 37 DEG C of incubations
The fluorescence intensity per hole is determined after 40 minutes, standard curve is updated to after calculating meansigma methodss and is obtained En Nuosha in measuring samples
Star concentration.Specific experiment result is as follows：Linear standard curve is y=-21.94ln (x)+20.74, R2=0.9905, see accompanying drawing
4.The half-inhibition concentration IC of the method is calculated by standard curve50(i.e. F/F0× 100%=50%) it is 0.26pg/mL.
Above method is not limited to the detection of enrofloxacin, can be also used for the detection of other kinds of veterinary drug, such as other
Fluoroquinolones, sulfonamides, beta receptor analeptic and tetracycline medication etc.
The test experience of 3.4 pairs of 19- nortestosterones
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add the anti-19- nortestosterones list of 0.1 μ g/mL
Per hole, 37 DEG C are incubated 120min, the PBS washings ELISA Plate of the 0.01M containing 0.05% polysorbas20 three times to the μ L of clonal antibody 100
Washed once with the PBS of 0.01M afterwards, add the μ L of bovine serum albumin 340 of 10mg/mL per hole, 37 DEG C of incubation 120min have
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of the 0.01M of 0.05% polysorbas20, add variable concentrations
19- nortestosterones, are added in ELISA Plate per the μ L of hole 50, add 50 μ L19- nortestosterones to be coupled bovine serum albumin coated
Quantum dot microsphere (competition antigen), after 37 DEG C of incubation 40min, is washed with the 0.01M phosphate buffers containing 0.05% polysorbas20
ELISA Plate washs afterwards ELISA Plate 1 time for three times with the phosphate buffer of 0.01M, is surveyed with SpectraMax i3x multi-function microplate readers
Determine ELISA Plate fluorescence intensity.Solution to be checked is added in the ELISA Plate for being coated with antibody per the μ L of hole 50, while adding 50 μ L competing
Strive antigen, 37 DEG C of incubations determine the fluorescence intensity per hole after 40 minutes, by calculating meansigma methodss after be updated to standard curve and obtain
19- nortestosterones concentration in measuring samples.Specific experiment result is as follows：Linear standard curve be y=-28.39ln (x)+
20.294, R2=0.9802, see accompanying drawing 5.The half-inhibition concentration IC of the method is calculated by standard curve50(i.e. F/F0×
100%=50%) it is 0.35pg/mL.
Above method is not limited to the detection of 19- nortestosterones, can be also used for the detection of other kinds of environmental hormone,
Such as gonadal hormone (progesterone, testosterone), steroid derivatives.
The test experience of 3.5 pairs of tripolycyanamide
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add the anti-melamine monoclonal of 0.1 μ g/mL
The μ L of antibody 100 are used per hole after the PBS washings ELISA Plate of 37 DEG C of incubation 120min, the 0.01M containing 0.05% polysorbas20 three times
The PBS of 0.01M washed once, and add the μ L of bovine serum albumin 340 of 10mg/mL per hole, and 37 DEG C of incubation 120min have 0.05%
Polysorbas20 0.01M PBS washing ELISA Plate three times after washed once with the PBS of 0.01M, add the melamine of variable concentrations
Amine, is added in ELISA Plate per the μ L of hole 50, adds 50 μ L tripolycyanamide to be coupled the coated quantum dot microsphere of bovine serum albumin (competing
Strive antigen), after 37 DEG C of incubation 40min, washed with the 0.01M phosphate buffers for containing 0.05% polysorbas20 and used after ELISA Plate three times
The phosphate buffer washing ELISA Plate of 0.01M 1 time, determines ELISA Plate fluorescence strong with SpectraMax i3x multi-function microplate readers
Degree.Solution to be checked is added in the ELISA Plate for being coated with antibody per the μ L of hole 50, while adding 50 μ L competition antigens, 37 DEG C of incubations
The fluorescence intensity per hole is determined after 40 minutes, standard curve is updated to after calculating meansigma methodss and is obtained melamine in measuring samples
Amine concentration.Specific experiment result is as follows：Linear standard curve is y=-17.11ln (x)+42.003, R2=0.9777, see accompanying drawing
6.The half-inhibition concentration IC of the method is calculated by standard curve50(i.e. F/F0× 100%=50%) it is 0.63pg/mL.
Above method is not limited to the detection of tripolycyanamide, can be also used for the detection of other violated food additive.3.6
Test experience to 1,5- dihydroxyvitamin Ds
The Protein G of 20 μ g/mL is added in ELISA Plate per the μ L of hole 100, and 4 DEG C overnight, with containing 0.05% polysorbas20
Washed once with the PBS of 0.01M after the PBS washing ELISA Plate three times of 0.01M, add anti-1, the 25- dihydroxy dimension of 0.1 μ g/mL
Per hole, 37 DEG C are incubated 120min, the PBS detersive enzymes of the 0.01M containing 0.05% polysorbas20 to the raw μ L of element D monoclonal antibodies 100
Washed once with the PBS of 0.01M after target three times, add the μ L of bovine serum albumin 340 of 10mg/mL per hole, 37 DEG C of incubations
120min, have 0.05% polysorbas20 0.01M PBS washing ELISA Plate three times after washed once with the PBS of 0.01M, add
1, the 25- dihydroxyvitamin Ds of variable concentrations, are added in ELISA Plate per the μ L of hole 50, add 50 μ L1,25- dihydroxyvitamins
D is coupled the coated quantum dot microsphere (competition antigen) of bovine serum albumin, after 37 DEG C of incubation 40min, with containing 0.05% polysorbas20
0.01M phosphate buffers washing ELISA Plate wash ELISA Plate 1 time for three times with the phosphate buffer of 0.01M afterwards, use
SpectraMax i3x multi-function microplate readers determine ELISA Plate fluorescence intensity.By solution to be checked per the μ L of hole 50 be added to be coated with it is anti-
In the ELISA Plate of body, at the same add 50 μ L competition antigen, 37 DEG C incubation 40 minutes after determine per hole fluorescence intensity, by calculate
Standard curve is updated to after meansigma methodss and obtains 1,25- dihydroxyvitamin D concentration in measuring samples.Specific experiment result is as follows：
Linear standard curve is y=-24.3ln (x)+26.373, R2=0.9844, see accompanying drawing 7.The party is calculated by standard curve
The half-inhibition concentration IC of method50(i.e. F/F0× 100%=50%) it is 0.38pg/mL.
Above method is not limited to the detection of 1,5- dihydroxyvitamin Ds, can be also used for other kinds of physiologically active
The detection of chemical substance.
Embodiment 2
A kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method includes following step
Suddenly：
1) coated antibody in ELISA Plate；
2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared；
3) by step 2) products therefrom and small haptens are coupled, that is, obtain competing antigen；
4) solution to be measured is added to step 1 with the competition antigen) ELISA Plate of antibody is coated with, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, following condition is met：
Step 2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared by the following method：With chloroform
For solvent, the quantum dot concentration for preparing oleic acid moieties modification is for 25mg/mL, PMAO concentration for 15mg/mL, PMMA concentration
The mixed solution of 15mg/mL, keep 25min, then will take sodium dodecyl sulfate aqueous solution mix with the mixed solution to
The quantum dot concentration of wherein oleic acid moieties modification is 3mg/mL, and chloroform is removed after mix homogeneously, and solid-liquid separation takes solid phase, that is, obtains
It is marked with the fluorescent microsphere of the quantum dot of carboxyl modified.
The quantum dot is CdSe/ZnS quantum dots.
The excitation wavelength of the quantum dot is 450nm, launch wavelength is 620nm.
The removal of chloroform is realized using revolving.
Step 3) specifically include following operation：Using bovine serum albumin coating be marked with carboxyl modified quantum dot it is glimmering
Light microsphere, product and small haptens are coupled, that is, obtain competing antigen.
The utilization bovine serum albumin coating is marked with the fluorescent microsphere of the quantum dot of carboxyl modified, including following step
Suddenly：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos third of the quantum dot of carboxyl modified will be marked with phosphate buffer
Base) phosphinylidyne diimine, bovine serum albumin mixing, into solution the quality volume fraction of bovine serum albumin be 0.8%%, in
35 DEG C of reaction 25min, then add 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 3 times, add every time after 35 DEG C
Reaction 25min, adds and is fully completed rear solid-liquid separation and takes solid phase, is dissolved in sodium bicarbonate solution after washing.
The solid-liquid separation is that 8min is centrifuged with the rotating speed of 12000rpm.
The condition of the antigen antibody reaction is to react 25min at 35 DEG C.
Embodiment 3
A kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method includes following step
Suddenly：
1) coated antibody in ELISA Plate；
2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared；
3) by step 2) products therefrom and small haptens are coupled, that is, obtain competing antigen；
4) solution to be measured is added to step 1 with the competition antigen) ELISA Plate of antibody is coated with, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, following condition is met：
Step 2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared by the following method：With chloroform
For solvent, the quantum dot concentration for preparing oleic acid moieties modification is for 35mg/mL, PMAO concentration for 25mg/mL, PMMA concentration
The mixed solution of 25mg/mL, keep 35min, then will take sodium dodecyl sulfate aqueous solution mix with the mixed solution to
The quantum dot concentration of wherein oleic acid moieties modification is 4mg/mL, and chloroform is removed after mix homogeneously, and solid-liquid separation takes solid phase, that is, obtains
It is marked with the fluorescent microsphere of the quantum dot of carboxyl modified.
The removal of chloroform is realized using revolving.
Step 3) specifically include following operation：Using bovine serum albumin coating be marked with carboxyl modified quantum dot it is glimmering
Light microsphere, product and small haptens are coupled, that is, obtain competing antigen.
The utilization bovine serum albumin coating is marked with the fluorescent microsphere of the quantum dot of carboxyl modified, including following step
Suddenly：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos third of the quantum dot of carboxyl modified will be marked with phosphate buffer
Base) phosphinylidyne diimine, bovine serum albumin mixing, into solution the quality volume fraction of bovine serum albumin be 1.2%, in 39
DEG C reaction 35min, then adds 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 5 times, adds every time anti-after 39 DEG C
35min is answered, is added and is fully completed rear solid-liquid separation and takes solid phase, be dissolved in sodium bicarbonate solution after washing.
The solid-liquid separation is that 12min is centrifuged with the rotating speed of 15000rpm.
The condition of the antigen antibody reaction is to react 35min at 39 DEG C.
Embodiment 4
A kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method includes following step
Suddenly：
1) coated antibody in ELISA Plate；
2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared；
3) by step 2) products therefrom and small haptens are coupled, that is, obtain competing antigen；
4) solution to be measured is added to step 1 with the competition antigen) ELISA Plate of antibody is coated with, antigen-antibody knot
Reaction is closed, the fluorescence intensity of ELISA Plate is then detected.
On the basis of above technical scheme, following condition is met：
The quantum dot is CdSe/ZnS quantum dots.
The condition of the antigen antibody reaction is to react 35min at 39 DEG C.
Step 3) specifically include following operation：Using bovine serum albumin coating be marked with carboxyl modified quantum dot it is glimmering
Light microsphere, product and small haptens are coupled, that is, obtain competing antigen.
Embodiment 5
A kind of small haptens detection method based on direct competitive fluorescence immunoassay, the method belongs to direct competitive
ELISA method, wherein the carrier being coupled with small haptens is the fluorescent microsphere of the quantum dot for being marked with carboxyl modified.
Embodiments of the invention have been described in detail above, but the content is only presently preferred embodiments of the present invention,
Not to limit the present invention.All any modification, equivalent and improvement made in the application range of the present invention etc., all should
It is included within protection scope of the present invention.
Claims (10)
1. a kind of small haptens detection method based on direct competitive fluorescence immunoassay, it is characterised in that the method belongs to
Direct competive ELISA method, wherein with small haptens be coupled carrier be the quantum dot for being marked with carboxyl modified fluorescence it is micro-
Ball.
2. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 1,
It is characterized in that comprising the following steps：
1) coated antibody in ELISA Plate；
2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared；
3) by step 2) products therefrom and small haptens are coupled, that is, obtain competing antigen；
4) solution to be measured is added to step 1 with the competition antigen) ELISA Plate of antibody is coated with, antigen-antibody combines anti-
Should, then detect the fluorescence intensity of ELISA Plate.
3. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 2,
It is characterized in that step 2) fluorescent microsphere of the quantum dot for being marked with carboxyl modified is prepared by the following method：With chlorine
Imitate as solvent, it is 25～35mg/mL, PMAO that the quantum dot concentration for preparing oleic acid moieties modification is 15～25mg/mL, PMMA concentration
Concentration is the mixed solution of 15～25mg/mL, keep 25～35min, then will take sodium dodecyl sulfate aqueous solution with it is described
It is 3～4mg/mL that mixed solution mixes the quantum dot concentration modified to wherein oleic acid moieties, and chloroform, solid-liquid are removed after mix homogeneously
Separation takes solid phase, that is, the fluorescent microsphere of the quantum dot for obtaining being marked with carboxyl modified.
4. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 3,
It is characterized in that the quantum dot is CdSe/ZnS quantum dots.
5. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 3,
It is characterized in that the excitation wavelength of the quantum dot be 450nm, launch wavelength be 620nm.
6. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 3,
It is characterized in that the removal of chloroform is realized using revolving.
7. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 2,
It is characterized in that step 3) specifically include following operation：The quantum dot of carboxyl modified is marked with using bovine serum albumin coating
Fluorescent microsphere, product and small haptens are coupled, that is, obtain competing antigen.
8. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 7,
It is characterized in that the utilization bovine serum albumin coating is marked with the fluorescent microsphere of the quantum dot of carboxyl modified, including following step
Suddenly：The fluorescent microsphere and 1- ethyls-(3- dimethylaminos third of the quantum dot of carboxyl modified will be marked with phosphate buffer
Base) phosphinylidyne diimine, bovine serum albumin mixing, into solution the quality volume fraction of bovine serum albumin be 0.8%～
1.2%, 25～35min is reacted in 35～39 DEG C, then add 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine 3～5
It is secondary, add react 25～35min after 35～39 DEG C every time, add and be fully completed rear solid-liquid separation and take solid phase, dissolve after washing
In sodium bicarbonate solution.
9. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 8,
It is characterized in that the solid-liquid separation is that 8～12min is centrifuged with the rotating speed of 12000～15000rpm.
10. a kind of small haptens detection method based on direct competitive fluorescence immunoassay according to claim 2,
It is characterized in that the condition of the antigen antibody reaction is to react 25～35min at 35～39 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110987884A (en) * | 2019-11-20 | 2020-04-10 | 复旦大学 | Light emission detection sensitivity enhancing method based on solid optical micro-resonant cavity |
| CN112067815A (en) * | 2020-09-03 | 2020-12-11 | 南昌大学 | Homogeneous immunization method for detecting small molecule hapten |
| CN113391076A (en) * | 2021-07-20 | 2021-09-14 | 深圳健丰源科技有限公司 | Immunodetection method of 25-hydroxy vitamin D and application thereof |
| CN117723749A (en) * | 2024-02-07 | 2024-03-19 | 南昌大学 | Dynamic light scattering immunosensory detection method based on molecular adhesive |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100291474A1 (en) * | 2006-05-10 | 2010-11-18 | 3M Innovative Properties Company | Compositions and coatings containing fluorescent, inorganic nanoparticles |
| CN101893623A (en) * | 2010-06-22 | 2010-11-24 | 上海师范大学 | Rapid detection method employing ultrasensitive quantum dot microsphere immunity-chromatograph test paper strips |
| CN102253214A (en) * | 2011-07-06 | 2011-11-23 | 清华大学深圳研究生院 | Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit |
| CN104991070A (en) * | 2015-06-25 | 2015-10-21 | 浙江大学 | Method for detecting fungaltoxin by using quantum dot cation exchange signal amplification technology |
| CN105602545A (en) * | 2015-12-24 | 2016-05-25 | 天津大学 | Preparation method of monodisperse quantum dot micro spheres with optical gain property |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN105842441A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for ochratoxin A |
-
2016
- 2016-11-02 CN CN201610944123.XA patent/CN106568949B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100291474A1 (en) * | 2006-05-10 | 2010-11-18 | 3M Innovative Properties Company | Compositions and coatings containing fluorescent, inorganic nanoparticles |
| CN101893623A (en) * | 2010-06-22 | 2010-11-24 | 上海师范大学 | Rapid detection method employing ultrasensitive quantum dot microsphere immunity-chromatograph test paper strips |
| CN102253214A (en) * | 2011-07-06 | 2011-11-23 | 清华大学深圳研究生院 | Quantum dot-based method for detecting ciprofloxacin by immunofluorescence and special kit |
| CN104991070A (en) * | 2015-06-25 | 2015-10-21 | 浙江大学 | Method for detecting fungaltoxin by using quantum dot cation exchange signal amplification technology |
| CN105602545A (en) * | 2015-12-24 | 2016-05-25 | 天津大学 | Preparation method of monodisperse quantum dot micro spheres with optical gain property |
| CN105759045A (en) * | 2016-03-18 | 2016-07-13 | 南昌大学 | Method for detecting aflatoxin B1 |
| CN105842441A (en) * | 2016-03-18 | 2016-08-10 | 南昌大学 | Detection method for ochratoxin A |
Non-Patent Citations (5)
| Title |
|---|
| 任美玲: "量子点荧光微球免疫层析试纸条定量检测玉米中的黄曲霉毒素B1", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 刘富华等: "量子点制备与表面修饰", 《化学通报》 * |
| 戴尽波: "量子点标记免疫技术在食品中小分子有害物检测中的应用", 《食品科学》 * |
| 董春红等: "双亲性高分子PMAO超声乳化法改性量子点的制备", 《天津市生物医学工程学会第三十三届学术年会论文集》 * |
| 金丽等: "无机/有机掺杂CdTe/PMMA的合成", 《吉林化工学院院报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110987884A (en) * | 2019-11-20 | 2020-04-10 | 复旦大学 | Light emission detection sensitivity enhancing method based on solid optical micro-resonant cavity |
| CN112067815A (en) * | 2020-09-03 | 2020-12-11 | 南昌大学 | Homogeneous immunization method for detecting small molecule hapten |
| CN113391076A (en) * | 2021-07-20 | 2021-09-14 | 深圳健丰源科技有限公司 | Immunodetection method of 25-hydroxy vitamin D and application thereof |
| CN117723749A (en) * | 2024-02-07 | 2024-03-19 | 南昌大学 | Dynamic light scattering immunosensory detection method based on molecular adhesive |
| CN117723749B (en) * | 2024-02-07 | 2024-06-04 | 南昌大学 | Dynamic light scattering immunosensory detection method based on molecular adhesive |
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