CN106996923A - The method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously - Google Patents
The method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of method for detecting a variety of environmental hormones simultaneously based on the surface plasma resonance technology that regenerated liquid optimizes, based on spr sensor, with reference to immunosuppressive method, according to the difference of adhesion between different antigen-antibodies, by using the actified solution gradient elution of varying strength, with reference to the real-time monitoring of spr sensor, detected while single pass censorchip surface realizes a variety of environmental hormones.It is of the invention compared with existing surface plasma detection technique, detection efficiency improves more than 2 times, testing cost reduces more than 2 times, time shortens more than 2 times, reusable hundreds of times of chip, solve traditional SPR single channels using the method for the present invention and be unable to the problem of Multiple detection, and can be applied to accurately to detect the small-molecular-weights such as bisphenol-A, phthalic acid ester (<1000Da) material.
Description
Technical field
The invention belongs to detection method field, more particularly to a kind of surface plasma resonance skill optimized based on regenerated liquid
The method that art detects a variety of environmental hormones simultaneously.
Background technology
In the last few years, environmental hormone had been widely used in as the hormonal substance beyond a kind of living organism
In daily life.Although environmental hormone has brought certain economic benefit, it is in environment and food
Residual it is serious affect human body health.Moreover, most environmental hormones belong to persistence organic pollutant, it is difficult to drop
Solution, and with certain cumulative toxicity, therefore, the monitoring for environmental hormone is imperative with analyzing.
At present, environmental hormone is by high performance liquid chromatography, liquid chromatograph mass spectrography and ELISA mostly
Etc. (ELISA) method is tested and analyzed, but be due to instrumentation it is technical it is strong, time-consuming, sample pre-treatments are cumbersome, matrix
Amount of samples wastes and can not reused etc. defect in complicated component, ELISA, limits the application of these conventional methods.
Accordingly, it would be desirable to develop a kind of relatively reliable, efficient, sensitive detection means.
Surface plasma resonance biological sensor (Surface Plasmon Resonance, SPR), with without mark,
The features such as highly sensitive, quick detection, be one of ideal tools of environmental hormone detection.With reference to immunoassay technology, antibody is utilized
And the principle of the combination of antigen, the protide things such as qualitative or quantitative analysis, and antibody are carried out to specific target molecule
Matter has larger molecular weight, and higher SPR can be caused to respond value changes, can be widely applied to small-molecule substance (such as double
Phenol A, plasticiser etc.) environmental hormone detection.Further, since environmental hormone species is various so that traditional detection process is complicated
Cumbersome, the spr sensor of single sense channel can not be met while detecting the requirement of a variety of environmental hormones.Therefore, in the urgent need to opening
Send out the novel detection method based on SPR technique.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of surface plasma resonance optimized based on regenerated liquid
The method that technology detects a variety of environmental hormones simultaneously.
The present invention is achieved by the following technical solutions：
A kind of method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously, including
Following steps：
(1) fixation of censorchip surface coating antigen or antibody：By the coating antigen or antibody of each environmental hormone residual component
It is 50-150 μ L to mix to the volume of mixed liquor, and the concentration of each coating antigen or antibody in the mixed liquor is 5-20 μ
G/mL, recycles active ester method (EDC/NHS) that the mixed liquor is fixed on into the censorchip surface and fixes；
(2) standard liquid is prepared：The hydroxyethyl piperazine second sulphur phthalate buffer for being 6-8 with 0.01-0.1mol/LpH
(HBS buffer solutions) configures the standard sample storing solution of each residual component, and the concentration of the standard sample storing solution is 0.1-
1mg/mL；
Each standard sample storing solution is measured, its corresponding standard stock solution is prepared；With hydroxyethyl piperazine second thiosulfonic acid salt buffer
Standard stock solution is configured to the standard liquid of various concentrations and mixes and incubate with the corresponding antibodies or coating antigen of excessive fixed concentration by liquid
Change, obtain the determinand mixed liquor of various concentrations；
(3) regenerated liquid eluting power is tested：Test HCl solution, NaOH solution, magnesium chloride solution and glycine-HCI molten
The elution profile for each environmental hormone antibody of censorchip surface that liquid is obtained for step (1), selects the optimal regeneration of each component
Condition, and contrast the power of its regenerated liquid eluting power；
(4) standard working curve is set up：
One pack system is detected：Measured using surface plasma resonance spectrum sensor, be passed through that 50-100 μ L steps (2) obtain is mixed
Liquid is closed, standard working curve is drawn in the response signal change of recording surface plasma resonance sensor, and it is bent to carry out multinomial
Line is fitted, and obtains the equation of one pack system regression curve；Measure the standard working curve and one pack system of other environmental hormones successively again
The equation of regression curve；
Multicomponent is detected simultaneously：Measured using surface plasma resonance spectrum sensor, standard prepared by plurality of step (2)
Solution mixes hatching with excessive antibody or coating antigen, takes the mixed liquor 50-100 μ L after hatching, is passed through sensor, records following table
The overall response signal change of surface plasma resonance sensor；Then according to the test result of step (3), washed according to regenerated liquid
Sample introduction is eluted the order that de- ability grows from weak to strong successively, records the response value changes of each remaining antibody or coating antigen；According to not
The response signal value of remaining antibody or coating antigen draws standard and works bent after mixed liquor and its corresponding regeneration after hatching with concentration
Line, and polynomial curve fitting is carried out, obtain multicomponent regression curve equation；
(5) quantitative detection：Detection simultaneously is carried out for unknown sample of the unknown content containing a variety of environmental hormones, to described
Excessive antibody or coating antigen is separately added into the prepare liquid of unknown sample, sample introduction after mixing hatching records overall response
Change；Sample introduction is eluted the order grown from weak to strong according to regenerated liquid eluting power successively, each remaining antibody in memorization COMS clip surface or
The response value changes of coating antigen；The response of remaining antibody or coating antigen is substituted into the mark for the multi-analyte immunoassay that step (4) is obtained
Quasi- working curve and corresponding single group water content detection standard working curve, calculate the concentration value of each environmental hormone；The like, it is real
Referring now to the quantitative detection of environmental hormone；
(6) being passed through the glycine-HCI cushioning liquid that 0.01-0.02mol/L, pH are 1.2-2.5 regenerates chip, regenerates
Chip afterwards is used to measure next time.
Preferably, in step (2), described pH value is 7.4.
Preferably, in step (5), the mixing brooding time of standard sample storing solution and excessive antibodies or coating antigen is 5-
10 minutes.
The step of the above method in (1), when censorchip surface fix be the mixed liquor of coating antigen when, subsequently walking
Carry out just using antibody during mixing hatching in rapid.Conversely, the step of the above method in (1), being fixed when censorchip surface
When being the mixed liquor of antibody, carry out just using coating antigen during mixing hatching in subsequent step.
By the method for the present invention, the single channel of same chip can connect a variety of coating antigens simultaneously.Furthermore, it is possible to using
The regenerated liquid of different eluotropic strengths is to chip gradient elution, while realizing that the detection of environmental hormone and chip regenerate.According to single
Detection curve equation and the curvilinear equation detected simultaneously, it is possible to achieve detected while a variety of environmental hormones.The step (4)
(5) excessive antibody-solutions are passed through in, using the mode of antibody binding chip, surface plasma resonance sensor is improved and rings
It should be worth, it is adaptable to measure the less small-molecular-weight environmental hormone of response signal, its molecular weight can<1000Da.
The present invention uses spr sensor gradient regeneration method, and by regenerated liquid according to the order that grows from weak to strong, gradient elution is passed
The antibody of the different bond strengths of sensor surfaces absorption, the characteristics of being monitored in real time based on SPR, chip list after analysis regenerated liquid elution
Face antibody and the situation of response value changes, a variety of environmental hormones can be detected simultaneously.
The present invention utilizes immunosuppressive method, and environmental hormone to be measured is premixed with excessive antibody, not anti-in mixed liquor
The antibody and the coating antigen of sensor chip surface answered are specifically bound, and cause signal intensity；If target in testing sample
Molecule is few, then remaining unreacted antibody is more in mixed liquor so that the antibody of chip surface absorption is more, now caused table
Surface plasma resonance response signal is bigger, on the contrary then smaller.Because the response value changes of antibody can reflect target to be measured point
The change in concentration of son, and the molecular weight of antibody is larger, its caused response is changed greatly, therefore, and small-molecule substance can be achieved
Highly sensitive detection.Moreover, when being detected to a variety of environmental hormones to be measured, corresponding antibodies are combined with chip surface, pass through
SPR obtains overall response value changes；, can be by using varying strength according to the difference of adhesion between different antigen-antibodies
The mode of solution gradient regeneration is eluted, the order grown from weak to strong according to regenerated liquid is eluted, one by one recursion, finally by SPR
The real-time monitoring of sensor is realized the quick of a variety of environmental hormones and detected simultaneously.
Advantages of the present invention and beneficial effect are：
1st, the present invention uses immunosuppressive method, by coating antigen modification in chip surface, substantially increases sensing chip
Service life, and solve SPR detection small molecular weight materials response is small, the low problem of sensitivity, realize small point
The highly sensitive detection of subring environment hormone；
2nd, the present invention is realized in the single inspection of surface plasma resonance sensor by the way of regenerated liquid gradient elution
Survey in passage, while detecting a variety of environmental hormones, compared to the detection of single substance, substantially increase sample detection efficiency, shorten
Sample detection time；
3rd, the present invention can not only realize the purpose of the various detection of conventional one-channel spr sensor, and can be further
The flux of multi-channel detection is improved, while improving the service efficiency of sensing chip, the testing cost of environmental hormone is reduced.
Embodiment
The present invention is described in further detail by following examples.It should be noted that：Following embodiments are illustrative, are not
Limited, it is impossible to limit protection scope of the present invention with following embodiments.
Two kinds of typical environment hormones of selection are detection object, respectively bisphenol-A and phthalic acid ester (DEHP).
The coating antigen that chip decorating molecule is two kinds of target molecules is selected, i.e. bovine serum albumin(BSA)-bisphenol-A and ox blood is pure
Albumen-phthalic acid ester.
The identification molecule of selection is mouse antibodies, i.e. bisphenol-A antibody and phthalic acid ester antibody.
Concrete operation step is as follows：
(1) sensor surface coating antigen is fixed：Chip surface carboxyl groups are activated first with EDC/NHS methods, then by bisphenol-A
Mixed with the coating antigen of phthalic acid ester, it is 150 μ L to make cumulative volume, concentration of two kinds of coating antigens in the mixed liquor is
20 μ g/mL, the sense channel of sensor is then passed through with 30 μ L/min speed, the mixed liquor of two kinds of coating antigens is directly modified
In sensor chip surface, finally closed using pH for 8.5 monoethanolamine.
(2) standard liquid is prepared：2 kinds of standard items 10mg of bisphenol-A and phthalic acid ester are weighed respectively, are dissolved in
0.01mol/L, pH value are 7.4 HBS buffer solutions, are settled to 10mL, as 1mg/mL Standard Reserving Solution；And measure respectively
2 kinds of Standard Reserving Solutions, the standard stock solution for preparing 2 kinds of residual components is respectively 100ng/mL；
When carrying out one pack system detection, the μ L of 100 μ g/mL bisphenol-As antibody 10 are taken, are mixed with the bisphenol-A standard stock solution of different volumes
Close, 100 μ L be diluted to HBS buffer solution mixed liquors, finally give bisphenol A concentration in mixed liquor be followed successively by 0,5,10,20,30,
40ng/mL；In a similar way, the μ L of 100 μ g/mL phthalic acid esters antibody 10, the phthalic acid with different volumes are taken
Ester standard stock solution is mixed, and is diluted to 100 μ L with HBS buffer solution mixed liquors, is finally given phthalic ester concentration in mixed liquor
Be followed successively by 0,5,10,20,30,40ng/mL.
Two kinds of samples are detected simultaneously when, 10 μ L bisphenol-As antibody (100 μ g/mL), 10 μ L phthalic acid esters antibody (10 μ are taken
G/mL bisphenol-A, phthalic acid ester standard liquid) with different volumes is mixed, and 100 μ L are diluted to HBS buffer solution mixed liquors,
Wherein bisphenol-A, phthalic ester concentration be respectively 0,5,10,20,30,40ng/mL, it is standby.
(3) screening of regenerated liquid：Following actified solution is optimized selection, the 0.01mol/L's of different pH value is sweet
Propylhomoserin-hydrochloric acid solution (Gly-HCl, pH value is respectively 1.2,1.5,2.0,2.5), concentration be respectively 0.015mol/L,
0.025mol/L, 0.03mol/L, 0.035mol/L and 0.05mol/L NaOH solution, 5mol/L magnesium chloride solution
(MgCl2) and 0.1mol/L HCl solution；Flow rate set is 30 μ L/min when sample in experiment is added, and is tested respectively, is seen
Respective regeneration effect is examined, optimal condition of protoplast isolation is selected, and judge the power of two kinds of regenerated liquid eluting powers.
(4) Specification Curve of Increasing：
First, the detection of one pack system, is passed through concentration mixed for 0,5,10,20,30,40ng/mL bisphenol-As standard liquid and antibody
The μ L of liquid 100 are closed, are measured using surface plasma resonance spectrum sensor, the change of surface plasma resonance sensor signal is recorded
Change, draw standard working curve, and carry out polynomial curve fitting, obtain the equation of one pack system regression curve；It is passed through successively again
Concentration be 0,5,10,20,30, the μ L of 40ng/mL phthalic acid esters mixed liquor 100, using surface plasma resonance spectrum sensor
Measurement, records surface plasma resonance sensor signal intensity, draws standard working curve, and carry out polynomial curve plan
Close, obtain the equation of one pack system regression curve.
Secondly, detected while two kinds of samples, the standard liquid of bisphenol-A and phthalic acid ester, corresponding antibodies are abundant
Mixing hatching, is passed through 100 μ L biased samples to be measured, records the overall response signal change of surface plasma resonance sensor
(T)；Then eluted using the weaker regenerated liquid of eluting power (NaOH), wherein phthalic acid ester antibody is thoroughly eluted
Fall, bisphenol-A antibody still remains chip surface, thus there is certain response signal change (R)；With bisphenol-A in mixed liquor
The change of concentration, response R can also change therewith, record bisphenol-A concentration of standard solution and its response R relation, draw mark
Quasi- working curve, and polynomial curve fitting is carried out, obtain multicomponent regression curve equation.
(5) 0.01mol/L (pH is 1.2) glycine-HCI solution is passed through, the antibody elution that chip surface is remained is complete
It is standby into sensing chip regeneration.
(6) after sensing chip regeneration, 10 μ L bisphenol-A antibody are added to actual unknown concentration testing sample (unknown sample)
(100 μ g/mL), 10 μ L phthalic acid esters antibody (100 μ g/mL), mixing hatching；It is passed through surface plasma resonance sensor
In, global response value changes (T) are recorded, are then regenerated with NaOH solution, residual bisphenol-A antibody response (R) are obtained, by R generations
Enter its corresponding curvilinear equation, obtain bisphenol A component content；Then bisphenol A concentration is updated to the mark during detection of its one pack system
Directrix curve equation, obtains the response (C) of bisphenol-A；Adjacent benzene two is obtained using global response value T and bisphenol-A response C difference
Formic acid esters response (G), then substitutes into G standard curve during the single detection of phthalic acid ester, so that it is determined that O-phthalic
The content of acid esters, test limit is up to ng/mL grades.
(7) 0.01mol/L (pH is 1.2) glycine-HCI solution is passed through again, and the antibody that chip surface is remained is washed
It is de-, sensing chip regeneration is completed, for measurement next time.
Detection sample, compared with existing surface plasma detection technique, detection efficiency are carried out according to the method for embodiment
More than 2 times are improved, testing cost reduces more than 2 times, and the time shortens more than 2 times, and reusable hundreds of times of chip is used
The method of the present invention solves traditional SPR single channels and is unable to the problem of Multiple detection, and can be applied to accurately detect bis-phenol
The small-molecular-weights such as A, phthalic acid ester (<1000Da) material.
The present invention is carried out in detail for the accurate of narration and conveniently in embodiment by taking bisphenol-A, phthalic acid ester as an example
Description, but the present disclosure applies equally to the measure of other environmental hormones, the artificial female hormone of such as diethyl, dioxin, organotin
Etc. the accurate detection and qualitative analysis of environmental hormone, therefore the above is within the scope of the present invention, it should explanation
It is that, in the case where not departing from the core of the present invention, any simple deformation, modification or other skilled in the art can
The equivalent substitution of creative work is not spent to each fall within protection scope of the present invention.
Claims (3)
1. a kind of method that a variety of environmental hormones are detected based on the surface plasma resonance technology that regenerated liquid optimizes simultaneously, its feature
It is to comprise the following steps：
(1) fixation of censorchip surface coating antigen or antibody：The coating antigen of each environmental hormone residual component or antibody are mixed
Volume to mixed liquor is 50-150 μ L, and the concentration of each coating antigen or antibody in the mixed liquor is 5-20 μ g/mL,
Recycle active ester method that the mixed liquor is fixed on into the censorchip surface to fix；
(2) standard liquid is prepared：The hydroxyethyl piperazine second sulphur phthalate buffer for being 6-8 with 0.01-0.1mol/LpH configuration is each
The standard sample storing solution of residual component, the concentration of the standard sample storing solution is 0.1-1mg/mL；
Each standard sample storing solution is measured, its corresponding standard stock solution is prepared；Will with hydroxyethyl piperazine second sulphur phthalate buffer
Standard stock solution is configured to the standard liquid of various concentrations and mixes hatching with the corresponding antibodies or coating antigen of excessive fixed concentration, obtains
To the determinand mixed liquor of various concentrations；
(3) regenerated liquid eluting power is tested：Test HCl solution, NaOH solution, magnesium chloride solution and glycine-HCI solution pair
The elution profile of each environmental hormone antibody of censorchip surface obtained in step (1), selects the optimal condition of protoplast isolation of each component,
And contrast the power of its regenerated liquid eluting power；
(4) standard working curve is set up：
One pack system is detected：Measured using surface plasma resonance spectrum sensor, be passed through the mixing that 50-100 μ L steps (2) are obtained
Liquid, the response signal change of recording surface plasma resonance sensor, draws standard working curve, and carry out polynomial curve
Fitting, obtains the equation of one pack system regression curve；The standard working curve and one pack system for measuring other environmental hormones successively again are returned
Return the equation of curve；
Multicomponent is detected simultaneously：Measured using surface plasma resonance spectrum sensor, standard liquid prepared by plurality of step (2)
Hatching is mixed with excessive antibody or coating antigen, the mixed liquor 50-100 μ L after hatching are taken, sensor, record lower surface etc. is passed through
The overall response signal change of gas ions resonance sensor；Then according to the test result of step (3), according to regenerated liquid eluting power
Sample introduction is eluted the order grown from weak to strong successively, records the response value changes of each remaining antibody or coating antigen；According to various concentrations
The response signal value of remaining antibody or coating antigen draws standard working curve after mixed liquor and its corresponding regeneration after hatching, goes forward side by side
Row polynomial curve fitting, obtains multicomponent regression curve equation；
(5) quantitative detection：Detection simultaneously is carried out for unknown sample of the unknown content containing a variety of environmental hormones, to described unknown
Excessive antibody or coating antigen is separately added into the prepare liquid of sample, sample introduction after mixing hatching records overall response value changes；
Sample introduction is eluted the order grown from weak to strong according to regenerated liquid eluting power successively, each remaining antibody in memorization COMS clip surface or coating antigen
Response value changes；The response of remaining antibody or coating antigen is substituted into the standard work for the multi-analyte immunoassay that step (4) is obtained
Curve and corresponding single group water content detection standard working curve, calculate the concentration value of each environmental hormone；The like, realize for
The quantitative detection of environmental hormone；
(6) being passed through the glycine-HCI cushioning liquid that 0.01-0.02mol/L, pH are 1.2-2.5 regenerates chip, after regeneration
Chip is used to measure next time.
2. according to the method described in claim 1, it is characterised in that：In step (2), described pH value is 7.4.
3. method according to claim 1 or 2, it is characterised in that：In step (5), standard sample storing solution resists with excessive
The mixing brooding time of body or coating antigen is 5-10 minutes.
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Cited By (1)
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| CN110646381A (en) * | 2018-06-27 | 2020-01-03 | 北京中龙益诚科技有限公司 | Surface plasma resonance immunization method for detecting beta 2 receptor stimulant in pig urine |
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Application publication date: 20170801 |
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