CN109596604A - A kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity and application - Google Patents
A kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity and application Download PDFInfo
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- CN109596604A CN109596604A CN201811579226.6A CN201811579226A CN109596604A CN 109596604 A CN109596604 A CN 109596604A CN 201811579226 A CN201811579226 A CN 201811579226A CN 109596604 A CN109596604 A CN 109596604A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
The invention discloses a kind of adjustable chemiluminescence Fiber imunosensors of range of linearity, it is prepared by the following method: 1) impregnating one end of optical fiber with chloroform respectively, peel off acrylic protective layer；It immerses in hydrofluoric acid again, removes quartzy coat, rinsed with 0.1 M NaOH and water；2) it immerses in 0.1M hydrochloric acid and impregnates, wash, immerse 0.1M NaOH and impregnate, wash, it is dry；Using the 3-ADMS alcohol solution dipping of volume fraction 1%, ethanol washing is dry；Fibre Optical Sensor response fibre core is immersed into the phosphate buffer solution containing crosslinking agent, is inserted into the solution of comlete antigen and reacts, PBS washes away unbonded comlete antigen；3) bunchy is assembled, is fixed with hot shortness's casing；And its application in terms of detecting food veterinary drug residue；The invention has the advantages that: range of linearity controllable adjustment, transducer sensitivity is high, and low in cost, easy to operate, external interference resistance is strong.
Description
Technical field
The invention belongs to food safety detection analysis technical fields, and in particular to a kind of adjustable chemiluminescence light of the range of linearity
Fine immunosensor and application.
Background technique
Food safety work be the major issue for being related to national economy and social development, wherein food veterinary drug residue by
Gradually become the hot issue of people's growing interest, there is an urgent need to develop the letter for being directed to the detection of animal derived food veterinary drug residue
Single, general and inexpensive analytical technology, to realize the quick and precisely quantitative analysis of the various residues of veterinary drug in daily bread.Mesh
Before, significant challenge that detection of veterinary drugs in food is faced first is that different types of residue of veterinary drug level difference is very big.For example, chlorine is mould
Element is the antibiotic being forbidden to use, and in milk sample, the minimum law enforcement limitation of chloramphenicol (CAP) is 300 pg/mL, needs to examine
Survey method has very high sensitivity.And the maximum residue limit (MRL) of sulphadiazine (SDZ) and neomycin (NEO) is respectively
100 ng/mL and 1. 5 μ g/mL, this just needs detection method to have the wider range of linearity.It is greatly more for concentration span
Object detection of veterinary drugs in food not only needs analysis means to have a high sensitivity, at the same its to need to provide sufficiently wide detection linear
Range generally requires large-scale precision analysis instrument and professional operation skill.In order to meet a variety of beasts of various concentration detection range
The remaining testing requirements of medicine, there is an urgent need to develop the simple and inexpensive analysis means with controllable detection range.
Silica fibre is a kind of using the silica glass material of high refractive index as fibre core, with the organic or inorganic material of low-refraction
Material is the optical fiber of foreskin.Since its intrinsic optical property and mechanical strength are big, good bandability, easily with light source couples etc.
Feature, silica fibre show unique advantage in building chemical/biological chemical sensor.For example, researchers will
The immune response of high specific and ultra sensitive chemical shine and are integrated into fiber optic tip, for the fast of biomarker or microorganism
Speed, the quantitative analysis of high specific and low consumption sample amount.However, the surface area of fiber core is small (diameter is usually several hundred microns),
Lead to the small in the fixed amount on its surface of reactive protein such as antigen/antibody or enzyme, limit sensitivity raising and linear model
The expansion enclosed.How while guaranteeing highly sensitive detection, realize that detection range controllable adjustment is of great significance, at present still
Have no that Sensitive Detection of the adjustable Fiber imunosensor of the range of linearity for a variety of residues of veterinary drug is reported.
Summary of the invention
Object of the present invention is to and provide to solve the problems, such as that detection of veterinary drugs in food sensitivity is not high, the detection range of linearity is small
A kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity and application.
A kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity, it is prepared by the following method:
1) optical fiber pre-processes: one end of optical fiber being impregnated 10 ~ 15 minutes with chloroform respectively, immersion length is 0.5 ~ 1.5cm, is peelled off
The acrylic protective layer for the part impregnated；One end of protective layer will be removed, then immersed rotten in the hydrofluoric acid that concentration is 20 ~ 30%
Erosion 18 ~ 25 minutes removes and adulterates quartzy coat, rinsed respectively with 0.1 M NaOH and water, obtains Fibre Optical Sensor response fibre core；
2) it fixed comlete antigen: by sensing response fibre core obtained by step 1), immerses in 0.1M hydrochloric acid and impregnates 28 ~ 35 minutes, use water
Washing, then sensing response fibre core is immersed into 0.1M NaOH and is impregnated 55 ~ 65 minutes, it is washed with water, it is dry；Use volume fraction 1%
3-ADMS alcohol solution dipping Fibre Optical Sensor respond fibre core 55 ~ 65 minutes, ethanol washing is dry；Fibre Optical Sensor is responded fine
Core immerses 0.1 7.0 phosphate buffer solution of M, pH, impregnates 1.3 ~ 2 hours, insertion containing 8 ~ 12 μ g/mL CAP-BSA, 15 ~
It reacts 1.3 ~ 1.8 hours in the solution of 25 μ g/mL SDZ-BSA and 75 ~ 85g/mL NEO-BSA comlete antigens, is washed away not with PBS
In conjunction with comlete antigen, obtain the optical fiber in conjunction with antigen；
3) assembling optical fiber beam: the optical fiber that step 2 is obtained is 1 ~ 2:4 by length 0.5cm, 1.0cm and 1.5cm number of fibers ratio
The ratio of ~ 6:7 ~ 9 assembles bunchy, is fixed with hot shortness's casing, obtains a kind of adjustable chemiluminescence optical fiber of the range of linearity
Immunosensor；
Phosphate buffer solution described in step 2, including 0.015 M 1- (3- dimethylamino-propyl) -3- ethyl carbon two are sub-
Amine hydrochlorate (EDC) and 0.03 M N- hydroxysuccinimide (NHS)；The drying is dry under nitrogen flowing；
Comlete antigen described in step 2 is 10 μ g/mL CAP-BSA, 20 μ g/mL SDZ-BSA and 80g/mL NEO-BSA；
The ratio of length 0.5cm, 1.0cm and 1.5cm number of fibers described in step 3) is 1:5:8；
Hydrofluoric acid described in step 1), concentration are 20 ~ 30%.
A kind of adjustable chemiluminescence Fiber imunosensor of range of linearity is in detection milk veterinary drug residue side
The application in face；
The veterinary drug is chloramphenicol, sulphadiazine and neomycin.
The present invention is by two kinds of simple modes, i.e. adjusting Fibre Optical Sensor response core length and optical fiber radical, to regulate and control
Signal strength and the detection range of linearity.Comlete antigen controllably and is quantitatively fixed on to the Fibre Optical Sensor response with different length
On fibre core, optical fiber is then assembled into bunchy with different radicals；Comlete antigen and the biotinylated detection of object competitive binding
Antibody.Signal conversion unit is being self-assembly of by streptavidin and biotinylated horseradish peroxidase
Compound, can be implemented in combination with by the strong affinity between streptavidin and biotin target analyte detection letter
Number be further amplified.The introducing of chemiluminescent substrate will cause the chemiluminescence reaction of quick catalysis, and signal is gathered by fiber optic bundle
Collection and conduction, to be read by small-sized and economic photon-counting detector.For three kinds of different veterinary drugs of detectable concentration range
CAP, SDZ, NEO, we detect CAP using the shorter fiber optic bundle of small number of Fibre Optical Sensor response core length, therefore
The CAP of only a small amount of comlete antigen and low concentration competition detection antibody.For the SDZ of intermediate concentration, using in medium radical
The Fibre Optical Sensor response fibre core of equal length forms fiber optic bundle.Due to the high concentration of NEO, we are using large number of with longer
The optical fiber of Fibre Optical Sensor response core length gathers into bundles, make its provide the NEO of a large amount of comlete antigens and high concentration carry out it is competing
Strive reaction.To realize the controllable adjustment of the range of linearity, and to milk sample by the simple process and assembling to optical fiber
In three kinds of residues of veterinary drug carried out quantitative detection.
The present invention provides a kind of adjustable chemiluminescence Fiber imunosensors of range of linearity, it is by following methods system
Standby: 1) one end of optical fiber is impregnated with chloroform respectively, peels off acrylic protective layer；It immerses in hydrofluoric acid again, removes quartz and apply
Coating is rinsed with 0.1 M NaOH and water；2) it immerses in 0.1M hydrochloric acid and impregnates, wash, immerse 0.1M NaOH and impregnate, washing,
It is dry；Using the 3-ADMS alcohol solution dipping of volume fraction 1%, ethanol washing is dry；Fibre Optical Sensor response fibre core is immersed
Phosphate buffer solution containing crosslinking agent, is inserted into the solution of comlete antigen and reacts, and PBS washes away unbonded comlete antigen；
3) bunchy is assembled, is fixed with hot shortness's casing；And its application in terms of detecting food veterinary drug residue；Advantage of the present invention
Be: range of linearity controllable adjustment, transducer sensitivity is high, and low in cost, easy to operate, external interference resistance is strong.
Detailed description of the invention
Fig. 1 present invention detects the schematic diagram of chloramphenicol, sulphadiazine, neomycin；
The phenogram of Fig. 2 Fiber imunosensor；
The optimization figure of Fig. 3 experiment condition；
Fig. 4 linear sensor range adjusts figure；
The quantitative detection matched curve of Fig. 5 chemiluminescence immunoassay sensor measurement CAP, SDZ, NEO prepared by the present invention；
The immune specificity assessment figure for passing sensor of Fig. 6 optical fiber.
Specific embodiment
The preparation of 1 chemiluminescence Fiber imunosensor of embodiment
1, optical fiber pre-processes
Several optical fiber (10 cm of total length) are divided into three groups, one end of every group of optical fiber is impregnated 10 minutes with chloroform respectively, is immersed
Length is respectively set to 0.5cm, 1.0cm and 1.5cm；The acrylic protective layer for the part that optical fiber impregnated gently is peelled off with knife；
One end of protective layer will be removed, then immerses in the hydrofluoric acid that concentration is 24% and corrode 20 minutes, adulterates quartzy coat to remove,
The fiber core of certain length is exposed, i.e. the length of Fibre Optical Sensor response fibre core is respectively 0.5 cm, 1.0 cm and 1.5 cm；
It is rinsed Fibre Optical Sensor response section 3 times with 0.1 M NaOH and water respectively.
2, fixed comlete antigen
The Fibre Optical Sensor of above-mentioned pretreated optical fiber is responded into fibre core, immerses in 0.1M hydrochloric acid and impregnates 30 minutes except decontamination
Object is contaminated, after being washed with water 3 times, then Fibre Optical Sensor response fibre core is immersed into 0.1M NaOH and is impregnated 60 minutes, to activate Fibre Optical Sensor
The hydroxyl of fibre core is responded, wash 3 times and is dried under nitrogen flowing；Fibre Optical Sensor is impregnated using 1% 3-ADMS ethanol solution (v/v)
Response fibre core 60 minutes, by silanization to introduce amino, ethanol washing 3 times is simultaneously dry in nitrogen stream；Fibre Optical Sensor is rung
It answers fibre core to immerse and contains 0.015 M 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and 0.03 M N-
The phosphate buffer solution (0.1 M, pH 7.0) of hydroxysuccinimide (NHS) is impregnated 1.5 hours, and insertion contains immediately after
It is reacted in the solution for having comlete antigen (0 μ g/mL of CAP-BSA:10 μ g/mL, SDZ-BSA:20 μ g/mL, NEO-BSA:8)
1.5 hours, unbonded comlete antigen is washed away with PBS；In order to prove that comlete antigen has successfully been fixed to capillary tube inner wall, I
To the step, treated that capillary has carried out detailed characterizations using ultraviolet, infrared, as shown in Figure 2；Use the oxygen of 10 mg/mL
Change type glutathione (GSSG) solution closes residual activity site 2 hours, to reduce non-specific adsorption.
3, assembling optical fiber beam
The optical fiber of above-mentioned processing is assembled into beam by following standard: chloramphenicol sensor: 0.5cm Fibre Optical Sensor responds fibre core 1；
Sulphadiazine sensor: 1.0cm Fibre Optical Sensor responds fibre core 5；Neomycin sensor: 1.5cm Fibre Optical Sensor responds fibre core 8
Root；It is fixed with hot shortness's casing, -4 DEG C save for use.
The range of linearity of 2 chemiluminescence Fiber imunosensor of embodiment is adjusted
1, condition optimizing
CAP-BSA, SDZ-BSA, NEO-BSA concentration are investigated, B-Ab of CAP, B-Ab of SDZ, B-Ab of NEO are dense
Degree, Immune competition reaction time, the ratio and SA of Streptavidin (SA) and biotinylated horseradish peroxidase (B-HRP)
Influence of the concentration to signal strength, and choosing the corresponding condition of peak signal is optimal conditions, as shown in Figure 3；Meanwhile from figure
3 can be seen that the concentration for merely changing envelope antigen (CAP-BSA, SDZ-BSA, NEO-BSA), can not significantly increase
Plus signal intensity, therefore cannot function as the effective means of linear adjustment range.
2, the influence of Fibre Optical Sensor response core length
Using the fibre optical sensor that Fibre Optical Sensor response core length is 0.5cm, 1.0cm, 1.5cm to the CAP of series of concentrations,
SDZ, NEO are measured, using sample concentration as abscissa, with blank signal (I0) it with the difference of sample signal (I) is ordinate
Mapping.
3, the influence of optical fiber radical
For the CAP of series of concentrations, the optical fiber that 1,5,8 inner fiber sensing response core length is 0.5cm is assembled into respectively
Beam is measured；For the SDZ of series of concentrations, the optical fiber group for being respectively 1.0cm by 1,5,8 Fibre Optical Sensor response core length
Beam is dressed up to be measured；For the NEO of series of concentrations, the light for being respectively 1.5cm by 1,5,8 Fibre Optical Sensor response core length
Fibre assembling bunchy is measured；Using sample concentration as abscissa, with blank signal (I0) it with the difference of sample signal (I) is vertical sit
It is denoted as figure；Range of linearity regulating effect is as shown in Figure 4.
4, the production of standard curve
According to the concentration range of actual sample veterinary drug residue, the range of linearity for being suitble to CAP, SDZ, NEO is selected, with sample concentration
(pg/mL) logarithm is abscissa, with (I0-I)/ I0It maps for abscissa, as shown in Figure 5.This patent is passed by changing optical fiber
Sense response core length and radical, realize the significantly adjusting (pg/mL- μ g/mL) of the range of linearity, have detectable concentration range
The great multiple target object of span.
The detection method of 3 veterinary drug chloramphenicol of embodiment, sulphadiazine, neomycin
It is detected using chemiluminescence Fiber imunosensor prepared by embodiment 1, the specific steps are as follows:
1, detecting step
1) Immune competition reacts
By CAP, SDZ, NEO of a series of concentration corresponding biotinylated antibody (B-Ab of CAP:5 μ g/mL, B-Ab
Of SDZ:10 μ g/mL, B-Ab of CAP:40 μ g/mL) mixing, the optical fiber of corresponding Fiber imunosensor is passed immediately
Sense response core segment insertion mixed solution in, 37 DEG C reaction 30-60 minutes, extra veterinary drug and B- are then washed away with PBST
Ab。
2) signal conversion unit is introduced
SA and B-HRP is mixed according to the molar ratio of 1:4 in advance, reacts 30 minutes to obtain SA-B-HRP compound, so
The Fibre Optical Sensor response core segment of the Fiber imunosensor after above-mentioned combination B-Ab is immersed in the complex solution afterwards, instead
It answers 30 minutes, PBST washes away unbonded compound.
3) chemiluminescent process progresses
The Fibre Optical Sensor response core segment of Fiber imunosensor is immersed in freshly prepd chemiluminescent substrate solution and (is taken
Luminol and each 10 μ L of hydrogen peroxide are mixed immediately).
2, sample detection
1) actual sample to be measured is mixed with corresponding B-Ab solution, sensor prepared by embodiment 1 is inserted progress immediately
Immune competition reacts 1 hour, and PBST is rinsed.
2) the sensor is immersed to the compound SA- of the horseradish peroxidase formation of Streptavidin and biotin labeling
It is reacted 30 minutes in B-HRP solution, PBST washes away unbonded compound.
3) it inserts the sensors into freshly prepd chemistry and gives out light and (take luminol and each 100 μ L of hydrogen peroxide, immediately in substrate solution
Mixing), carry out data acquisition and the record of chemiluminescence signal.
4) chemiluminescence intensity measured is substituted into corresponding standard curve, it is dense finds out CAP, SDZ, NEO in actual sample
Degree.
4, interference measurement
1) it prepares and interferes solution containing 20ng/mL SDZ and 100 μ g/mL NEO, compare the CAP solution for 1000 pg/mL, point
Not with its chemiluminescence signal intensity of three kinds of sensor measurements；
2) the interference solution containing 1000pg/mL CAP and 100 μ g/mL is prepared, the SDZ solution for 20ng/mL is compareed, uses respectively
Three kinds of sensor measurement its chemiluminescence signal intensity；
3) the interference solution containing 1000pg/mL CAP and 20ng/mL SDZ is prepared, is compareed as the NEO solution of 100 μ g/mL, point
Not with its chemiluminescence signal intensity of three kinds of sensor measurements；
Concentration is calculated according to the signal strength measured, and is compared, as shown in fig. 6, between tri- kinds of veterinary drugs of CAP, SDZ, NEO
It interferes with each other in the range of -5.9%-9.0%, detection while illustrating to have no effect on three kinds of veterinary drugs in same sample.
The detection of 4 actual sample of embodiment
Take three kinds of commercially available milk samples, every kind of CAP, SDZ, NEO standard specimen for being separately added into different known concentrations calculates mark-on reclaims
Rate；As a result it is listed in the table below；The result shows that being existed with the recovery of standard addition that sensor prepared by this patent measures in actual sample
Between 85.0%-109.4%, illustrate the quantitative detection that can be used for actual sample veterinary drug residue.
* ND expression is not detected.
The Applicant declares that the present invention illustrates the process method of the present invention through the above embodiments, but the present invention not office
It is limited to above-mentioned processing step, that is, does not mean that the present invention must rely on the above process steps to be carried out.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to raw material selected by the present invention
Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
Claims (7)
1. a kind of adjustable chemiluminescence Fiber imunosensor of the range of linearity, it is prepared by the following method:
1) optical fiber pre-processes: one end of optical fiber being impregnated 10 ~ 15 minutes with chloroform respectively, immersion length is 0.5 ~ 1.5cm, is peelled off
The acrylic protective layer for the part impregnated；One end of protective layer will be removed, then immersed rotten in the hydrofluoric acid that concentration is 20 ~ 30%
Erosion 18 ~ 25 minutes removes and adulterates quartzy coat, rinsed respectively with 0.1 M NaOH and water, obtains Fibre Optical Sensor response fibre core；
2) it fixed comlete antigen: by sensing response fibre core obtained by step 1), immerses in 0.1M hydrochloric acid and impregnates 28 ~ 35 minutes, use water
Washing, then sensing response fibre core is immersed into 0.1M NaOH and is impregnated 55 ~ 65 minutes, it is washed with water, it is dry；Use volume fraction 1%
3-ADMS alcohol solution dipping Fibre Optical Sensor respond fibre core 55 ~ 65 minutes, ethanol washing is dry；Fibre Optical Sensor is responded fine
Core immerses 0.1 7.0 phosphate buffer solution of M, pH, impregnates 1.3 ~ 2 hours, insertion containing 8 ~ 12 μ g/mL CAP-BSA, 15 ~
It reacts 1.3 ~ 1.8 hours in the solution of 25 μ g/mL SDZ-BSA and 75 ~ 85g/mL NEO-BSA comlete antigens, is washed away not with PBS
In conjunction with comlete antigen, obtain the optical fiber in conjunction with antigen；
3) assembling optical fiber beam: the optical fiber that step 2 is obtained is 1 ~ 2:4 by length 0.5cm, 1.0cm and 1.5cm number of fibers ratio
The ratio of ~ 6:7 ~ 9 assembles bunchy, is fixed with hot shortness's casing, obtains a kind of adjustable chemiluminescence optical fiber of the range of linearity
Immunosensor.
2. the adjustable chemiluminescence Fiber imunosensor of a kind of range of linearity according to claim 1, feature exist
In: phosphate buffer solution described in step 2, including 0.015 M 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Hydrochloride (EDC) and 0.03 M N- hydroxysuccinimide (NHS)；The drying is dry under nitrogen flowing.
3. the adjustable chemiluminescence Fiber imunosensor of a kind of range of linearity according to claim 2, feature exist
In: comlete antigen described in step 2 is 10 μ g/mL CAP-BSA, 20 μ g/mL SDZ-BSA and 80g/mL NEO-BSA.
4. the adjustable chemiluminescence Fiber imunosensor of a kind of range of linearity according to claim 3, it is characterised in that:
The ratio of length 0.5cm, 1.0cm and 1.5cm number of fibers described in step 3) is 1:5:8.
5. the adjustable chemiluminescence Fiber imunosensor of a kind of range of linearity according to claim 1,2,3 or 4, special
Sign is: hydrofluoric acid described in step 1), and concentration is 20 ~ 30%.
6. the adjustable chemiluminescence Fiber imunosensor of a kind of range of linearity described in claim 1 is in detection milk herbal medicine
Remain the application of aspect.
7. application according to claim 6, it is characterised in that: the veterinary drug is chloramphenicol, sulphadiazine and neomycin.
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