CN107966484A - Application of the electrochemical immunosensor in sulphonamides multi-relict context of detection - Google Patents
Application of the electrochemical immunosensor in sulphonamides multi-relict context of detection Download PDFInfo
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Abstract
The invention discloses a kind of electrochemical immunosensor sulphonamides multi-relict context of detection application.The application is：First in glassy carbon electrode surface physical modification graphene chitosan complexes, sulphadiazine is modified by physical absorption again, cross-immune reaction can be produced with sulfa drugs based on sulphadiazine antibody, in conjunction with the detection method of indirect competition, the multi-residue determination to four kinds of sulfa drugs including sulphadiazine, sulphaguanidine, sulfamethazine and sulfacetamide can be achieved, test limit is between 0.006~0.1 ng/mL, and the range of linearity is between 0.01~1000 ng/mL.
Description
Technical field
The invention belongs to food safety detection and technical field of analytical chemistry, and in particular to and a kind of sulphadiazine/graphene-
Application of the electrochemical immunosensor of chitosan complexes modification in sulphonamides multi-relict context of detection.
Background technology
Sulfa drugs（Sulfonamides, SAs）It is a kind of material containing P-aminobenzene-sulfonamide structure, has pre-
Anti- and treatment bacterium infection effect.Because it is cheap, good antimicrobial effect, is widely used in animal-breeding.At present, sulphur
The abuse of amine drug can enter food chain by it in the accumulation of poultry livestock organisations, influence the health of consumer.European Union,
The U.S. and China set highest limit standard to sulfanilamide (SN) total content residual, must not surpass in all edible musculatures of animal
Cross 100 μ g/kg.
The detection method of sulfamido material was quickly grown in recent years, is mainly included immunodetection such as enzyme-linked immunization, is changed
Shine enzyme-linked immunization, spectrophotometry, high performance liquid chromatography, liquid-mass chromatography etc. are learned, the above method is widely applied at present
The content of sulfanilamide (SN) in food is monitored, detection method is sensitive, result is stablized, with a high credibility, but most presence need to use large-scale instrument
The shortcomings that device, complicated sample treatment, can not realize while detect the high flux screening of a variety of or a kind of compound.
Graphene（Graphene, GR）Material is excellent due to the specific surface area with superelevation and excellent electronic conduction ability etc.
Point, is usually used in preparing high performance nanocomposite.Chitosan（Chitosan, CS）It is that one kind contains great amount of hydroxy group and amino
Natural polymers, all there is stronger adsorption capacity to many ions, large biological molecule, fluorescence probe, and
And it is inexpensive nontoxic, there are good permeability and film forming, be widely used in the preparation of various sensors.Graphene-chitosan
（GR-CS）Compound is commonly used for the modification of electrode because having good electric conductivity and film forming at the same time.Therefore in glass-carbon electrode
Sulphadiazine/graphite simple and easy to get is made to adsorb sulphadiazine small molecule in surface modification graphene-chitosan complexes
Alkene-chitosan electrochemical immunosensor, realizes the multi-residue determination of sulfa drugs.
The content of the invention
It is an object of the invention to provide a kind of electrochemical immunosensor in sulphonamides multi-relict context of detection
Using the sensor includes substrate glass-carbon electrode, and sulphadiazine/graphene-chitosan complexes decorative layer.The sensing
Sulphadiazine antibody in sulfa drugs competition binding Incubating Solution in the sulphadiazine and Incubating Solution of device surface modification, so as to lead
Sensor sheet surface resistance is caused to change, by the knots modification of peak current with treating that the logarithm of test sample concentration establishes linear relationship with reality
Now to the multi-residue determination of sulfa drugs.
Sulfa drugs of the present invention includes sulphadiazine, sulphaguanidine, sulfamethazine and sulfacetamide（Knot
Structure formula is shown in formula 1）.
The structural formula of 1 four kinds of sulfa drugs of formula
The technical solution adopted in the present invention is：
（1）The preparation of electrochemical immunosensor：Pretreatment is first carried out to glass-carbon electrode polishing and to clean up, then by 6 μ L stones
The black suspended drop-coated of alkene-chitosan is placed in 37 DEG C of insulating boxs and dried in processed glassy carbon electrode surface, afterwards drop coating 10 again
The sulphadiazine solution of μ L0.2g/L, dries in 37 DEG C of insulating boxs, carries out DPV detections respectively；Finally insert electrodes into 500 microlitres
40 ~ 50mim is closed in 0.5%BSA solution, that is, obtains sulphadiazine/graphene-chitosan electrochemical immunosensor.
（2）The preparation of sulfa drugs standard solution：It is 2 with volume ratio：3 ethanol：Aqueous solvent prepare it is a series of including
The sulfa drugs solution containing various concentrations including blank sample is as standard solution.By sulphadiazine antibody 0.1mol/L,
The phosphate buffer solution of PH=7.4（PBS）1000 times of dilution is used as antibody standard liquid.
（3）The detection of a variety of sulfa drugs：By step（1）The electrochemical immunosensor immerses cumulative volume respectively
For 50 μ L the sulphadiazine antibody containing 8 μ L and a series of various concentrations sulfa drugs standard liquid PBS Incubating Solutions in 37
DEG C insulating box is incubated 40min, is rinsed after incubation with PBS, in the K of 2 mmol/L3[Fe(CN)6] KCl solution（0.1 mol/L）
Middle carry out differential pulse voltammetry（DPV）Scanning.With DPV response currents changing value Δ I before and after incubation to sulfa drugs concentration pair
Numerical value logC maps, and obtains Δ I-logC working curves, and Δ I-logC equations of linear regression are obtained using linear regression method.Will be to be measured
Sample presses step（2）Prepare liquid is configured to, same method detection obtains Δ Ix, substitute into Δ I-logC equations of linear regression, you can
Draw the concentration of sulfa drugs in sample to be tested.
The sulfa drugs includes sulphadiazine, sulphaguanidine, sulfamethazine and sulfacetamide.
Compared with prior art, beneficial effects of the present invention are：
Electrochemical immunosensor provided by the invention sulphonamides multi-relict context of detection application, based on sensor sheet
Graphene-chitosan complexes of face modification prepare more mature, cross immunity of the sulphadiazine antibody to sulfa drugs
Reaction, sulphadiazine can be adsorbed in electrode surface, realize to phonetic including sulphadiazine, sulphaguanidine, sulfanilamide (SN) dimethyl well
The multi-residue determination of four kinds of sulfa drugs of pyridine and sulfacetamide.The sensor cost is low, making is simple, stability is good,
High sensitivity, and the method has relatively low detection limit（0.006~0.1 ng/ml）The wider range of linearity（0.1
~1000 ng/mL）.When detecting actual sample, sample pre-treatments are simple, detection speed is fast, high sensitivity, there is good city
Field value.
Brief description of the drawings
Fig. 1 is the DPV curve maps that electrochemical immunosensor is detected sulphadiazine.
Fig. 2 is the changing value Δ I and sulphadiazine log concentration value logC of response currentSDWorking curve diagram.
Fig. 3 is the DPV curve maps that electrochemical immunosensor is detected sulphaguanidine.
Fig. 4 is the changing value Δ I and sulphaguanidine log concentration value logC of response currentSGWorking curve diagram.
Fig. 5 is the changing value Δ I and sulphadiazine log concentration value logC of pork liver mark-on sulphadiazine response currentSDWork
Make curve map.
Fig. 6 is the changing value Δ I and sulphadiazine log concentration value logC of flesh of fish mark-on sulphadiazine response currentSDWork
Make curve map.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings
The preparation of 1 electrochemical immunosensor of embodiment
First carry out pretreatment to glass-carbon electrode to clean up, then by 6 μ L graphenes-chitosan solution, drop coating is in processed glass
Carbon electrodes, are placed in 37 DEG C of insulating boxs and dry, then by 10 μ L sulphadiazines drop coatings to electrode, dry in the air in 37 DEG C of insulating boxs
It is dry, DPV detections are carried out respectively；40 ~ 50mim of closing in 500 microlitres of 0.5%BSA solution is finally inserted electrodes into, it is phonetic to obtain sulfanilamide (SN)
The electrochemical immunosensor of pyridine/graphene-chitosan-modified.
Embodiment 2 is to sulphadiazine（SD）Detection
The electrochemical immunosensor of sulphadiazine/graphene-chitosan-modified is prepared in the method for embodiment 1, is dipped in
The sulphadiazine antibody containing 8 μ L and a series of PBS Incubating Solutions of the sulphadiazine standard liquid of various concentrations that cumulative volume is 50 μ L
In 37 DEG C of insulating boxs be incubated 40min, rinsed after incubation with PBS, then the K in 2 mmol/L3[Fe(CN)6] KCl solution（0.1
mol/L）Middle progress DPV scannings, respectively record various concentrations under peak current I, DPV curve map as shown in Figure 1, in figure curve from
Top to bottm concentration is followed successively by 1000 ng/mL, 300 ng/mL, 100 ng/mL, 30 ng/mL, 10 ng/mL, 3 ng/mL, 1
Ng/mL, 0.3 ng/mL, 0.1 ng/mL, 0.03 ng/mL and 0.01 ng/mL.Define peak current when concentration to be detected is zero
It is worth for I0, Δ I is the difference of detection sample peak current and blank concentration peak current, according to Δ I to sulphadiazine log concentration value
logCSD（ng/mL）Mapping, obtains Δ I-logCSDWorking curve, working curve diagram using linear regression method as shown in Fig. 2, obtained
Δ I ~ logCSDEquation of linear regression is Δ I（μA）= 16.0292-2.1374 logCSD（ng/mL）, when sulphadiazine concentration
Its Δ I and logC in the range of 0.01~1000 ng/mLSDIt is directly proportional, linearly dependent coefficient 0.9932.Surveyed with blank standard specimen
3 times of numeraire deviation（3σ）Limited as sample detection, be repeated 10 times experiment and draw, method described above detection sulphadiazine
Sample detection is limited to 0.006 ng/mL.Sample to be tested is incubated under these conditions and scanning draws response current I；Root
According to changing value △ I and △ I ~ logC equations of linear regression of response current, the dense of sulfa drugs in sample to be tested can be calculated
Spend C.
Embodiment 3 is to sulphaguanidine（SG）Detection
The electrochemical immunosensor of sulphadiazine/graphene-chitosan-modified is prepared in the method for embodiment 1, is dipped in
In the sulphadiazine antibody containing 8 μ L and a series of PBS Incubating Solutions of the sulphaguanidine standard liquid of various concentrations that cumulative volume is 50 μ L
37 DEG C of insulating boxs are incubated 40min, are rinsed after incubation with PBS, then the K in 2 mmol/L3[Fe(CN)6] KCl solution（0.1
mol/L）Middle progress DPV scannings, respectively record various concentrations under peak current I, DPV curve map as shown in figure 3, in figure curve from
Top to bottm concentration is followed successively by 1000 ng/mL, 300 ng/mL, 100 ng/mL, 30 ng/mL, 10 ng/mL, 3 ng/mL, 1
Ng/mL, 0.3 ng/mL, 0.1 ng/mL, 0.03 ng/mL and 0.01 ng/mL.Define peak current when concentration to be detected is zero
It is worth for I0, Δ I is the difference of detection sample peak current and blank concentration peak current, according to Δ I to sulphaguanidine log concentration value
logCSG（ng/mL）Mapping, obtains Δ I ~ logCSGWorking curve, working curve diagram using linear regression method as shown in figure 4, obtained
Δ I ~ logCSGEquation of linear regression is Δ I（μA）= 22.8231-3.7066 logCSG（ng/mL）, when sulphaguanidine concentration exists
Its Δ I and logC in the range of 0.01~1000 ng/mLSGIt is directly proportional, linearly dependent coefficient 0.9985.Measured with blank standard specimen
It is worth 3 times of standard deviation（3σ）Limited as sample detection, be repeated 10 times experiment and draw, method described above detects the sample of sulphadiazine
Product examine surveys and is limited to 0.005 ng/mL.Sample to be tested is incubated under these conditions and scanning draws response current I；According to
Changing value △ I and △ I ~ logC of response currentSGEquation of linear regression, can calculate the concentration of sulfa drugs in sample to be tested
C。
The measure of mark-on sulphadiazine in 4 pork liver of embodiment
1）The pre-treatment of Pig Liver：Weigh the pig liver of rubbing（5±0.0050）G, it is homogeneous to add 20mL ethyl acetate
2min, centrifuges 5min with 4000r/min, takes upper liquid, repeats extraction and once merges supernatant.Add 0.1mol/L hydrochloric acid 4mL
It is concentrated to dryness with 40 DEG C of rotary evaporations, respectively with 0.1mol/L hydrochloric acid 2mL, n-hexane 3mL dissolved residues, merges cleaning solution and mix
Even, 4000r/min centrifugations 5min abandons upper strata n-hexane, wash flask with 3mL n-hexanes and is repeated to extract and once merges subnatant, and 40
The drying of DEG C nitrogen, with the phosphate buffer constant volume of 1mL pH7.4, concussion makes concentrate to be measured after dissolving.
2）The measure of mark-on sulphadiazine in Pig Liver：The pork liver sample of three same volumes is taken to add SD standard items to determine respectively
Hold to 100,10,1ng/mL as prepare liquid, the electricity of sulphadiazine/graphene-chitosan-modified is prepared in the method for embodiment 1
Chemo-immunity sensor, is dipped in sulphadiazine antibody containing 8 μ L and a series of various concentrations that cumulative volume is 50 μ L
In the PBS Incubating Solutions of prepare liquid, be incubated using method same as Example 2, DPV scanning and data processing, calculate
The concentration C of sulphadiazine, detects rate of recovery result such as table 1, working curve diagram is as shown in Figure 5.
Table 1 is the rate of recovery result of the sulphadiazine concentration in mark-on pork liver
| Sulphadiazine additive amount (ng/mL) | Sulfadiazine determination amount (ng/mL) | The rate of recovery（%） | Average recovery rate（%）±RSD（%） |
| 100.0 | 107.55、114.78、119.34 | 107.55、114.78、119.34 | 113.89±5.94 |
| 10.0 | 9.77、10.09、9.16 | 97.70、100.93、91.55 | 96.73±4.76 |
| 1.0 | 0.85、0.83、0.89 | 85.36、82.63、89.33 | 85.77±3.37 |
The measure of mark-on sulphadiazine in 5 flesh of fish of embodiment
1）The pre-treatment of flesh of fish sample：Weigh the structure of fish muscle of rubbing（5±0.0050）G, it is homogeneous to add 25mL ethyl acetate
2min, adds 5g anhydrous sodium sulfates homogeneous 2min, 3000r/min centrifugation 10min, takes supernatant, repeated with 25mL ethyl acetate
Extraction once merges supernatant, and 40 DEG C of rotary evaporations are concentrated to dryness.Washed respectively with 1% hydrochloric acid 2mL, methanol mL dissolved residues, merging
Liquid mixing is washed, repeats dissolving once, merges cleaning solution.5min is centrifuged with 3000r/min with after 8mL n-hexane extractions, abandons upper strata
Liquid, repeats extraction with 8mL n-hexanes and once merges subnatant, 40 DEG C of rotary evaporated to dryness, with the phosphate buffer of 1mL pH7.4
Constant volume, concussion make concentrate to be measured after dissolving.
2）The measure of mark-on sulphadiazine in flesh of fish sample：The flesh of fish sample of three same volumes is taken to add SD standard items to determine respectively
Hold to 100,10,1ng/mL as prepare liquid, the electricity of sulphadiazine/graphene-chitosan-modified is prepared in the method for embodiment 1
Chemo-immunity sensor, is dipped in sulphadiazine antibody containing 8 μ L and a series of various concentrations that cumulative volume is 50 μ L
In the PBS Incubating Solutions of prepare liquid, be incubated using method same as Example 2, DPV scanning and data processing, calculate
The concentration C of sulphadiazine, detects rate of recovery result such as table 2, working curve diagram is as shown in Figure 6.
Table 2 is the rate of recovery result of the sulphadiazine concentration in the mark-on flesh of fish
| Sulphadiazine additive amount (ng/mL) | Sulfadiazine determination amount (ng/mL) | The rate of recovery（%） | Average recovery rate（%）±RSD（%） |
| 100.0 | 106.85、103.35、110.47 | 106.85、103.35、110.47 | 106.89±3.56 |
| 10.0 | 8.56、8.90、9.36 | 85.62、89.01、93.57 | 89.40±3.99 |
| 1.0 | 1.00、1.05、1.02 | 100.05、105.17、102.29 | 102.50±2.57 |
Claims (3)
1. a kind of electrochemical immunosensor is in the application of sulphonamides multi-relict context of detection, it is characterised in that the biography
Sensor includes substrate glass-carbon electrode, and sulphadiazine/graphene-chitosan complexes decorative layer.
2. a kind of electrochemical immunosensor is in the application of sulphonamides multi-relict context of detection, it is characterised in that the biography
Sensor is by the sulfa drugs competition binding sulphadiazine antibody with containing in determinand, to realize the sensor to sulfamido
The multi-residue determination of medicine.
3. the sulphonamides multi-relict detection according to claim 1-2, it is characterised in that the sulfa drugs includes
Sulphadiazine, sulphaguanidine, sulfamethazine and sulfacetamide.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113325050A (en) * | 2021-05-31 | 2021-08-31 | 河南中泽生物工程有限公司 | Method for rapidly and highly sensitively detecting SM2 in animal-derived food |
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