CN106442427A - Surface plasmon resonance immunoassay method for detecting sulfonamides - Google Patents
Surface plasmon resonance immunoassay method for detecting sulfonamides Download PDFInfo
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- CN106442427A CN106442427A CN201610892262.2A CN201610892262A CN106442427A CN 106442427 A CN106442427 A CN 106442427A CN 201610892262 A CN201610892262 A CN 201610892262A CN 106442427 A CN106442427 A CN 106442427A
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
- G01N2021/5903—Transmissivity using surface plasmon resonance [SPR], e.g. extraordinary optical transmission [EOT]
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Abstract
The invention relates to a surface plasmon resonance immunosensor for detecting sulfonamides and a working method thereof. The main technical key points adopted by the invention are as follows: an amido-coupled sulfonamide coating antigen (sulfonamide-OVA) is modified onto the surface of a sensing chip, mixed liquid of sulfonamides to be detected and an antibody flows through the surface of a surface plasmon resonance chip, and according to an immunosuppression principle, four sulfonamides (sulfaquinoxaline, sulfachlorpyridazine, sulfamethoxazole and sulfamethoxypyridazine) are analyzed and detected. According to the invention, high specificity of immunoassay and high-sensitivity, real-time and rapid characteristics of surface plasmon resonance are effectively combined, and the detection process is simple and rapid. The established surface plasmon resonance immunosensor has the advantages of high detection accuracy, high sensitivity, reusability, good result repeatability and the like, and can be applied to analysis and detection of the four sulfonamides in milk, egg, chicken, beef, fish and other samples.
Description
Technical field
The present invention relates to sensing and technical field of immunoassay, especially relate to a kind of surface plasma of detection sulfanilamide
Resonance immunization method.
Background technology
Sulfa drugss (Sulfonamides) refer to the class chemotherapeutic agent with P-aminobenzene-sulfonamide structure
General name, can be by disturbing the folic acid metabolism of sensitive organism, and the growth to Gram-positive and negative bacterium, breeding are produced and substantially pressed down
Make and use, with broad-spectrum antiseptic feature.Sulfa drugss substantially tasteless, generally white or slightly yellow crystalline powder, property is steady
Fixed, it is easy to long-term preservation.Sulfa drugss due to its antibacterial stability and broad spectrum activity, can preserve for a long time, cheap the features such as wide
General clinical with fields such as livestock and poultry cultivations for physianthropy.Meanwhile, sulfa drugss there is also a lot of side effect, such as internal metabolism
Time length, the bacterial drug resistance that causes, it is possible to accumulate in vivo, draws harm immune system and urinary system etc., and may be with
Prototype or active metabolite form are discharged in environment and cause environmental pollution.Up to the present, countries in the world or tissue all pins
MRL to sulfa drugss in food has made clear and definite regulation, the detection of sulfa drug residue in food
Method be also concentrated mainly on Instrumental Analysis (liquid chromatograph, LC-MS etc.) and immunological method (enzyme linked immunological, electrochemiluminescent immunoassay,
Colloidal gold immunochromatographimethod etc.), these methods often have that testing cost is high, the detection poor or complex operation of automaticity, easy
The shortcomings of cross-contamination causes false positive.
Surface plasma resonance technology (Surface Plasmon Resonance, SPR) is use developed in recent years
In analysis bio-molecular interaction a new technique, can determine in real time the interphase interaction of molecule affinity parameters,
Kinetic parameter and thermodynamic parameter.At present, SPR technique is widely used in studying protein-protein, nucleic acid-protein
In the trace detection of matter, nucleic acid-nucleic acid and some micromolecular compounds.The key character of SPR technique be without the need for labelling and reality
When dynamic analysis, have unrivaled advantage in food safety field of fast detection.Pass in conjunction with the SPR immunity of immuno analytical method
Sensor combines SPR technique and immunoassay detects accurate, high-sensitive advantage, causes a large amount of food safety research worker
Extensive concern.Both combinations, divide to nuisance in the food of new accurate, highly sensitive, the repeatable, high degree of automation of exploitation
Analysis new detecting method provides new direction.
Content of the invention
In view of this, the invention is directed to a kind of easy to operate, accuracy and sensitivity is high, reusable
Sulfanilamide surface plasma body resonant vibration immunization method, for the survey of sulfa drugss in Carnis Gallus domesticus, egg, beef, milk, the flesh of fish
Fixed.
For reaching above-mentioned purpose, the technical scheme of the invention is realized in：
A kind of surface plasma body resonant vibration immunization method of detection sulfanilamide, its method and step is：
(1) chip is coated modification：Fixing plasmon resonance chip is placed in flow cell, is passed through EDC and NHS mixed liquor
Chip activation is carried out, is rinsed with 0.5%PBS, standby；Sulfanilamide-OVA solution is passed through, is rinsed with 0.5%PBS solution；It is passed through ethanol
Amine aqueous solution is closed, and 0.5%PBS solution is rinsed to baseline stability；
(2) determine：By sulphaquinoxaline, cistosulfa, sulfamethoxazole and sulfamethoxypyridazine analyte solution respectively
Mix with antibody, being passed through in flow cell carries out immune association reaction, grapher response value；
(3) regenerate：The antibody for being combined with hydrochloric acid eluting plasmon resonance chip surface, realizes chip regeneration, uses 0.5%
PBS is rinsed to baseline stability, is determined next time.
Preferably, in EDC and NHS mixed liquor, EDC concentration is 0.4mol L-1, NHS concentration is 0.1mol L-1, volume ratio
For 1：1, it is 30 μ L min that consumption is 170 microlitres, flow velocity-1.
Preferably, sulfanilamide-OVA solution concentration used is 50 μ g mL-1, consumption be 175 microlitres；Ethanolamine used is dense
Spend for 1.0mol L-1, pH is that 8.5, consumption is 126 microlitres, and flow velocity is 30 μ L min-1.
Preferably, the compound method of 0.5%PBS buffer used is the 20 times of dilutions of phosphate buffer with pH 7.4
Surfactant 10%PBS solution；PBS is with 30 μ L min-1Flow velocity flow through chip.
Preferably, the concentration range of step (2) sulphaquinoxaline, cistosulfa, sulfamethoxazole and sulfamethoxypyridazine
For 1.0-50.0ng mL-1, antibody concentration is 20 μ g mL-1；Mixed liquor sample introduction flow velocity is 30 μ L min-1, time 3min.
Preferably, step (3) concentration of hydrochloric acid solution is 0.1mol L-1, the flow velocity that 0.5%PBS is rinsed is 30 μ L min-1,
1 minute time.
In course of reaction of the present invention, coutroi velocity is because that closed process and continuous mode are secured in chip surface
Carry out on the basis of coating antigen (sulfanilamide-OVA), the purpose of coutroi velocity is to avoid being washed down, be also coating antigen and antibody
Protein binding provides stable environment.
Advantages of the present invention and good effect:
(1) continuous mode of the present invention adopts immunosuppressant principle detection of complex biological sample (egg, Carnis Gallus domesticus, milk, cattle
Meat, the flesh of fish) in sulfa drugss content, with high accuracy and sensitivity.
(2) surface plasma body resonant vibration of unmarked, in-situ analysis is combined by the present invention with immuno analytical method,
The surface plasma body resonant vibration immunosensor of preparation has strong operability, good stability, reusable feature.
Description of the drawings
Four kinds of sulfa drugss suppression ratio curves of Fig. 1-Fig. 4
The substrate impact of five kinds of selected samples of Fig. 5 eliminates result
Specific embodiment
It should be noted that in the case of not conflicting, the embodiment in the invention and the feature in embodiment can
To be mutually combined.
The SPR technique of unmarked, real-time dynamic monitoring is combined by the present invention with immuno analytical method, constructs sulfanilamide
SPR immunosensor.Its specific embodiment is：
First, chip modification
1. CM-5 type surface plasma resonance chip is fixed in circulation draw-in groove, is passed through PBS (0.5%P20) buffering
Liquid is to baseline stability, and setting flow velocity is 30 μ L min-1, temperature be 25 DEG C.
2. 170 μ L concentration are passed through for 0.4mol L-1EDC and 0.1mol L-1NHS mixed liquor (1/1, V/V), use PBS
(0.5%P20) wash buffer, flow velocity is 30 μ L min-1；
3. 175 μ L concentration are passed through for 50 μ g mL-1Sulfanilamide-OVA be coated original solution, with PBS (0.5%P20) buffer rush
Wash, flow velocity is 30 μ L min-1；
4. 126 μ L concentration are passed through for 1.0mol L-1Ethanolamine solutions (pH=8.5), are rushed with PBS (0.5%P20) buffer
It is washed till baseline stability.
2nd, continuous mode
1. Specification Curve of Increasing
By variable concentrations (0ng mL-1、1.0ng mL-1、5.0ng mL-1、10.0ng mL-1、15.0ng mL-1、20.0ng
mL-1、25.0ng mL-1、30.0ng mL-1、40.0ng mL-1、50.0ng mL-1) four kinds of sulfa drugss sulphaquinoxaline,
Cistosulfa, sulfamethoxazole and sulfamethoxypyridazine respectively with concentration be 20 μ g mL-1Sulfanilamide antibody mix homogeneously, standing
10min；Mixed solution is passed through in flow cell, flow velocity is 30 μ L min-1, grapher response value；According to each concentration of gained
Response value draws suppression ratio standard curve, such as Fig. 1-Fig. 4.
2. sample pre-treatments
Milk：Plain chocolate sample 10mL is measured in centrifuge tube, 10000r min-1Centrifugation 30min.Aspirate supernatant, surpasses
10 times of dilutions of pure water, ultrafiltration.Collect gained sample liquid PBS (pH=7.4) and dilute 10 times.
Egg：Weigh the egg 2.0g after mixing to be placed in centrifuge tube.10mL acetonitrile is added, whirlpool is mixed, ultrasound
20min, 10000r min-1Centrifugation 10min.Aspirate supernatant, PBS (pH=7.4) diluted for use.
Beef, Carnis Gallus domesticus, the flesh of fish：After accurately weighing crushing, meat sample 2.0g is placed in centrifuge tube, adds 10mL acetonitrile, and whirlpool is mixed
Even, ultrasonic 20min, 10000r min-1Centrifugation 10min.Aspirate supernatant, PBS (pH=7.4) diluted for use.
Milk, egg, Carnis Gallus domesticus, extraction standard curve such as Fig. 5 of beef and the flesh of fish.
3. continuous mode
By sulfa drugss (sulphaquinoxaline, cistosulfa, sulfamethoxazole and each a quarter of sulfamethoxypyridazine)
Add concentration and be respectively 10.0ng g-1、20.0ng g-1、30.0ng g-1(or ng mL-1) egg, Carnis Gallus domesticus, milk, beef and
The flesh of fish sample carry out sample pre-treatments, each extracting solution respectively with concentration be 20 μ g mL-1Sulfanilamide antibody mix homogeneously, stands 10min
Flow cell is injected afterwards, and grapher response value calculates the response rate.As a result show, the sulfonamides standard substance in 5 kinds of selected samples
Overall recovery is between 84.8%-106.2%, and relative standard deviation (RSD) (table 1) within 7.1%.
The response rate result of three interpolation concentration of sulfa drugss in 1 milk of table, egg, Carnis Gallus domesticus, beef and flesh of fish sample
4. regenerative process
180 μ LHCl solution are injected in flow cell, modification chip regeneration is realized, concentration of hydrochloric acid solution is 0.1mol L-1, the flow velocity that 0.5%PBS is rinsed is 30 μ L min-1, 1 minute time, replication 300 times, instrument signal decay 3.7%.
The preferred embodiment of the invention is the foregoing is only, not in order to limit the invention, all at this
Within the spirit and principle of innovation and creation, any modification, equivalent substitution and improvement that is made etc., should be included in the invention
Protection domain within.
Claims (6)
1. a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that comprise the following steps：
(1) chip is coated modification：Fixing plasmon resonance chip is placed in flow cell, being passed through EDC and NHS mixed liquor is carried out
Chip activation, is rinsed with 0.5%PBS, standby；Sulfanilamide-OVA solution is passed through, is rinsed with 0.5%PBS solution；It is passed through ethanolamine molten
Liquid is closed, and 0.5%PBS solution is rinsed to baseline stability；
(2) determine：By sulphaquinoxaline, cistosulfa, sulfamethoxazole and sulfamethoxypyridazine analyte solution respectively with anti-
Body mixes, and being passed through in flow cell carries out immune association reaction, grapher response value；
(3) regenerate：The antibody for being combined with hydrochloric acid eluting plasmon resonance chip surface, realizes chip regeneration, uses 0.5%PBS
Rinse to baseline stability, determined next time.
2. according to claim 1 a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that：
In EDC and NHS mixed liquor, EDC concentration is 0.4mol L-1, NHS concentration is 0.1mol L-1, volume ratio is 1：1, consumption is 170
Microlitre, flow velocity is 30 μ L min-1.
3. according to claim 1 a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that：Institute
Sulfanilamide-OVA solution concentration is 50 μ g mL-1, consumption be 175 microlitres；Ethanolamine concentration used is 1.0mol L-1, pH
For 8.5, it is 30 μ Lmin that consumption is 126 microlitres, flow velocity-1.
4. according to claim 1 a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that：Institute
The compound method of 0.5%PBS buffer is the 20 times of dilution surfactant 10%PBS of phosphate buffer with pH 7.4
Solution；PBS is with 30 μ L min-1Flow velocity flow through chip.
5. according to claim 1 a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that：Step
Suddenly (2) sulphaquinoxaline, cistosulfa, the concentration range of sulfamethoxazole and sulfamethoxypyridazine are 1.0-50.0ng mL-1,
Antibody concentration is 20 μ g mL-1；Mixed liquor sample introduction flow velocity is 30 μ L min-1, time 3min.
6. according to claim 1 a kind of detection sulfanilamide surface plasma body resonant vibration immunization method, it is characterised in that：Step
Suddenly (3) concentration of hydrochloric acid solution is 0.1mol L-1, the flow velocity that 0.5%PBS is rinsed is 30 μ L min-1, 1 minute time.
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| CN201610892262.2A CN106442427A (en) | 2016-10-13 | 2016-10-13 | Surface plasmon resonance immunoassay method for detecting sulfonamides |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030422A (en) * | 2018-06-25 | 2018-12-18 | 北京中龙益诚科技有限公司 | The surface plasma body resonant vibration immunization method of sulphadiazine, melamine and aflatoxin B1 in a kind of quantitative detection milk |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936983A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method for detecting sulfonamide compound and special quantum dot fluorescent immune kit thereof |
| CN103884682A (en) * | 2014-03-20 | 2014-06-25 | 暨南大学 | Surface plasma resonance biochip, and preparation method and application thereof |
| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
-
2016
- 2016-10-13 CN CN201610892262.2A patent/CN106442427A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101936983A (en) * | 2010-08-03 | 2011-01-05 | 中国农业大学 | Method for detecting sulfonamide compound and special quantum dot fluorescent immune kit thereof |
| CN103884682A (en) * | 2014-03-20 | 2014-06-25 | 暨南大学 | Surface plasma resonance biochip, and preparation method and application thereof |
| CN106018347A (en) * | 2016-05-06 | 2016-10-12 | 中国科学院电子学研究所 | Surface plasma resonance sensing chip and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| SABINA REBE RAZ ET AL.: "Label-Free and Multiplex Detection of Antibiotic Residues in Milk Using Imaging Surface Plasmon Resonance-Based Immunosensor", 《ANALYTICAL CHEMISTRY》 * |
| 周宏敏等: "利用表面等离子共振技术快速检测牛奶中的磺胺甲噁唑", 《食品科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109030422A (en) * | 2018-06-25 | 2018-12-18 | 北京中龙益诚科技有限公司 | The surface plasma body resonant vibration immunization method of sulphadiazine, melamine and aflatoxin B1 in a kind of quantitative detection milk |
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Application publication date: 20170222 |