CN101532980B - Enzyme immunosensor for detecting Shigella species and its preparation method and application - Google Patents
Enzyme immunosensor for detecting Shigella species and its preparation method and application Download PDFInfo
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- CN101532980B CN101532980B CN2009100975778A CN200910097577A CN101532980B CN 101532980 B CN101532980 B CN 101532980B CN 2009100975778 A CN2009100975778 A CN 2009100975778A CN 200910097577 A CN200910097577 A CN 200910097577A CN 101532980 B CN101532980 B CN 101532980B
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Abstract
The invention discloses a sensitive, rapid, accurate, high-specificity and economical enzyme immunosensor for detecting Shigella species, comprising screen printing electrodes, a sensitive membrane containing enzyme marked Shigella species antibody- carbon nano-tube- shitosan mixture is coated on the working electrode of the screen printing electrodes. In the invention, the chemical amplification function of the multi-wall carbon nano-tube and enzyme is combined with the specificity of the immunosensor, theenzyme immunosensor has the specificity of immunoreactivity and the sensitivity of the electrochemistry analysis and can accurately detect the lower-content substance. The invention also discloses a preparation method and application of the enzyme immunosensor, the Shigella species can be directly detected, with the sensitive, rapid, high-specificity advantages, enzyme immunosensor is cheap and suitable for detecting the Shigella species in the foundation and in situ.
Description
Technical field
The present invention relates to the food-borne pathogens rapid detection technical field, particularly detect enzyme immunosensor and preparation method thereof and the utilization of Shigella based on the antigen-antibody immune response.
Background technology
In the annual hundreds of millions of food origin disease patient in the whole world, the 70%th, food that various invasive organisms pollute and drinking-water causes owing to eaten.Shigella (Shigella) is one of main pathogens that causes acute infectious diarrhea, few can cause infection to 10 bacteriums, infected person know from experience occur feeling sick, vomiting, stomachache, diarrhoea symptoms such as (serious pus and blood stools), even threat to life.More than 100 ten thousand people death because of infecting Shigella is arranged every year, and mostly the dead is the children of developing country.
The Shigella fast detecting is to carry out the health check-up and one of most critical measure of handling public health emergency of healthy population in enormous quantities, and quick enforcement prevention, treatment are had earthshaking meaning.
At present, Shigella detection method commonly used comprises National Standard Method, enzyme-linked immunosorbent assay (ELISA), PCR and RT-PCR technology, genetic chip and Nucleic Acid Probe Technique.National Standard Method is drawn materials conveniently, but sample pretreatment is complicated, operating process is more loaded down with trivial details, and sense cycle is longer, and susceptibility is low; ELISA needs special instrument and equipment, detects complex steps, expense is high, detection time is long, and is prone to false positive; The required instrument and equipment of PCR, genetic chip and Nucleic Acid Probe Technique is expensive, testing process is complicated, to testing environment and operating personnel's professional technique have relatively high expectations, employed reagent all has bigger harm to human body and environment, also is prone to false positive.Therefore, to set up a kind of sensitivity, Shigella detection method quick, easy, that specificity is high and economic be food production or marketing enterprise, Quality Control personnel, import and export the powerful guarantee with food security that presses for of commodity inspection, government administration section.
Electrochemical analysis can realize on-the-spot the detection, does not receive the influence of color sample, turbidity, and sample can be not treated, need not to separate, and used instrument and equipment is simple relatively, and these all make electrochemistry immuno-sensing become a kind of use detection method comparatively widely.In recent years, in the food-borne pathogens detection range, immunosensor more and more causes people's attention.It is with the analytical approach that Electrochemical Detection and traditional immunological technique combine, and existing excellent specificity has lower detectability again.And compare with other technologies, this sensor requires lowly, simple to operate to instrument and equipment, has shortened detection time more widely.Adopt the disposable electrode of silk-screen printing technique preparation further to reduce the detection cost, fast, accurately detecting the important effect of bringing into play in the food-borne pathogens on a large scale.
Since Iijima in 1991 found multi-walled carbon nano-tubes (multi-wall carbon nanotube), multi-walled carbon nano-tubes caused extensive concern because of its good electricity, mechanical property and potential application prospect.Multi-wall carbon nano-tube composite material has excellent conducting performance and helps keeping biomolecule activity, is very suitable for the preparation and the application of biology sensor.
Summary of the invention
In order to overcome the above problems; The invention provides a kind of sensitivity, fast, accurately, the enzyme immunosensor of the high and economic detection Shigella of specificity; Comprise screen printing electrode, be coated with the sensitive membrane of embedding " enzyme labeling Shigella antibody-CNT-shitosan " potpourri on the working electrode of said screen printing electrode.
Preferably, described CNT is a multi-walled carbon nano-tubes.
Further, described Shigella is a shigella flexneri.
Further, a kind of in the described enzyme labeling Shigella antibody Shigella antibody that is horseradish peroxidase, alkaline phosphatase, lactic acid dehydrogenase, glucose oxidase, PA ase, hydrolysis of urea enzyme labeling or their potpourri.
On the other hand, the invention provides the preparation method of above-mentioned enzyme immunosensor, it is characterized in that may further comprise the steps:
1) be dispersed in the shitosan colloidal sol CNT is ultrasonic, the CNT-chitosan complexes of homogeneous;
2) enzyme labeling Shigella antibody and CNT-chitosan complexes are fully mixed, place 12h, make " enzyme labeling Shigella antibody-CNT-shitosan " potpourri for 4 ℃;
3) " enzyme labeling Shigella antibody-CNT-shitosan " potpourri is coated on the working electrode surface of screen printing electrode, dry under the room temperature;
4) electrode after filming is soaked in the PBS that contains 0.2% bovine serum albumin(BSA) 4 ℃ of held 1h.
The present invention also provides a kind of method of using above-mentioned enzyme immunosensor to detect Shigella, it is characterized in that may further comprise the steps:
1) sample to be tested drips the working electrode surface in enzyme immunosensor, hatches 30min for 28 ℃;
2) immunosensor places electrolytic cell, and liquid is for containing 0.6mmol/L H at the bottom of the detection of electrolytic cell
2O
2With the acetate buffer of 1.0mmol/L thionine, the pH value is 7.0;
3) set suitable sweep parameter, working electrode is applied voltage, detect the current value between auxiliary electrode and working electrode;
4) measure the current information input data Collection & Processing System that obtains;
5) information processing result output.
Preferably, data Collection & Processing System adopts cyclic voltammetry as immunoelectrode sign, qualitative judgement and quantitative detection method.
Immunosensor of the present invention, the membrane material of embedding enzyme labeling Shigella antibody and CNT can also use teflon, N except that shitosan, dinethylformamide, Nafion, tygon, agarose etc.;
Detecting sample can be diarrhoea patient's ight soil, anal swab, vomitus and polluted soil, water source, food, tableware equal samples, and sampling must be carried out under sterile working, and the sample of being gathered must be representative.Freezing sample should keep freezing state, and non-freezing sample needs 0～5 ℃ of preservation.As can not in time inspect by ready samples, should sample be stored in 30% GBS.
Detection method principle among the present invention: in the electrochemical reaction system, substrate for enzymatic activity H
2O
2Generate corresponding enzymatic reaction thing H
2O, enzyme becomes oxidation state by going back ortho states; This moment, the enzymatic electron mediator thionine of oxidation state became oxidation state by going back ortho states, and enzyme self is then become by oxidation state goes back ortho states; The electron mediator of oxidation state becomes again after electrode surface obtains electronics goes back ortho states, produces response current simultaneously.
(Cyclic voltammetry is CV) as immunosensor sign, qualitative judgement and quantitative detection method to adopt cyclic voltammetry commonly used.When containing Shigella in the detected material, bacterial antigens react with the enzyme labeling Shigella antibody that is embedded on the working electrode, generate antigen antibody complex; When immunosensor places electrolytic cell; Antigen antibody complex spatially hindered thionine to multi-walled carbon nano-tubes and horseradish peroxidase activated centre near; Make mediator and substrate be difficult near multi-walled carbon nano-tubes and horseradish peroxidase; Greatly hindered electron transport; Cause the redox reaction of thionine to weaken, significantly reduce before making in the response curve reduction peak current than immune response, can judge whether contain Shigella and the Shigella concentration that is contained in the determinand according to the variable quantity of response curve peak value.
As shown in Figure 1, be the immunosensor of apparatus of the present invention current-responsive curve map in different phase.Place electrolytic cell to detect end liquid (0.6mmol/L H in different phase same sensor
2O
2+ 1.0mmol/L thionine+0.1mol/L pH 7.0 hac buffers) detect response current, more same immunosensor changes at the response current that different phase produces.
Wherein curve a is the current-responsive curve of naked screen printing electrode in detecting end liquid.What naked screen printing electrode occurred is a pair of stable thionine redox peak.
Curve b is the current-responsive curve of CNT-chitosan film modified electrode in detecting end liquid.Its redox peak-to-peak signal significantly strengthens, and is because multi-walled carbon nano-tubes can increase electron transfer speed and transmit due to the electronics effectively.
Curve c is the current-responsive curve of immunosensor in detecting end liquid that enzymic-labelled antibody-CNT-chitosan complexes is modified, and the redox peak-to-peak signal in the curve further increases.
Curve d is that immunosensor is hatched the current-responsive curve of back in detecting end liquid with the detection sample.Can find out; The redox peak current obviously reduces; Show immune complex that the Ag-Ab immune response generates spatially hindered thionine to the activated centre of CNT and enzyme near; Greatly hindered electron transport, caused the redox reaction of thionine to weaken, thereby make reduction peak current significantly reduce.
With Δ I=Ip1-Ip2 (be before and after the immune response catalytic current reduce value) as the foundation that whether contains shigella flexneri in the judgement sample.Positive when K >=0.3 μ A, contain Shigella in the expression tested sample; Negative when K＜0.3 μ A, do not contain Shigella in the expression tested sample.
The enzyme immunosensor of detection Shigella of the present invention combines immunological technique with Electrochemical Detection, be a kind of current mode enzyme immunosensor.It combines the chemical enlarging function of multi-walled carbon nano-tubes and enzyme with the specificity of immunosensor, merge the advantage of the two, makes its sensitivity that possesses immunoreactive specificity and electrochemical analysis simultaneously, can carry out the detection of low content material exactly.The enzyme immunosensor of detection of the present invention Shigella is disposable use, easily preparation and test operation is simple and convenient, cheap, high specificity, highly sensitive, the response time short.
The method of the above-mentioned enzyme immunosensor of preparation of the present invention; Adopt multi-walled carbon nano-tubes-glycan substrate directly enzymic-labelled antibody to be fixed on the working electrode surface of screen printing electrode; Greatly simplified the preparation process of immunosensor; Detection to shigella flexneri has specificity, and the selectivity that detects is good, highly sensitive, accuracy is better.
The present invention detects the method for Shigella; Simple to operate; Can qualitatively judge the disposable enzyme immunosensor with the detection by quantitative bacterial concentration, can realize direct detection Shigella, have sensitivity, fast, the specificity advantages of higher; And cheap, be applicable to basic unit or on-the-spot detection Shigella.
The present invention is applicable to medical diagnosis, food industry and field of environment protection fast detecting Shigella.
For further specifying characteristics of the present invention and effect, the present invention is further described below in conjunction with accompanying drawing.
Description of drawings
Fig. 1 is that the sensor of apparatus of the present invention detects the current-responsive curve in the end liquid in different phase in electrolytic cell;
Fig. 2 shigella flexneri normalized current-concentration curve.
Embodiment
Embodiment 1: the preparation enzyme immunosensor
Be in 1% the shitosan colloidal sol with the ultrasonic 10mL of the being dispersed in mass and size of 2.0mg multi-walled carbon nano-tubes percentage, obtain the multi-walled carbon nano-tubes-chitosan complexes of homogeneous;
Multi-walled carbon nano-tubes-chitosan complexes is fully mixed with the anti-shigella flexneri antibody-solutions equal-volume of mark horseradish peroxidase; Mixed liquor is placed 12h for 4 ℃;
Get the working electrode surface that the above-mentioned mixing drop of 3 μ L is applied to disposable screen printing electrode, and, make it drying and forming-film in room temperature held 3～5h;
Immunoelectrode after filming is soaked in the PBS PBS that contains 0.2%BSA (bovine serum albumin(BSA)); 4 ℃ of held 1h; Rinse well with redistilled water; Remove and the shitosan antibody membrane of the non-strong bonded of electrode and sealing non-specific adsorption site, the enzyme immunosensor that makes.
Enzyme immunosensor is dipped in the PBS solution (0.01mol/L pH 7.4) to be preserved subsequent use down at 4 ℃.
Embodiment 2: the method that detects Shigella
Enzyme immunosensor described in the embodiment 1 is linked to each other with electrochemical workstation (CHI 1030A) through lead, and computing machine links to each other with electrochemical workstation.
Put into electrolytic cell to enzyme immunosensor, liquid is for containing 0.6mmol/L H at the bottom of the detection of electrolytic cell
2O
2With the acetate buffer of 1.0mmol/L thionine, the pH value is 7.0.Set the CV location parameter of electrochemical workstation: electric potential scanning scope-0.1～-0.6V, sweep speed is 0.1V/s, and the working electrode of enzyme immunosensor is applied voltage, the current value I p1 that records between auxiliary electrode and working electrode is 1.946 μ A.
The anal swab of getting vomiting, diarrhoea patient with sterile working in the centrifugal 5min of 8000r/min, takes off a layer bacterium mud after enrichment culture, separation and Culture, be diluted to sample to be tested with the PBS of pH 7.4.Sample to be tested is dripped the working electrode surface in immunosensor, hatch 30min under 28 ℃, make bacterium and the antibody of immunosensor working electrode surface in the sample produce immune response.Put into electrolytic cell to immunosensor.The sweep speed of setting electrochemical workstation is 0.1V/s, and working electrode is applied voltage, and the current value I p2 that detects between auxiliary electrode and working electrode is 1.051 μ A.
The current information of said determination acquisition is imported the data Collection & Processing System of electrochemical workstation; Adopt cyclic voltammetry commonly used as immunosensor sign, qualitative judgement and quantitative detection method; Last computing machine output process information result, Δ I=1.946 μ A-1.051 μ A=0.895 μ A＞0.3 μ A shows and contains shigella flexneri in the tested sample.
Adopt the detection method of Shigella among immunosensor described in the embodiment 1 and the embodiment 2, the variable concentrations Shigella is measured, production standard electric current-concentration curve specifically is with shigella flexneri standard sample preparation 10
10The PBS solution of cfu/mL, and stepwise dilution to 10
4Cfu/mL.Produce typical curve, as shown in Figure 2.It detects lower limit: 10
4Cfu/mL; Between linear zone: 10
4Cfu/mL～10
10Cfu/mL; Linearly dependent coefficient: R
2=99.54%.
Get vomiting, diarrhoea patient's anal swab with sterile working, sample preparation and detection method get Δ I=0.9312 μ A at last with embodiment 2, reference standard electric current-concentration curve, and calculating the shigella flexneri concentration that contains in the tested sample is 3 * 10
7Cfu/mL.
Claims (6)
1. detect the enzyme immunosensor of Shigella, it is characterized in that comprising screen printing electrode, be coated with the sensitive membrane of embedding " enzyme labeling Shigella antibody-CNT-shitosan " potpourri on the working electrode of said screen printing electrode; A kind of in the Shigella antibody that described enzyme labeling Shigella antibody is horseradish peroxidase, alkaline phosphatase, lactic acid dehydrogenase, glucose oxidase, PA ase, hydrolysis of urea enzyme labeling or their potpourri.
2. detect the enzyme immunosensor of Shigella according to claim 1, it is characterized in that described CNT is a multi-walled carbon nano-tubes.
3. like the enzyme immunosensor of the said detection of claim 2 Shigella, it is characterized in that described Shigella is a shigella flexneri.
4. the method for preparing the said enzyme immunosensor of claim 1 is characterized in that may further comprise the steps:
1) be dispersed in the shitosan colloidal sol CNT is ultrasonic, the CNT-chitosan complexes of homogeneous;
2) enzyme labeling Shigella antibody and CNT-chitosan complexes are fully mixed 4
oC places 12 h, makes " enzyme labeling Shigella antibody-CNT-shitosan " potpourri; A kind of in the Shigella antibody that described enzyme labeling Shigella antibody is horseradish peroxidase, alkaline phosphatase, lactic acid dehydrogenase, glucose oxidase, PA ase, hydrolysis of urea enzyme labeling or their potpourri;
3) " enzyme labeling Shigella antibody-CNT-shitosan " potpourri is coated on the working electrode surface of screen printing electrode, drying and forming-film under the room temperature;
4) electrode after filming is soaked in the PBS that contains 0.2% bovine serum albumin(BSA) 4
oC held 1 h.
5. detect the method for Shigella, it is characterized in that may further comprise the steps:
1) sample to be tested drips in the working electrode surface of enzyme immunosensor according to claim 1, and 28
oC is hatched 30 min;
2) immunosensor places electrolytic cell, and liquid is for containing 0.6 mmol/L H at the bottom of the detection of electrolytic cell
2O
2With the acetate buffer of 1.0 mmol/L thionines, the pH value is 7.0;
3) set suitable sweep parameter, working electrode is applied voltage, detect the current value between auxiliary electrode and working electrode;
4) measure the current information input data Collection & Processing System that obtains;
5) information processing result output.
6. the method for detection Shigella as claimed in claim 5 is characterized in that data Collection & Processing System adopts cyclic voltammetry as immunoelectrode sign, qualitative judgement and quantitative detection method.
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| CN102608189B (en) * | 2012-03-30 | 2014-07-09 | 山东理工大学 | Method for manufacturing nanometer magnetic ferroferric oxide modified immunosensor |
| CN102854320B (en) * | 2012-10-09 | 2014-08-06 | 天津商业大学 | Preparation method of bacillus cereus electrochemical nano-immunosensor |
| CN102998348A (en) * | 2012-11-27 | 2013-03-27 | 重庆医科大学 | Preparation method of dehydrogenase-electrochemical biosensor |
| CN103018438A (en) * | 2012-12-31 | 2013-04-03 | 山东理工大学 | Method for preparing immunosensor for detecting chlorpyrifos pesticide residue |
| CN104089997B (en) * | 2014-05-29 | 2016-04-13 | 北京盈盛恒泰科技有限责任公司 | A kind of electrochemical immunosensor and its preparation method and application |
| CN104198734B (en) * | 2014-09-01 | 2016-06-08 | 深圳出入境检验检疫局食品检验检疫技术中心 | The detection method of a kind of streptococcus aureus |
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| EP1261869A2 (en) * | 2000-03-02 | 2002-12-04 | The Procter & Gamble Company | Sensor device for diarrhea diagnosis using fecal markers |
| JP2005345243A (en) * | 2004-06-02 | 2005-12-15 | Toshiba Corp | Nucleic acid detecting substrate and nucleic acid detecting method using it |
| CN101008036A (en) * | 2007-01-15 | 2007-08-01 | 深圳市第二人民医院 | Shigella gene quick diagnosis kit based on loop-mediated equal-temperature amplification technology |
| CN101113471A (en) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting food-derived pathogenic enterobacteria by composite fluorescence PCR technique |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1261869A2 (en) * | 2000-03-02 | 2002-12-04 | The Procter & Gamble Company | Sensor device for diarrhea diagnosis using fecal markers |
| JP2005345243A (en) * | 2004-06-02 | 2005-12-15 | Toshiba Corp | Nucleic acid detecting substrate and nucleic acid detecting method using it |
| CN101008036A (en) * | 2007-01-15 | 2007-08-01 | 深圳市第二人民医院 | Shigella gene quick diagnosis kit based on loop-mediated equal-temperature amplification technology |
| CN101113471A (en) * | 2007-06-07 | 2008-01-30 | 天津出入境检验检疫局动植物与食品检测中心 | Method for detecting food-derived pathogenic enterobacteria by composite fluorescence PCR technique |
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