CN102520187B - Manufacture method and application of immune sensor based on polyaniline nano-particle composite membrane - Google Patents
Manufacture method and application of immune sensor based on polyaniline nano-particle composite membrane Download PDFInfo
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Abstract
The invention relates to a manufacture method and application of an immune sensor based on a polyaniline nano-particle composite membrane. By manufacturing the polyaniline nano-particle composite membrane, self-conductivity of materials is improved, and the membrane can be successfully applied to the immune sensor so as to greatly reduce detection limits of the sensor and achieve better stability. Representation and measurement of the sensor are performed through a cyclic voltammetry method and an alternate current impedance method, a staphylococcus aureus enterotoxin B detection standard curve is built, the linear range ranges from 0.1ng/ml to 8ng/ml, correlation coefficient R2=0.9932, and detection limit is 0.033ng/ml(S/N=3). The manufacture method and the application are good in specificity, can be used repeatedly, are good in stability, can be applied to fast detection of staphylococcus aureus enterotoxin B in dairy products and has wide application prospect, and dairy product detection recovery rate ranges from 84% to 111%.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof based on polyaniline nano gold composite membrane immunosensor, the invention belongs to dairy food quality detection technique field.
Background technology
Staphylococcus aureus (Staphylococcus aureus) is that one is extensively present in natural gram-positive cocci.The pathogenecity of staphylococcus aureus depends primarily on the ability of its product enterotoxin and enzyme.Staphylococcus aureus enterotoxin (staphylococcal enterotoxin is called for short SE), is a kind of extracellular toxin of staphylococcus aureus secretion, is that one group of structure is relevant, the single chain polypeptide toxic protein that virulence is close.Press serological classification, mainly contain the serotypes such as A, B, Cs, D, E, be mainly present in meat, in the higher food of the protein contents such as newborn class.Ses thermal stability is high, in 70～80 ℃ of 30min, can not destroyed completely, still has pathogenicly, and is not degraded by trypsase.
The detection method of Staphylococcus aureus enterotoxin original adoption is exactly zoology test method, but because the source of animal used as test is more difficult, and sensitivity is lower, it is not very desirable causing testing result.The basis of euzymelinked immunosorbent assay (ELISA) is the enzyme labeling of immobilization and antigen or the antibody of antigen or antibody.Because the catalytic efficiency of enzyme is very high, indirectly amplify immunoreactive result, make assay method reach very high sensitivity.Euzymelinked immunosorbent assay (ELISA) is sensitive, simple, quick, and technician does not need special training, is applicable to detection and the evaluation of staphylococcal food poisoning.The many of research is double antibody sandwich method and indirect competitive, and its detection limit can reach 1ng/ml, and the range of linearity is good.The quality of enzyme-linked immunoassay method effect very major part depends on the preparation of antibody.
Since the nineties, risen rapidly take biosensor technology as basic different kind organism detection system, some are quick and adopt the detection method of modern technologies constantly to occur, and reach its maturity, and are constantly applied in each anatoxic detection and go.Utilizing surface plasma body resonant vibration (SPR) sensing technology is the mutual identifying of antigen/antibody for the most common monitoring target of the detection of staphylococcal enterotoxin, antigen-antibody reaction high specificity, highly sensitive, reproducible, the response time is often about tens minutes.Utilize SPR technology to measure SEB, its detection limit can reach ng/ml level, response time 10min left and right.
Summary of the invention
The object of this invention is to provide a kind of preparation method of the immunosensor for detection of Staphylococcus aureus enterotoxin.
The invention provides following technical scheme: polyaniline nano gold fluid drips is applied to through clean gold electrode surfaces, under room temperature, leave standstill, to be finished completely after, on the electrode surface of having modified, drip SEB antibody, hatch 20～100min, with BSA-PBS sealing 20～140Min, make based on Polyaniline-modified nm of gold self assembly immunosensor.
Described SEB antibody is on market, to buy or prepare, preparation method is shown in Jeremy M.Yarwood, John K.McCormick, Michael L.Paustian, Vivek Kapur, and Patrick M.Schlievert, Repression of the Staphylococcus aureus Accessory Gene Regulator in Serum and In Vivo, JOURNAL OF BACTERIOLOGY, Feb.2002, p.1095-1101.
Described polyaniline nano gold liquid is by HAuCl
4be dissolved in the water, treat its boiling, add sodium citrate solution, after reaction 5-15min, add successively sodium hydroxide solution, aniline solution, sodium peroxydisulfate solution, the 20-50min that seethes with excitement under stirring condition, stops heating, is stirred to room temperature.
Preferably, described polyaniline nano gold liquid preparation method is as follows: by the HAuCl of 250 μ L 4%
4be dissolved in 100mL water, treat its boiling, add the sodium citrate of 800 μ L 4%, after reaction 8min, add successively 0.2molL
-1sodium hydroxide solution 1200 μ L, 0.1molL
-1aniline solution 1000 μ L, 0.1molL
-1sodium peroxydisulfate solution 1500 μ L, the 30min that seethes with excitement under stirring condition, stop heating, be stirred to room temperature.
Preferably, gold electrode clean method is as follows: gold electrode (Φ=2mm) is placed in to Piranha solution and soaks 15Min, use successively the Al of 0.3,0.05 μ m
2o
3electrode surface is thrown into minute surface by burnishing powder, then with the nitric acid, absolute ethyl alcohol, the ultrapure water ultrasonic cleaning 5min that use 1: 1 (volume ratio), nitrogen dries up again, and 4 ℃ for subsequent use;
Preferably, on the electrode surface of having modified, drip 10ul 200～900ug/ml SEB antibody, hatch 20～100min for 37 ℃, at 1%BSA-PBS37 ℃, seal 20～140Min, make based on Polyaniline-modified nm of gold self assembly immunosensor.
When above-mentioned antibody concentration is 700ug/ml, the antibody of the material institute combination state that reaches capacity, if antibody concentration is too high, can cause the waste of antibody; The above-mentioned antibody incubation time is 60min, and in the time that antibody concentration is 60min, antibody can farthest be combined on material; Be 120min described off-period, and in the time that reach 120min off-period, 1%BSA-PBS can close the not binding site of antibody preferably.
The present invention also provides a kind of application of described immunosensor, step is as follows: take out the SEB immunoelectrode of having modified, 37 ℃ of SEB standard items that drip successively 10 μ L variable concentrations are hatched after 5～30min, adopt the variation of electrode interface of investigating of cyclic voltammetry and AC impedence method, and electrode table is characterized.
CV test condition: voltage 0.2-0.6V, sweep speed 0.1V/s; EIS test condition: initial amplitude 0.05V, frequency is 1～100kHz, time of repose 2s.Reaction medium liquid is 2.5mmolFe (CN)
6 3-/4-solution (all tests are all at room temperature carried out).
Described SEB standard items incubation time is 5～30min, preferably, described SEB standard items incubation time is 25min, in the time that incubation time reaches 25min, SEB antibody can fully react with SEB standard items.
The concentration of SEB standard items of the described variable concentrations dripping is successively 0.1,0.5,1,2,3,4,5,6,7,8ng/ml, mimic channel obtains resistance value, take SEB standard items concentration as horizontal ordinate, the size of resistance value is ordinate mapping, obtains the typical curve that SEB self assembly immunoelectrode detects.There is respectively good linear relationship in the concentration of the size of resistance value and SEB standard items, linear equation is respectively Y=1262.2x+1540.8 between 0.1～8ng/ml, and related coefficient is respectively R
2=0.9932, lowest detection is limited to 0.033ng/ml.
The analytical approach of SEB is as follows: electrochemical workstation CHI760, at initial amplitude 0.05V, under the condition that frequency is 1～100kHz, is 2.5mmolFe (CN) at reaction medium liquid
6 3-/4-in solution, measure, adopt EIS method, and calculate by best equivalence circuit, the resistance value using working electrode after BSA sealing is as blank AC impedance value R
et(Ab), take this electrode with contain variable concentrations SEB standard items in react after in same electrolytic solution the resistance value of being surveyed as R
et(Ab-Ag), obtain R in different SEB standard items concentration
etchanging value Δ R
et:
ΔR
et＝R
et(Ab-Ag)-R
et(Ab)
R
et(Ab): blank AC impedance value;
R
et(Ab-Ag): transmit resistance value with the reacted electrode electronics of SEB standard items;
Δ R
et: before and after reaction, utmost point electronics transmits the changing value of resistance;
Transmit the changing value of resistance and the mapping of the relation of SEB concentration of standard solution with electronics, obtain the examination criteria curve of SEB immunosensor.
The recovery of SEB:
Sample is diluted to after finite concentration with PBS (PH=7.4), and application self assembly immunosensor carries out mark-on to sample and reclaims mensuration.
Method of the present invention has following beneficial effect:
(1) polyaniline nano gold structure of composite membrane has improved the dispersiveness of nm of gold ion, has effectively stoped golden polymerization, thereby has made the more even of its dispersion.
(2) polyaniline nano gold structure of composite membrane has effectively promoted the transfer of electronics, and then effectively improves the conduction of material itself, greatly reduces detectability.
(3) self assembly immunosensor can, in conjunction with the advantage of multiple material, be given full play to the advantage of antigen and antibody specific combination, reduces greatly detectability.
Accompanying drawing explanation
Fig. 1 represents the assembling schematic diagram of self assembly immunosensor.
Embodiment
Embodiment 1: the preparation method of immunosensor
Steps A: by the HAuCl of 250 μ L 4%
4be dissolved in 100mL water, treat its boiling, add the sodium citrate of 800 μ L 4%, after reaction 8min, add successively 0.2molL
-1sodium hydroxide solution 1200 μ L, 0.1molL
-1aniline solution 1000 μ L, 0.1molL
-1sodium peroxydisulfate solution 1500 μ L, the 30min that seethes with excitement under stirring condition, stop heating, be stirred to room temperature;
Step B: gold electrode (Φ=2mm) is placed in to Piranha solution and soaks 15Min, use successively the Al of 0.3,0.05 μ m
2o
3electrode surface is thrown into minute surface by burnishing powder, then with the nitric acid, absolute ethyl alcohol, the ultrapure water ultrasonic cleaning 5min that use 1: 1 (volume ratio), nitrogen dries up again, and 4 ℃ for subsequent use;
Step C: the polyaniline nano gold fluid drips that steps A is obtained is applied to thorough clean gold electrode surfaces in step B, under room temperature, leave standstill, to be finished completely after, on the electrode surface of having modified, drip 10ul 700ug/ml SEB antibody, hatch 60min for 37 ℃, at 1%BSA-PBS37 ℃, seal 120Min, make based on Polyaniline-modified nm of gold self assembly immunosensor.
Embodiment 2: the preparation method of immunosensor
Steps A: by the HAuCl of 250 μ L 4%
4be dissolved in 100mL water, treat its boiling, add the sodium citrate of 800 μ L 4%, after reaction 8min, add successively 0.2molL
-1sodium hydroxide solution 1200 μ L, 0.1molL
-1aniline solution 1000 μ L, 0.1molL
-1sodium peroxydisulfate solution 1500 μ L, the 30min that seethes with excitement under stirring condition, stop heating, be stirred to room temperature;
Step B: gold electrode (Φ=2mm) is placed in to Piranha solution and soaks 15Min, use successively the Al of 0.3,0.05 μ m
2o
3electrode surface is thrown into minute surface by burnishing powder, then with the nitric acid, absolute ethyl alcohol, the ultrapure water ultrasonic cleaning 5min that use 1: 1 (volume ratio), nitrogen dries up again, and 4 ℃ for subsequent use;
Step C: the polyaniline nano gold fluid drips that steps A is obtained is applied to thorough clean gold electrode surfaces in step B, under room temperature, leave standstill, to be finished completely after, on the electrode surface of having modified, drip 10ul 200ug/ml SEB antibody, hatch 20min for 37 ℃, at 1%BSA-PBS37 ℃, seal 20Min, make based on Polyaniline-modified nm of gold self assembly immunosensor.
Embodiment 3: the preparation method of immunosensor
Steps A: by the HAuCl of 250 μ L 4%
4be dissolved in 100mL water, treat its boiling, add the sodium citrate of 800 μ L 4%, after reaction 8min, add successively 0.2molL
-1sodium hydroxide solution 1200 μ L, 0.1molL
-1aniline solution 1000 μ L, 0.1molL
-1sodium peroxydisulfate solution 1500 μ L, the 30min that seethes with excitement under stirring condition, stop heating, be stirred to room temperature;
Step B: gold electrode (Φ=2mm) is placed in to Piranha solution and soaks 15Min, use successively the Al of 0.3,0.05 μ m
2o
3electrode surface is thrown into minute surface by burnishing powder, then with the nitric acid, absolute ethyl alcohol, the ultrapure water ultrasonic cleaning 5min that use 1: 1 (volume ratio), nitrogen dries up again, and 4 ℃ for subsequent use;
Step C: the polyaniline nano gold fluid drips that steps A is obtained is applied to thorough clean gold electrode surfaces in step B, under room temperature, leave standstill, to be finished completely after, on the electrode surface of having modified, drip 10ul 900ug/ml SEB antibody, hatch 100min for 37 ℃, at 1%BSA-PBS37 ℃, seal 140Min, make based on Polyaniline-modified nm of gold self assembly immunosensor.
Embodiment 4:
Take out the SEB immunoelectrode of having modified, 37 ℃ of SEB standard items that drip successively 10 μ L variable concentrations are hatched after 5～30min, adopt the variation of electrode interface of investigating of cyclic voltammetry and AC impedence method, and electrode table is characterized.
CV test condition: voltage 0.2-0.6V, sweep speed 0.1V/s; EIS test condition: initial amplitude 0.05V, frequency is 1～100kHz, time of repose 2s.Reaction medium liquid is 2.5mmolFe (CN)
6 3-/4-solution (all tests are all at room temperature carried out).
Described SEB standard items incubation time is 5～30min, preferably, described SEB standard items incubation time is 25min, in the time that incubation time reaches 25min, SEB antibody can fully react with SEB standard items.
The concentration of SEB standard items of the described variable concentrations dripping is successively 0.1,0.5,1,2,3,4,5,6,7,8ng/ml, mimic channel obtains resistance value, take SEB standard items concentration as horizontal ordinate, the size of resistance value is ordinate mapping, obtains the typical curve that SEB self assembly immunoelectrode detects.There is respectively good linear relationship in the concentration of the size of resistance value and SEB standard items, linear equation is respectively Y=1262.2x+1540.8 between 0.1～8ng/ml, and related coefficient is respectively R
2=0.9932, lowest detection is limited to 0.033ng/ml.
The analytical approach of SEB is as follows: electrochemical workstation CHI760, at initial amplitude 0.05V, under the condition that frequency is 1～100kHz, is 2.5mmolFe (CN) at reaction medium liquid
6 3-/4-in solution, measure, adopt EIS method, and calculate by best equivalence circuit, the resistance value using working electrode after BSA sealing is as blank AC impedance value R
et(Ab), take this electrode with contain variable concentrations SEB standard items in react after in same electrolytic solution the resistance value of being surveyed as R
et(Ab-Ag), obtain R in different SEB standard items concentration
etchanging value Δ R
et:
ΔR
et＝R
et(Ab-Ag)-R
et(Ab)
R
et(Ab): blank AC impedance value;
R
et(Ab-Ag): transmit resistance value with the reacted electrode electronics of SEB standard items;
Δ R
et: before and after reaction, utmost point electronics transmits the changing value of resistance;
Transmit the changing value of resistance and the mapping of the relation of SEB concentration of standard solution with electronics, obtain the examination criteria curve of SEB immunosensor.
The recovery of SEB:
Sample is diluted to after finite concentration with PBS (PH=7.4), and application self assembly immunosensor carries out mark-on to sample and reclaims mensuration.
Obtaining the recovery of skimmed milk powder under the mark-on concentration of 4ng/ml according to said determination method and analytical approach is 92%.
Embodiment 5:
Obtaining the recovery of skimmed milk under the mark-on concentration of 0.5ng/ml according to embodiment 4 assay methods and analytical approach is 84%.
Embodiment 6:
Obtaining the recovery of fresh cow milk under the mark-on concentration of 4ng/ml according to embodiment 4 assay methods and analytical approach is 111%.
Claims (4)
1. the preparation method based on polyaniline nano gold composite membrane immunosensor, it is characterized in that, polyaniline nano gold fluid drips is applied to through clean gold electrode surfaces, under room temperature, leave standstill, to be finished completely after, on the electrode surface of having modified, drip SEB antibody, hatch 20～100min, with BSA-PBS sealing 20～140min, make based on Polyaniline-modified nm of gold self assembly immunosensor;
Described polyaniline nano gold liquid preparation method is as follows: by the HAuCl of 250 μ L4%
4be dissolved in 100mL water, treat its boiling, add the sodium citrate of 800 μ L4%, after reaction 8min, add successively 0.2molL
-1sodium hydroxide solution 1200 μ L, 0.1molL
-1aniline solution 1000 μ L, 0.1molL
-1sodium peroxydisulfate solution 1500 μ L, the 30min that seethes with excitement under stirring condition, stop heating, be stirred to room temperature;
Gold electrode clean method is as follows: gold electrode is placed in to Piranha solution and soaks 15min, use successively the A1 of 0.3,0.05 μ m
2o
3electrode surface is thrown into minute surface by burnishing powder, then use nitric acid, absolute ethyl alcohol, the ultrapure water ultrasonic cleaning 5min of volume ratio l:l:1, and nitrogen dries up, and 4 ℃ for subsequent use;
Described SEB antibody dripping quantity is 200～900 μ g/ml.
2. preparation method according to claim 1, is characterized in that, described SEB antibody dripping quantity is 700ug/ml.
3. the application of the immunoelectrode that described in a claim 1, method builds, it is characterized in that, step is as follows: take out the SEB immunoelectrode of having modified, 37 ℃ of SEB standard items that drip successively l0 μ L variable concentrations are hatched after 5～30min, adopt the variation of electrode interface of investigating of cyclic voltammetry and AC impedence method, and electrode table is characterized.
4. application according to claim 3, is characterized in that, described SEB standard items incubation time is 25min.
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| CN103308579A (en) * | 2013-05-09 | 2013-09-18 | 东华大学 | Preparation method of polyaniline/precious metal nanometer composite material modified electrode |
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| 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 |
| CN104630869B (en) * | 2015-01-22 | 2017-07-14 | 江南大学 | A kind of DNA sensor for detecting staphylococcus aureus and its preparation and application |
| CN108020587B (en) * | 2017-11-30 | 2020-08-28 | 江苏大学 | Method for detecting staphylococcus aureus in milk with double signal amplification effects |
| CN111638326B (en) * | 2020-05-20 | 2023-05-09 | 江苏大学 | Enzyme-free polymer immune probe and preparation method and application thereof |
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