CN102565161A - Two-dimensional molecular engram array type quality sensing detection method for staphylococcus enterotoxin - Google Patents
Two-dimensional molecular engram array type quality sensing detection method for staphylococcus enterotoxin Download PDFInfo
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Abstract
The invention discloses a two-dimensional molecular engram array type quality sensing detection method for staphylococcus enterotoxin, which comprises the following steps: (1) performing surface active treatment on a quartz oscillating electrode; (2) preparing an initial sol-gel; (3) preparing an engram sol-gel of a template containing SEA (staphylococcus enterotoxin A) and SEB (staphylococcus enterotoxin B); (4) performing coating treatment on the surface of the quartz oscillating electrode; (5) removing protein molecules SEA and SEB of the template; and (6) constructing a molecular engram array quality sensor for the SEA and SEB detection. The conventional SEA and SEB macromolecular proteins in the staphylococcus enterotoxin which is the important virulence factor of sitotoxismus are quickly detected. The method is simple; no mark is required; the detection speed is high; the external interference is small; the cost is low; the result is stable and reliable; the time of the whole detecting process is short; and the method has a wide application prospect in the fields of food safety, hygiene detection, environment monitoring, clinical examination, biological terror monitoring, and the like.
Description
Technical field
The present invention relates to the detection method of Staphylococcus aureus enterotoxin in the food, particularly relate to the detection method of SEA (SEA) and B (SEB).
Background technology
Staphylococcus aureus is common food poisoning pathogenic bacteria, can cause the multiple disease of humans and animals, and its pathogenic enterotoxin main and its secretion is closely related.Staphylococcal enterotoxin (SEs) is one group of high heat-proof low-molecular-weight soluble albumen, and wherein SEB is the important virulence factor of food poisoning, and is classified as one of main toxin of biological warfare agent.SEB albumen is to thermally-stabilised, and the least concentration that causes food poisoning is a 200ng/100g food, eat by mistake by the people of its contaminated food products behind 4～6h, to cause nausea, vomit and suffer from diarrhoea, therefore, and the detection SEB and other multiple SEs of research rapid sensitive, like SEA, SEC
1, SEC
2Deng method seem particularly important, desirable food inspection means should reach the ng/mL level to the detection of SEB, and sample does not need too much processing.
At present, the method for the detection of SEs has single, double to agar diffusion method, reverse indirect blood coagulation (RPHA), solid-phase radioimmunoassay (RIA) and enzyme linked immunosorbent assay immunological methods such as (ELISA).The required Antibody Preparation technology of serological method is numerous and diverse, and the cycle is long, tires to receive the influence of numerous biodynes.Though RIA, ELISA and RPHA detection sensitivity can reach the ng level, its requirement for experiment condition is harsh, drawbacks limit such as false positive appearance has been arranged in the reaction its range of application.The gene probe detection method that grows up again in recent years because half life period of labelled with radioisotope probe is short and drawbacks limit such as radiocontamination should technology apply.Along with science and technology development; Molecular imprinting (molecular imprinting technique has appearred; MIT); This technology is claimed molecular brand (molecular imprinting) again, belongs to host-guest chemistry category in the supramolecular chemistry, is an interleaving techniques that comes from subjects such as high polymer chemistry, biological chemistry, material science.This technology has the process of the polymkeric substance of specific selectivity;, template molecule can form the multiple action point with monomer as much as possible when contacting with polymer monomer; If through polymerization; These multiple action points fixing or " freezing " get off, after template molecule is removed, just formed the hole that on space and binding site, is complementary with template molecule in the polymkeric substance with multiple action point.Molecularly imprinted polymer (the molecularly imprinted polymers that obtains; MIPs) have the function of simulating biological antibody, be called " artificial antibody " by image become one of hot subject of current research; Its preparation process is simple; With low cost, physicochemical property are stable, and have the precordainment of effect, specific recognition property and extensive practicality.But traditional imprinted polymer is three-dimensional block; General density is bigger; Macromolecular template is difficult to reach and leave binding site; Bad mass transfer and the reservation of permanent template have a negative impact to recognition performance, because the organic solvent of using always in conventional non-physiologic trace condition and the experiment all can make albuminous degeneration, make three-dimensional engram technology also be not suitable for discerning macro-molecular protein again.
Two dimension protein molecular trace only makes the surface of template protein molecular trace at polymkeric substance; Imprinted sites also is in the surface of film; This can overcome sterically hindered influence, is convenient to remove template and protein molecular near recognition site, and the solvent great majority that use are mild and water-soluble solvent; And the damping fluid of using always in the Bioexperiment, can make the target proteins quality guarantee hold its physiologically active and normal space structure.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of two-dimentional molecular engram array mass sensitivity detection method of staphylococcal enterotoxin is provided.
Technical scheme of the present invention is summarized as follows:
A kind of two-dimentional molecular engram array mass sensitivity detection method of staphylococcal enterotoxin comprises the steps:
(1) the quartz crystal oscillator electrode active surface is handled; The quartz crystal oscillator electrode of three surface gold-plating is immersed in the ethanolic solution of 11-sulfydryl alkyd of 0.05-0.2M, soak 8-16h, take out, with the absolute ethyl alcohol flushing, nitrogen dries up, and is labeled as A, B and C electrode respectively;
(2) preparation of initial collosol and gel:
With 2～3mL tetraethyl orthosilicate, 100～200 μ l phenyltrimethoxysila,e, 100～200 μ l MTMSs, 400～600 μ l concentration are the HCl WS of 0.05-0.2M and the H of 2～3ml
2O mixes, and is prepared into initial collosol and gel in ultrasonic 20～30 minutes;
(3) contain the preparation of the trace collosol and gel of SEA or SEB template:
Get the initial collosol and gel of 2～4mL and 100～200 μ l concentration are the SEA phosphate buffer of 0.1～0.3mg/mL, mix, ultrasonic 20-40 minute, obtain to contain the trace collosol and gel of SEA template;
Get the initial collosol and gel of 2～4mL and 100～200 μ l concentration are the SEB phosphate buffer of 0.1～0.3mg/mL, mix, ultrasonic 20-40 minute, obtain to contain the trace collosol and gel of SEB template;
(4) the quartz crystal oscillator electrode surface is carried out coated:
1. with 1000～3000 rev/mins rotating speed the trace collosol and gel of the said SEA of containing template is evenly coated said A electrode surface 20～30 seconds; With 1000～3000 rev/mins rotating speeds the trace collosol and gel of the said SEB of containing template is evenly coated said B electrode surface 20～30 seconds; Left standstill 10～15 minutes; Water rinses out the said gel of surface physics absorption, dries up with nitrogen;
2. repeating step is 1. 3-5 time; Obtaining surface combination has the quartz crystal oscillator electrode of molecular imprinted polymer membrane, dry under the room temperature;
3. the initial collosol and gel that step (2) is obtained was evenly coated the C electrode surface 20～30 seconds with 1000～3000 rev/mins rotating speeds, left standstill 10～15 minutes, and water rinses out the said gel of surface physics absorption, dries up with nitrogen;
4. repeating step is 3. 3-5 time; Dry under the room temperature;
(5) remove template protein molecular SEA and SEB:
With 40～50 ℃ distilled waters the electrode that step in the step (4) 2. obtains is washed, remove template albumen, dry up, obtain being coated with the quartz crystal oscillator electrode A and the B of molecular engram film, and place under the room temperature subsequent use after the drying with nitrogen;
With 40～50 ℃ distilled waters the C electrode that step in the step (4) 4. obtains is washed, dry up, obtain contrast electrode, and place under the room temperature subsequent use after the drying with nitrogen;
(6) structure of the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin and liquid to be measured detect:
The quartz crystal oscillator electrode A that is coated with molecular engram film and B and contrast electrode C are placed three addition pools respectively; And be connected on the quartz crystal micro-array sensors E4; Construct the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin; In each addition pool, add 200 μ l PBS; Treat after its frequency stabilization respectively in the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film, B, to add 200 μ l liquid to be measured respectively, detect change of frequency, detect staphylococcal enterotoxin SEA or SEB in the liquid to be measured until stable;
Said liquid to be measured be formulated as a kind of of following two kinds of methods:
Method one: directly get fluid sample and be liquid to be measured or to be 1: 0.5～1.5 ratio by volume mix the back for liquid to be measured or be that 1: 0.5～1.5 ratio is mixed the back with fluid sample and filtered with filter with PBS by volume with fluid sample with PBS, getting filtrates is liquid to be measured;
Method two: the solid sample after will pulverizing in the ratio of 1g: 0.5～1.5ml be mixed into liquid to be measured with PBS or will pulverize in the ratio of 1g: 0.5～1.5ml after solid sample mix with PBS, filter with filter, getting filtrating and being liquid to be measured.
Advantage of the present invention: method of the present invention is carried out fast detecting to common SEA and SEB high molecular weight protein in the important virulence factor staphylococcal enterotoxin of sitotoxismus.It is numerous and diverse to have abandoned conventional Staphylococcus aureus enterotoxin serology and immunological method Antibody Preparation technology, and the deficiency that the cycle is long, experimental procedure is loaded down with trivial details, cost is high, preparation method simply, do not need any mark; Detect fast; Receive external interference less, with low cost, the result obtains reliable and stable; Whole testing process was accomplished in 30 minutes, was with a wide range of applications in fields such as food security, health detection, environmental monitoring, clinical examination, bio-terrorism monitorings.
The present invention is a transducer with monolithic array formula mass sensitivity device; Synthesize the two-dimentional molecularly imprinted polymer of SEA and SEB molecule on the quartz crystal oscillator surface through organic-silylation self assembly sol-gal process original position, and wash template off, the two-dimentional molecular engram array mass sensor that the multiple staphylococcal enterotoxin of structure detects; Replace biological antibody as recognition component; Through molecular recognition, the testing molecule in the film adsorption sample, and then make the quality of MIPs change; Cause that the quartz crystal oscillator vibration frequency changes, and detects target to be measured in the sample with this.
Description of drawings
Fig. 1 is the time dependent curve of quartz crystal oscillator frequency that is coated with SEA, SEB molecular engram film and non-blotting membrane.
Fig. 2 for the SEA of variable concentrations in the PBS damping fluid and SEB to being coated with concentration-frequency that quartz crystal oscillator produced (Hz) change curve (n=3) of SEA, SEB molecular engram film and non-molecular engram film.
Fig. 3 measures for the specificity of the two-dimentional molecular engram array mass sensor that staphylococcal enterotoxin detects
(a) SEB, the SEC of adding 10 μ g/ml in the SEA reaction tank
1, BSA, OVA and PBS quartz crystal oscillator frequency (Hz) change; (b) SEB, the SEC of adding 100 μ g/ml in the SEB reaction tank
1, BSA, OVA and PBS quartz crystal oscillator frequency (Hz) change.
Embodiment
Below in conjunction with specific embodiment the present invention is further described.
Experiment material
The sulfydryl undecanoic acid
TEOS(Tetraethyl?orthosilicate)
PTMOS(phenyltrimethoxysilane)
MTMOS(methyl?trimethoxysilane)
Absolute ethyl alcohol
Hydrochloric acid
SEA
SEB
It is following to test used instrument
Dissipative type quartz crystal micro-array sensors (E4, Q-Sense)
Embodiment 1
The two-dimentional molecular engram array mass sensitivity detection method of multiple Staphylococcus aureus enterotoxin comprises the steps: in a kind of food
(1) the quartz crystal oscillator electrode active surface is handled; The quartz crystal oscillator electrode of three surface gold-plating is immersed in the ethanolic solution of 11-sulfydryl alkyd of 0.1M, soak 12h, take out, with the absolute ethyl alcohol flushing, nitrogen dries up, and is labeled as A, B and C electrode respectively;
(2) preparation of initial collosol and gel:
With the 2.5mL tetraethyl orthosilicate, 150 μ l phenyltrimethoxysila,e, 150 μ l MTMSs, 500 μ l concentration are the HCl WS of 0.1M and the H of 2mL
2O mixes, and is prepared into initial collosol and gel in ultrasonic 25 minutes;
(3) contain the preparation of trace collosol and gel of the template of SEA or SEB:
Get the initial collosol and gel of 3mL and 150 μ l concentration are the SEA phosphate buffer of 0.1mg/mL, mix, ultrasonic 30 minutes, obtain to contain the trace collosol and gel of SEA template;
Get the initial collosol and gel of 3mL and 150 μ l concentration are the SEB phosphate buffer of 0.1mg/mL, mix, ultrasonic 30 minutes, obtain to contain the trace collosol and gel of SEB template;
(4) the quartz crystal oscillator electrode surface is carried out coated:
1. with 2000 rev/mins rotating speed the trace collosol and gel of the said SEA of containing template is evenly coated said A electrode surface 25s; With 2000 rev/mins rotating speeds the trace collosol and gel of the said SEB of containing template is evenly coated said B electrode surface 25s; Left standstill after the coating 13 minutes; Water rinses out the said gel of surface physics absorption, dries up with nitrogen;
2. repeating step is 1. 4 times; Obtaining surface combination has the quartz crystal oscillator electrode of molecular imprinted polymer membrane, and drying is 24 hours under the room temperature;
3. the initial collosol and gel that step (2) is obtained is evenly coated C electrode surface 25s with 2000 rev/mins rotating speeds, leaves standstill 13 minutes, and water rinses out the said gel of surface physics absorption, dries up with nitrogen;
4. repeating step is 3. 4 times; Drying is 24 hours under the room temperature;
(5) remove template protein molecular SEA and SEB:
With 45 ℃ distilled waters step in the step (4) is 2. obtained electrode and wash, remove template albumen, dry up, obtain being coated with the quartz crystal oscillator electrode A and the B of molecular engram film, and place under the room temperature subsequent use behind the dry 24h with nitrogen;
With 45 ℃ distilled waters the C electrode that step in the step (4) 4. obtains is washed, dry up, obtain contrast electrode, and place under the room temperature subsequent use behind the dry 24h with nitrogen;
(6) structure of the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin and liquid to be measured detect:
The quartz crystal oscillator electrode A that is coated with molecular engram film and B and contrast electrode C are placed three addition pools respectively; And be connected on the quartz crystal micro-array sensors E4; Construct the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin; In each addition pool, add 200 μ l PBS; Treat after its frequency stabilization respectively in the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film, B, to add 200 μ l liquid to be measured respectively, detect change of frequency, detect staphylococcal enterotoxin SEA or SEB in the liquid to be measured until stable;
The preparation of liquid to be measured:
The ratio that is 1: 1 is by volume mixed milk with PBS.
By 10 quartz crystal oscillator electrode A and 10 quartz crystal oscillator electrode B that are coated with molecular engram film that are coated with molecular engram film of above-mentioned same method preparation.
Embodiment 2
The two-dimentional molecular engram array mass sensitivity detection method of multiple Staphylococcus aureus enterotoxin comprises the steps: in a kind of food
(1) the quartz crystal oscillator electrode active surface is handled; The quartz crystal oscillator electrode of three surface gold-plating is immersed in the ethanolic solution of 11-sulfydryl alkyd of 0.05M, soak 16h, take out, with the absolute ethyl alcohol flushing, nitrogen dries up, and is labeled as A, B and C electrode respectively;
(2) preparation of initial collosol and gel:
With the 2mL tetraethyl orthosilicate, 200 μ l phenyltrimethoxysila,e, 100 μ l MTMSs, 600 μ l concentration are the HCl WS of 0.05M and the H of 3mL
2O mixes, and is prepared into initial collosol and gel in ultrasonic 20 minutes;
(3) contain the preparation of trace collosol and gel of the template of SEA or SEB:
Get the initial collosol and gel of 2mL and 100 μ l concentration are the SEA phosphate buffer of 0.2mg/mL, mix, ultrasonic 20 minutes, obtain to contain the trace collosol and gel of SEA template; Get the initial collosol and gel of 2mL and 100 μ l concentration are the phosphate buffer of the SEB of 0.2mg/mL, mix, ultrasonic 20 minutes, obtain to contain the trace collosol and gel of SEB template;
(4) the quartz crystal oscillator electrode surface is carried out coated:
1. with 1000 rev/mins rotating speed the trace collosol and gel of the said SEA of containing template is evenly coated said A electrode surface 20 seconds; With 1000 rev/mins rotating speeds the trace collosol and gel of the said SEB of containing template is evenly coated said B electrode surface 20 seconds; Left standstill after the coating 15 minutes; Water rinses out the said gel of surface physics absorption, dries up with nitrogen;
20 seconds 2. repeating steps 1. 3 times; Obtaining surface combination has the quartz crystal oscillator electrode of two-dimentional molecular imprinted polymer membrane, and drying is 24 hours under the room temperature;
3. the initial collosol and gel that step (2) is obtained was evenly coated the C electrode surface 20 seconds with 1000 rev/mins rotating speeds, left standstill 15 minutes, and water rinses out the said gel of surface physics absorption, dries up with nitrogen;
4. repeating step is 3. 3 times; Drying is 24 hours under the room temperature;
(5) remove template protein molecular SEA and SEB
With 40 ℃ distilled waters the electrode that step in the step (4) 2. obtains is washed, remove template albumen, dry up, obtain being coated with the quartz crystal oscillator electrode A and the B of molecular engram film, and place under the room temperature subsequent use behind the dry 24h with nitrogen; With 40 ℃ distilled waters the C electrode that step in the step (4) 4. obtains is washed, dry up, obtain contrast electrode C, and place under the room temperature subsequent use behind the dry 24h with nitrogen;
(6) structure of the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin and liquid to be measured detect
Quartz crystal oscillator electrode A that is coated with molecular engram film and B contrast electrode C are placed three addition pools respectively; And be connected on the quartz crystal micro-array sensors E4; Construct the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin; In each addition pool, add 200 μ l PBS; Treat after its frequency stabilization respectively in the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film, B, to add 200 μ l liquid to be measured respectively, detect change of frequency, detect staphylococcal enterotoxin SEA or SEB in the liquid to be measured until stable;
The preparation of liquid to be measured:
The ratio that is 1: 0.5 is by volume mixed leftovers soup with PBS, filter with filter, gets filtrating and is liquid to be measured.
(present embodiment also can be that 1: 05 ratio is mixed leftovers soup with PBS by volume, filters with filter, gets filtrating and is liquid to be measured.)
Embodiment 3
The two-dimentional molecular engram array mass sensitivity detection method of multiple Staphylococcus aureus enterotoxin comprises the steps: in a kind of food
(1) the quartz crystal oscillator electrode active surface is handled; The quartz crystal oscillator electrode of three surface gold-plating is immersed in the ethanolic solution of 11-sulfydryl alkyd of 0.2M, soak 8h, take out, with the absolute ethyl alcohol flushing, nitrogen dries up, and is labeled as A, B and C electrode respectively;
(2) preparation of initial collosol and gel:
With the 3mL tetraethyl orthosilicate, 100 μ l phenyltrimethoxysila,e, 200 μ l MTMSs, 400 μ l concentration are the HCl WS of 0.2M and the H of 2ml
2O mixes, and is prepared into initial collosol and gel in ultrasonic 30 minutes;
(3) contain the preparation of the trace collosol and gel of SEA or SEB template:
Get the initial collosol and gel of 4mL and 200 μ l concentration are the SEA phosphate buffer of 0.3mg/mL, mix, ultrasonic 40 minutes, obtain to contain the trace collosol and gel of SEA template; Get the initial collosol and gel of 4mL and 200 μ l concentration are the phosphate buffer of the SEB of 0.3mg/mL, mix, ultrasonic 40 minutes, obtain to contain the trace collosol and gel of SEB template;
(4) the quartz crystal oscillator electrode surface is carried out coated:
1. with 3000 rev/mins rotating speed the trace collosol and gel of the said SEA of containing template is evenly coated said A electrode surface 30 seconds; With 3000 rev/mins rotating speeds the trace collosol and gel of the said SEB of containing template is evenly coated said B electrode surface 30 seconds; After the coating; Left standstill 10 minutes, water rinses out the said gel of surface physics absorption, dries up with nitrogen;
2. repeating step is 1. 5 times; Obtaining surface combination has the quartz crystal oscillator electrode of molecular imprinted polymer membrane, and drying is 24 hours under the room temperature;
3. the initial collosol and gel that step (2) is obtained was evenly coated the C electrode surface 30 seconds with 1000～3000 rev/mins rotating speeds, left standstill 10 minutes, and water rinses out the said gel of surface physics absorption, dries up with nitrogen;
4. repeating step is 3. 5 times; Drying is 24 hours under the room temperature;
(5) remove template protein molecular SEA and SEB
With 50 ℃ distilled waters the electrode that step in the step (4) 2. obtains is washed, remove template albumen, dry up, obtain being coated with the quartz crystal oscillator electrode A and the B of molecular engram film, and place under the room temperature subsequent use behind the dry 24h with nitrogen;
With 50 ℃ distilled waters the C electrode that step in the step (4) 4. obtains is washed, dry up, obtain contrast electrode, and place under the room temperature subsequent use behind the dry 24h with nitrogen;
(6) structure of the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin and liquid to be measured detect
The quartz crystal oscillator electrode A that is coated with molecular engram film and B and contrast electrode C are placed three addition pools respectively; And be connected on the quartz crystal micro-array sensors E4; Construct the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin; In each addition pool, add 200 μ l PBS; Treat after its frequency stabilization respectively in the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film, B, to add 200 μ l liquid to be measured respectively, detect change of frequency, detect in the liquid to be measured two kinds of staphylococcal enterotoxin SEA or SEB until stable;
The preparation of liquid to be measured:
Sauce system food is ground, and again in 1g: the jam product sample after the ratio of 1ml will be pulverized mixes with PBS with the filter filtration, getting filtrating and being liquid to be measured.
(also can again in 1g: the jam product sample after the ratio of the ratio of 0.5ml or 1g: 1.5ml will be pulverized mixes with PBS, the same present embodiment of other step)
Electrode and addition pool all use 40 ℃ deionized water to wash repeatedly before each the mensuration, dry up with nitrogen then.
Embodiment 4
The two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin is measured the repeatability of staphylococcal enterotoxin:
The PBS that 200 μ l is contained 10 μ g/ml SEA injects the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film 10 times repeatedly; The PBS that 200 μ l is contained 10 μ g/ml SEB injects the addition pool that is soaked with the quartz crystal oscillator electrode B that is coated with molecular engram film 10 times repeatedly; Under identical conditions, measure the change of frequency (Δ f) of electrode, batch interior repeatability is detected with this.Next, use 3 quartz crystal oscillator electrode A and 3 quartz crystal oscillator electrode B that are coated with molecular engram film that are coated with molecular engram film of embodiment 1 preparation, under identical conditions, measure Δ f, repeatability between criticizing is detected with this.The result is as shown in table 1.
The two-dimentional molecular engram array mass sensor of table 1 staphylococcal enterotoxin detect staphylococcal enterotoxin batch in and batch between repeatability relatively
* compare P＞0.05. with △
Embodiment 5
The two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin detects the staphylococcal enterotoxin specificity and measures;
Choose SEC
1, BSA and OVA be used for the detection of the specificity of sensor.
In the addition pool of the quartz crystal oscillator electrode that is coated with the SEA molecular engram film, adding 200 μ l concentration respectively is the PBS solution of 100 μ g/mLSEB, the 100 μ g/mL SEC of 200 μ l
1PBS solution, the PBS solution of the 100 μ g/mL BSA of 200 μ l, the PBS solution of the 100 μ g/mL OVA of 200 μ l compares with the PBS of 200 μ l; Simultaneously, in the addition pool of the quartz crystal oscillator electrode that is coated with the SEB molecular engram film, add the PBS solution of SEA of the 100 μ g/mL of 200 μ l respectively, the 100 μ g/mL SEC of 200 μ l
1PBS solution, the PBS solution of the 100 μ g/mL BSA of 200 μ l, the PBS solution of the 100 μ g/mL OVA of 200 μ l compares with the PBS of 200 μ l; Visible from Fig. 3 (a), the change of frequency that the quartz crystal oscillator electrode that is coated with the SEA molecular engram film and SEA reaction cause is higher than far away and SEB, SEC
1, the variation that causes of BSA, OVA and PBS reaction; Being coated with the change of frequency that quartz crystal oscillator electrode and the SEB reaction of the molecular engram film of SEB causes among Fig. 3 (b) also is higher than far away and SEA, SEC
1, the variation that causes of BSA, OVA and PBS reaction.
Embodiment 6
The fast detecting of two kinds of staphylococcal enterotoxins in the milk sample
The testing result of two kinds of staphylococcal enterotoxins in table 2 milk sample (
n=3)
aDo not detect
In milk, add the SEA and the SEB of variable concentrations, do not carry out any pre-treatment, testing result is as shown in table 2.The recovery that SEA is detected is 97.00%～104.12%; The recovery that SEB is detected is 93.42%～114.00%.Whole testing process can be controlled in 30 minutes with interior completion.
This method is to common SEA and SEB high molecular weight protein in the important virulence factor staphylococcal enterotoxin of sitotoxismus; At the two-dimentional molecule engram film of quartz crystal oscillator electrode surface through synthetic SEA of organic-silylation self assembly sol-gal process original position and SEB molecule; With monolithic array formula mass sensitivity device is transducer; Make up a kind of high flux, low cost, unmarked two-dimentional molecularly imprinted polymer array mass sensor; Promote the food safety detection technology towards quick, sensitive, special, easy and cheaply direction develop; Can be applicable to fields such as food security, environmental monitoring and bioengineering, have good practical value and wide application prospect.
Claims (1)
1. the two-dimentional molecular engram array mass sensitivity detection method of a staphylococcal enterotoxin is characterized in that comprising the steps:
(1) the quartz crystal oscillator electrode active surface is handled; The quartz crystal oscillator electrode of three surface gold-plating is immersed in the ethanolic solution of 11-sulfydryl alkyd of 0.05-0.2M, soak 8-16h, take out, with the absolute ethyl alcohol flushing, nitrogen dries up, and is labeled as A, B and C electrode respectively;
(2) preparation of initial collosol and gel:
With 2～3mL tetraethyl orthosilicate, 100～200 μ l phenyltrimethoxysila,e, 100～200 μ l MTMSs, 400～600 μ l concentration are the HCl WS of 0.05-0.2M and the H of 2～3ml
2O mixes, and is prepared into initial collosol and gel in ultrasonic 20～30 minutes;
(3) contain the preparation of the trace collosol and gel of SEA or SEB template:
Get the initial collosol and gel of 2～4mL and 100～200 μ l concentration are the SEA phosphate buffer of 0.1～0.3mg/mL, mix, ultrasonic 20-40 minute, obtain to contain the trace collosol and gel of SEA template;
Get the initial collosol and gel of 2～4mL and 100～200 μ l concentration are the phosphate buffer of the SEB of 0.1～0.3mg/mL, mix, ultrasonic 20-40 minute, obtain to contain the trace collosol and gel of SEB template;
(4) the quartz crystal oscillator electrode surface is carried out coated:
1. with 1000～3000 rev/mins rotating speed the trace collosol and gel of the said SEA of containing template is evenly coated said A electrode surface 20～30 seconds; With 1000～3000 rev/mins rotating speeds the trace collosol and gel of the said SEB of containing template is evenly coated said B electrode surface 20～30 seconds; Left standstill 10～15 minutes; Water rinses out the said gel of surface physics absorption, dries up with nitrogen;
2. repeating step is 1. 3-5 time; Obtaining surface combination has the quartz crystal oscillator electrode of molecular imprinted polymer membrane, dry under the room temperature;
3. the initial collosol and gel that step (2) is obtained was evenly coated the C electrode surface 20～30 seconds with 1000～3000 rev/mins rotating speeds, left standstill 10～15 minutes, and water rinses out the said gel of surface physics absorption, dries up with nitrogen;
4. repeating step is 3. 3-5 time; Dry under the room temperature;
(5) remove template protein molecular SEA and SEB:
With 40～50 ℃ distilled waters the electrode that step in the step (4) 2. obtains is washed, remove template albumen, dry up, obtain being coated with the quartz crystal oscillator electrode A and the B of molecular engram film, and place under the room temperature subsequent use after the drying with nitrogen;
With 40～50 ℃ distilled waters the C electrode that step in the step (4) 4. obtains is washed, dry up, obtain contrast electrode, and place under the room temperature subsequent use after the drying with nitrogen;
(6) structure of the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin and liquid to be measured detect:
The quartz crystal oscillator electrode A that is coated with molecular engram film and B and contrast electrode C are placed three addition pools respectively; And be connected on the quartz crystal micro-array sensors E4; Construct the two-dimentional molecular engram array mass sensor of staphylococcal enterotoxin; In each addition pool, add 200 μ l PBS; Treat after its frequency stabilization respectively in the addition pool that is soaked with the quartz crystal oscillator electrode A that is coated with molecular engram film, B, to add 200 μ l liquid to be measured respectively, detect change of frequency, detect staphylococcal enterotoxin SEA or SEB in the liquid to be measured until stable.
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| CN114712305A (en) * | 2022-04-22 | 2022-07-08 | 中南大学 | BSA (bovine serum albumin) gel sterilization material for in-situ loading of dihydromyricetin as well as preparation method and application of BSA gel sterilization material |
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| CN101836107A (en) * | 2007-09-10 | 2010-09-15 | 香港大学 | Electronic tongue sensor |
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| Title |
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| 《Biosensors and Bioelectronics》 20060115 Zhaohui Zhang et al Molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol-gel process for protein recognition 第2.2-2.4节，图3 1 第21卷, 第7期 * |
| WEIYAO ET AL: "Recognition of Staphylococcus enterotoxin via Molecularly imprinted beads", 《JOURNAL OF SEPARATION SCIENCE》, vol. 31, no. 2, 29 February 2008 (2008-02-29), pages 413 - 418 * |
| YANG LUO ET AL: "Rapid and Simultaneous Quantification of 4 Urinary Proteins by Piezoelectric Quartz Crystal Microbalance Immunosensor Array", 《CLINICAL CHEMISTRY》, vol. 52, no. 12, 31 December 2006 (2006-12-31) * |
| ZHAOHUI ZHANG ET AL: "Molecularly imprinted thin film self-assembled on piezoelectric quartz crystal surface by the sol–gel process for protein recognition", 《BIOSENSORS AND BIOELECTRONICS》, vol. 21, no. 7, 15 January 2006 (2006-01-15) * |
| 刘虹 等: "检测 B型葡萄球菌肠毒素的 Love波免疫传感器研究", 《传感器与微系统》, vol. 25, no. 3, 31 March 2006 (2006-03-31), pages 28 - 31 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114712305A (en) * | 2022-04-22 | 2022-07-08 | 中南大学 | BSA (bovine serum albumin) gel sterilization material for in-situ loading of dihydromyricetin as well as preparation method and application of BSA gel sterilization material |
| CN114712305B (en) * | 2022-04-22 | 2023-10-24 | 中南大学 | In-situ dihydromyricetin-loaded BSA gel sterilization material, and preparation method and application thereof |
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