CN105203524A - Method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food - Google Patents
Method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food Download PDFInfo
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- CN105203524A CN105203524A CN201510632911.0A CN201510632911A CN105203524A CN 105203524 A CN105203524 A CN 105203524A CN 201510632911 A CN201510632911 A CN 201510632911A CN 105203524 A CN105203524 A CN 105203524A
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Abstract
The invention discloses a method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food. The method includes: adopting a silver-coated gold core-shell nano material as a substrate, and adding a sulfhydrylation modified salmonella aptamer into the silver-coated gold core-shell nano material for incubation so as to fix the salmonella aptamer on the substrate; mixing a detected object with the silver-coated gold core-shell nano material modified with the salmonella aptamer, adding a salmonella aptamer with Raman signal ROX modification for incubation, and then performing Raman spectroscopy scanning; realizing detection of salmonella in food on the basis of specific combining of the aptamer and salmonella. The method is high in sensitivity and specificity and convenient to operate and has wide application prospect in the field of food safety detection.
Description
Technical field
The present invention relates to field of detection of food safety, be specifically related to a kind of method strengthening Raman spectrum detection Salmonella in Food based on aptamers identified surface.
Background technology
Salmonella belongs to a kind of enteric bacilli pathogenic bacteria, Gram-negative.Salmonella is widespread in nature, especially easily polluted source, food and livestock products, and the health for human and animal all forms high risks.Human or animal has eaten and namely can have been caused food poisoning by salmonella-polluted food.Salmonellal acute infectious disease, clinical manifestation is mainly divided into the outer focal infection of gastroenteritis type (i. e. is poisoning), typhoid fever type, septicemia type and enteron aisle.Wherein common with gastroenteritis type, can cause nausea, vomit, suffer from abdominal pain, suffer from diarrhoea and the clinical signs group such as heating.According to statistics, in China's food posioning, 70% ~ 80% is by salmonellal, and in food poisoning all over the world, the U.S., Chinese salmonella food poisoning rank first.Salmonellosis is one of zoonosis main on public hygienics as can be seen here.Therefore set up accurate, sensitive, Salmeterol fluticasone propionate technology is significant for food security fast.
Traditional Micro biological Tests program very complicated, time and effort consuming, the accuracy of assay and reliability depend on specialized capability and the experience of tester to a great extent, and institute is objective not, sensitive, quick in this way; The fluorescence real-time quantitative PCR technology that new development is got up, although can proving time be shortened, high specificity, highly sensitive, somewhat expensive, easily to pollute, require high to operating environment; Immunological method conventional now, such as enzyme-linked immunosorbent assay, dot enzyme-linked immuno absorption, immunomagnetic isolation technology, immunofluorescence label etc., have high specificity, highly sensitive, be easy to the advantages such as observation, but the time of Dispersal risk is long, and cost is high, and unstable.In the last few years, because aptamers (aptamer) has plurality of advantages than antibody: cost is low, good stability, be easy to modify etc., therefore by the promising alternative molecule as antibody molecule, receive the concern in a lot of field.
Raman scattering, owing to containing the abundant information of molecule vibrational-rotational level, has become the appearance of the powerful of species analysis, particularly Surface enhanced raman spectroscopy (SERS) technology, makes its detection sensitivity obtain great raising.In recent years, the feature such as SERS spectrum, good stability little with its high resolving power, high sensitivity, solution interference is widely used in the even biological analysis of physics, material, Surface Science, environmental chemistry, biological chemistry, organic chemistry and research.
Summary of the invention
The object of the invention is that providing a kind of strengthens based on aptamers identified surface the method that Raman spectrum detects Salmonella in Food.The present invention adopts the core-shell type nano material of silver-colored coated gold as substrate, the salmonella aptamers that sulfhydrylation is modified is joined and hatches above-mentioned preparation in the core-shell type nano material of the coated gold of silver of gained, thus salmonella aptamers be fixed in substrate.The core-shell type nano material of measured object with the coated gold of the silver being modified with salmonella aptamers is mixed, adds the salmonella aptamers with Raman signal ROX modification and hatch, then carry out Raman spectroscopy scans.Based on the specific binding of aptamers and salmonella, realize the detection to Salmonella in Food.Concrete detection schematic diagram as shown in Figure 1.This method is highly sensitive, high specificity, easy to operate, will have broad application prospects in field of detection of food safety.
Realize concrete grammar of the present invention:
Strengthen based on aptamers identified surface the method that Raman spectrum detects Salmonella in Food, comprise the following steps:
1) Raman strengthens the synthesis of substrate: adopt trisodium citrate reduction method first to prepare nm of gold, then add a certain amount of ascorbic acid and silver nitrate, prepare the core-shell type nano material substrate of silver-colored coated gold.
2) immobilization of aptamers: the aptamers that a certain amount of sulfhydrylation is modified is joined and hatches above-mentioned preparation in the core-shell type nano material of the coated gold of silver of gained, thus aptamers is fixed in substrate.
3) detection of salmonella: mixed by the core-shell type nano material of liquid to be measured with the coated gold of the silver being modified with aptamers, adds a certain amount of aptamers having Raman signal ROX and modify, then carries out Raman spectrum detection.
Concrete: step 1) the synthesis concrete operations that strengthen substrate of described Raman are get the chlorauric acid solution of 0.5mL1%, join in 49.5mL ultrapure water, heats and stir, to seething with excitement; Then add rapidly the citric acid three sodium solution of 1.5mL1%, continue to boil 15min, synthesis of nano gold grain.Then get the nm of gold 2mL prepared, add 1mL ultrapure water, 0.4mLL-ascorbic acid (0.1mol/L), Keep agitation 5min, dropwise adds 1.2mL silver nitrate (1 × 10
-3mol/L), Keep agitation 1 hour, prepares the core-shell type nano material of silver-colored coated gold.
Step 2) described in aptamers immobilization be operating as, get silver-colored coated gold core-shell type nano particle 190 μ L, add 10 μ L aptamers (10 μm of ol/L), hatch 12 hours for 4 DEG C.
Step 3) described in detection, the coated gold core-shell type nano material of silver that the salmonella sample of variable concentrations and aptamers are modified in incubated at room temperature 45min, then add ROX modify aptamers continue to hatch 45min.Afterwards in the centrifugal 5min of 3000r/min, and clean twice.Sample is resuspended in upper Raman spectrometer in damping fluid and detects.
The invention has the advantages that:
1. the inventive method is using aptamers as recognition component, compared to using antibody as recognition component in immunoassay, aptamers good stability, preparation cost is low, be easy to mark and after mark, do not affect its activity, to target thalline, there is high affinity and high selectivity simultaneously, improve the accuracy of detection to a great extent.
2. the present invention strengthens substrate using the core-shell nano-composite material of the coated gold of silver as Raman, has the spectral characteristic of noble metal, and sample preparation fast, low cost and other advantages.
3. the detection method provided in the present invention, compared with the detection method of existing salmonella, has highly sensitive feature, and its detectability can reach 17cfu/mL.
Accompanying drawing explanation
Fig. 1 strengthens the schematic diagram of Raman detection salmonella based on aptamers identified surface
The transmission electron microscope figure (TEM) of the coated gold core-shell type nano particle of Fig. 2 silver
The salmonellal Surface enhanced raman spectroscopy figure (A) of Fig. 3 variable concentrations, the linear relationship chart (B) of relative raman scattering intensity and salmonella concentration
Embodiment
More being convenient to make content of the present invention understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention being not limited only to this.
Embodiment 1
1) Raman strengthens the synthesis of substrate
First the chlorauric acid solution getting 0.5mL massfraction 1%, in 100mL round-bottomed flask, adds ultrapure water 49.5mL, heats and stirs, to boiling; Then add rapidly the citric acid three sodium solution that 1.5mL massfraction is 1%, after continuing to boil 15min, solution colour becomes claret, stops reaction, naturally cools to room temperature.Then get the nm of gold 2mL for preparing in 50mL round-bottomed flask, add 1mL ultrapure water, 0.4mLL-ascorbic acid (0.1mol/L), room temperature with constant stirs 5min, then dropwise adds 1.2mL silver nitrate (1 × 10
-3mol/L), Keep agitation 1 hour, solution is become orange-yellow from claret, cessation reaction, and gained solution is that Raman strengthens the coated gold core-shell type nano particle of substrate-Yin.Fig. 2 is the transmission electron microscope figure (TEM) of silver-colored coated gold core-shell type nano particle, and as can be seen from the figure, the particle diameter of nano particle is about 20nm, and the thickness of silver-colored shell is about 4nm.
2) immobilization of aptamers
Get silver-colored coated gold core-shell type nano particle 190 μ L, add 10 μ L aptamers (10 μm of ol/L), make aptamers ultimate density be 500nmol/L, be placed in 4 DEG C of shaking tables, 75r/min, hatches 12h.With the centrifugal 15min of 12000r/min, abandon supernatant, and by PBS buffer solution for cleaning 2 times, be resuspended in 100 μ LPBS damping fluids for subsequent use.
3) detection of salmonella in damping fluid
Concentration is obtained for 1.7 × 10 with colony counting method
7the salmonella bacterium liquid of cfu/mL, then by these bacterium liquid gradient dilution to 1.7 × 10
6cfu/mL, 1.7 × 10
5cfu/mL, 1.7 × 10
4cfu/mL, 1.7 × 10
3cfu/mL, 1.7 × 10
2cfu/mL; Using step 2) compound substance of the coated gold of silver modified of the aptamers of synthesizing is as Raman Contrast agent and capture probe, and the testing sample getting this compound 175 μ L and 10 μ L is mixed in 37 DEG C and hatches 45min.Then add the aptamers that 15 μ L (10 μm of ol/L) are modified with ROX signaling molecule, hatch 45min in 37 DEG C.Afterwards in the centrifugal 5min of 3000r/min, and clean twice.Sample is resuspended to be detected with upper Raman spectrometer in damping fluid.Fig. 3 A is depicted as concentration range 17 ~ 1.7 × 10
5the salmonellal Raman spectrogram of cfu/mL.As seen from the figure, along with the increase of salmonella concentration, raman scattering intensity is also corresponding to be increased.With 1628cm
-1for quantitative characteristic peak, Fig. 3 B is depicted as salmonella linear diagram.Salmonella is 17 ~ 1.7 × 10
5in cfu/mL concentration range, with 1628cm
-1locating relative raman scattering intensity is good linear relationship, and linear equation is y=580.29x-737.59 (R=0.9925), and minimum detectability is 17cfu/mL.
Embodiment 2
1) Raman strengthens the synthesis of substrate
First the chlorauric acid solution getting 0.5mL massfraction 1%, in 100mL round-bottomed flask, adds ultrapure water 49.5mL, heats and stirs, to boiling; Then add rapidly the citric acid three sodium solution that 1.5mL massfraction is 1%, after continuing to boil 15min, solution colour becomes claret, stops reaction, naturally cools to room temperature.Then get the nm of gold 2mL for preparing in 50mL round-bottomed flask, add 1mL ultrapure water, 0.4mLL-ascorbic acid (0.1mol/L), room temperature with constant stirs 5min, then dropwise adds 1.2mL silver nitrate (1 × 10
-3mol/L), Keep agitation 1 hour, solution is become orange-yellow from claret, cessation reaction, and gained solution is that Raman strengthens the coated gold core-shell type nano particle of substrate-Yin.Fig. 1 is the transmission electron microscope figure (TEM) of silver-colored coated gold core-shell type nano particle, and as can be seen from the figure, the particle diameter of nano particle is about 20nm, and the thickness of silver is about 4nm.
2) immobilization of aptamers
Get silver-colored coated gold core-shell type nano particle 190 μ L, add 10 μ L aptamers (10 μm of ol/L), make aptamers ultimate density be 500nmol/L, be placed in 4 DEG C of shaking tables, 75r/min, hatches 12h.With the centrifugal 15min of 12000r/min, abandon supernatant, and by PBS buffer solution for cleaning 2 times, be resuspended in 100 μ LPBS damping fluids.
3) detection of salmonella in milk
Milk is as actual sample; Get 5mL milk, the centrifugal 10min of 7000r/min at 10 DEG C, remove upper-layer fat layer completely.Diluted by the sample deionized water 1:20 obtained, then through disposable injecting type frit, finally obtain solution to be measured, the salmonella of preparation variable concentrations adds in solution to be measured.Detect by the inventive method, and calculate the recovery, result is as shown in table 1.
Table 1 the inventive method detects the result of salmonella in milk
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (4)
1. one kind strengthens based on aptamers identified surface the method that Raman spectrum detects Salmonella in Food.It is characterized in that: adopt the core-shell type nano material of silver-colored coated gold as substrate, the salmonella aptamers that sulfhydrylation is modified is joined and hatches above-mentioned preparation in the core-shell type nano material of the coated gold of silver of gained, thus salmonella aptamers is fixed in substrate.The core-shell type nano material of measured object with the coated gold of the silver being modified with salmonella aptamers is mixed, adds the salmonella aptamers with Raman signal ROX modification and hatch, then carry out Raman spectrum detection.Within the scope of finite concentration, quantity and the Raman signal intensity of salmonella are proportionate, to reach the object quantitatively detected salmonella.
2. a kind ofly as claimed in claim 1 strengthen Raman spectrum based on aptamers identified surface and detect the method for Salmonella in Food, it is characterized in that: adopt trisodium citrate reduction method first to prepare nm of gold, then add a certain amount of ascorbic acid and silver nitrate, prepare the core-shell type nano material substrate of silver-colored coated gold.
3. a kind ofly as claimed in claim 1 strengthen Raman spectrum based on aptamers identified surface and detect the method for Salmonella in Food, it is characterized in that: the salmonella aptamers sequence that sulfhydrylation is modified is 5 '-SH-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3 ', the salmonella aptamers sequence with Raman signal ROX modification is 5 '-ROX-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAGGAAAAGA-3 '.
4. a kind ofly as claimed in claim 1 strengthen Raman spectrum based on aptamers identified surface and detect the method for Salmonella in Food, it is characterized in that: within the scope of finite concentration, quantity and the raman spectral signal intensity of salmonella are proportionate, and contrast 1628cm
-1glow peak signal intensity Criterion curve.
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| CN106052872A (en) * | 2016-06-01 | 2016-10-26 | 江南大学 | Oxytetracycline SERS detection method based on nanomaterial self-assembly |
| CN106124475A (en) * | 2016-06-17 | 2016-11-16 | 江苏大学 | A kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer |
| CN107505306A (en) * | 2017-08-10 | 2017-12-22 | 江南大学 | The method of salmonella in Raman spectrum quick detection milk based on heavy water mark |
| CN110146486A (en) * | 2019-05-31 | 2019-08-20 | 西安工程大学 | A kind of active pathogen in-situ detection method based on Ta@Ag micro-nano interface |
| CN110514829A (en) * | 2019-07-30 | 2019-11-29 | 华东理工大学 | A method of based on signal cascade dual amplification system with highly sensitive and quick detection food-borne pathogens |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN112710649A (en) * | 2021-01-15 | 2021-04-27 | 山东农业大学 | Method for detecting kanamycin sulfate by using dual-signal-enhanced surface-enhanced Raman spectroscopy |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105929150A (en) * | 2016-04-28 | 2016-09-07 | 江南大学 | Gold-sliver core-shell structure Raman enhancement effect-based staphylococcus aureus enterotoxin B immunodetection method |
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| CN106124475A (en) * | 2016-06-17 | 2016-11-16 | 江苏大学 | A kind of trace pesticide residues Raman spectrum method for detecting surface reinforcement based on aptamer |
| CN106124475B (en) * | 2016-06-17 | 2019-01-04 | 江苏大学 | A kind of trace pesticide residue Raman spectrum method for detecting surface reinforcement based on aptamer |
| CN107505306A (en) * | 2017-08-10 | 2017-12-22 | 江南大学 | The method of salmonella in Raman spectrum quick detection milk based on heavy water mark |
| CN110146486A (en) * | 2019-05-31 | 2019-08-20 | 西安工程大学 | A kind of active pathogen in-situ detection method based on Ta@Ag micro-nano interface |
| CN110146486B (en) * | 2019-05-31 | 2021-11-09 | 西安工程大学 | Active pathogenic bacteria in-situ detection method based on Ta @ Ag micro-nano interface |
| CN110514829A (en) * | 2019-07-30 | 2019-11-29 | 华东理工大学 | A method of based on signal cascade dual amplification system with highly sensitive and quick detection food-borne pathogens |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN112710649A (en) * | 2021-01-15 | 2021-04-27 | 山东农业大学 | Method for detecting kanamycin sulfate by using dual-signal-enhanced surface-enhanced Raman spectroscopy |
| CN113008864A (en) * | 2021-03-04 | 2021-06-22 | 天津中医药大学 | Method for detecting food-borne pathogenic bacteria by using surface-enhanced Raman spectrum sensor |
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Application publication date: 20151230 |