CN105203524A - Method based on aptamer recognition surface enhanced Raman spectroscopy for detecting salmonella in food - Google Patents

Abstract

The invention discloses a method for detecting Salmonella in food based on aptamer recognition surface-enhanced Raman spectroscopy. Using silver-coated gold core-shell nanomaterials as the substrate, thiol-modified Salmonella aptamers were added to the silver-coated gold core-shell nanomaterials prepared above for incubation, thereby immobilizing the Salmonella aptamers on on the base. The analyte was mixed with the silver-coated gold core-shell nanomaterial modified with the Salmonella aptamer, and the Salmonella aptamer modified with a Raman signal ROX was added to incubate, and then the Raman spectrum was scanned. Based on the specific binding of aptamer to Salmonella, the detection of Salmonella in food is realized. The method has high sensitivity, strong specificity and convenient operation, and will have broad application prospects in the field of food safety detection.

Description

A method for the detection of Salmonella in food based on surface-enhanced Raman spectroscopy based on aptamer recognition

technical field

The invention relates to the field of food safety detection, in particular to a method for detecting Salmonella in food based on aptamer recognition surface-enhanced Raman spectroscopy.

Background technique

Salmonella is a Gram-negative pathogenic enterobacteriaceae. Salmonella exists widely in nature, and it is especially easy to pollute water sources, food and animal products, and pose a great hazard to human and animal health. Food poisoning can occur when humans or animals eat food contaminated with Salmonella. The clinical manifestations of acute infectious diseases caused by Salmonella are mainly divided into gastroenteritis type (ie food poisoning), typhoid type, sepsis type and extraintestinal focal infection. Among them, gastroenteritis is the most common type, which can cause clinical symptoms such as nausea, vomiting, abdominal pain, diarrhea and fever. According to statistics, in my country's bacterial food poisoning, 70% to 80% are caused by Salmonella. Among food poisoning around the world, Salmonella food poisoning in the United States and China ranks first. It can be seen that salmonellosis is one of the most important zoonotic diseases in public health. Therefore, it is of great significance to establish an accurate, sensitive and rapid detection technology for Salmonella for food safety.

The traditional microbial testing procedures are cumbersome and time-consuming, and the accuracy and reliability of the test results largely depend on the professional quality and experience of the testers, so this method is not objective, sensitive, and fast enough; the newly developed fluorescent Although real-time quantitative PCR technology can shorten the inspection time, has strong specificity and high sensitivity, it is expensive, easy to pollute, and has high requirements for the operating environment; the commonly used immunological methods today, such as enzyme-linked immunosorbent assay, dot enzyme-linked immunosorbent assay Adsorption, immunomagnetic separation technology, immunofluorescence labeling, etc. have the advantages of strong specificity, high sensitivity, and easy observation, but the time to prepare antibodies is relatively long, the cost is high, and they are unstable. In recent years, because aptamers have many advantages over antibodies: low cost, good stability, easy modification, etc., they have been regarded as promising alternative molecules for antibody molecules and have attracted attention in many fields.

Raman scattering has become a powerful tool for material analysis because it contains rich information on molecular vibrational energy levels, especially the emergence of surface-enhanced Raman scattering (SERS) technology, which has greatly improved its detection sensitivity. In recent years, SERS spectroscopy has been widely used in the analysis and research of physics, materials, surface science, environmental chemistry, biochemistry, organic chemistry and even biology due to its high resolution, high sensitivity, low solution interference, and good stability. .

Contents of the invention

The purpose of the present invention is to provide a method for detecting Salmonella in food based on aptamer recognition surface-enhanced Raman spectroscopy. The present invention uses silver-coated gold core-shell nanomaterials as a substrate, and adds thiol-modified Salmonella aptamers to the above-prepared silver-coated gold core-shell nanomaterials for incubation, so that the Salmonella aptamers fixed on the base. The analyte was mixed with the silver-coated gold core-shell nanomaterial modified with the Salmonella aptamer, and the Salmonella aptamer modified with a Raman signal ROX was added to incubate, and then the Raman spectrum was scanned. Based on the specific binding of aptamer to Salmonella, the detection of Salmonella in food is realized. The specific detection schematic diagram is shown in Figure 1. The method has high sensitivity, strong specificity and convenient operation, and will have broad application prospects in the field of food safety detection.

Realize the concrete method of the present invention:

A method for detecting Salmonella in food based on aptamer recognition surface-enhanced Raman spectroscopy, comprising the following steps:

1) Synthesis of Raman-enhanced substrates: firstly prepare nano-gold by trisodium citrate reduction method, and then add a certain amount of ascorbic acid and silver nitrate to prepare silver-coated gold core-shell nanomaterial substrates.

2) Immobilization of aptamers: adding a certain amount of thiol-modified aptamers to the above-prepared silver-coated gold core-shell nanomaterials for incubation, thereby immobilizing the aptamers on the substrate.

3) Detection of Salmonella: Mix the liquid to be tested with the silver-coated gold core-shell nanomaterial modified with aptamers, add a certain amount of aptamers with Raman signal ROX modification, and then perform Raman spectrum detection .

Specifically: Step 1) The specific operation of the synthesis of the Raman-enhanced substrate is as follows: take 0.5mL of 1% chloroauric acid solution, add it to 49.5mL of ultrapure water, heat and stir until boiling; then quickly add 1.5mL of 1% The trisodium citrate solution was continuously boiled for 15 minutes to synthesize gold nanoparticles. Then take 2 mL of the prepared gold nanoparticles, add 1 mL of ultrapure water, 0.4 mL of L-ascorbic acid (0.1 mol/L), continue stirring for 5 min, add 1.2 mL of silver nitrate (1×10 ^-3 mol/L) dropwise, and continue stirring After 1 hour, a silver-coated gold core-shell nanomaterial was prepared.

The aptamer immobilization operation in step 2) is as follows: take 190 μL of silver-coated gold core-shell nanoparticles, add 10 μL of aptamer (10 μmol/L), and incubate at 4° C. for 12 hours.

For the detection described in step 3), Salmonella samples of different concentrations were incubated with aptamer-modified silver-coated gold core-shell nanomaterials at room temperature for 45 minutes, and then ROX-modified aptamers were added to continue incubation for 45 minutes. Then centrifuge at 3000r/min for 5min and wash twice. Samples were resuspended in buffer and detected by Raman spectrometer.

The advantages of the present invention are:

1. The method of the present invention uses aptamers as recognition elements. Compared with using antibodies as recognition elements in immunoassays, aptamers have good stability, low preparation cost, easy labeling, and do not affect their activity after labeling. The bacteria have high affinity and high selectivity, which greatly improves the accuracy of detection.

2. The present invention uses silver-coated gold core-shell nanocomposites as the Raman-enhanced substrate, which has the spectral characteristics of noble metals, and has the advantages of rapid sample preparation and low cost.

3. Compared with the existing detection method of Salmonella, the detection method provided in the present invention has the characteristics of high sensitivity, and its detection limit can reach 17cfu/mL.

Description of drawings

Figure 1 Schematic diagram of surface-enhanced Raman detection of Salmonella based on aptamer recognition

Figure 2 Transmission electron microscope image (TEM) of silver-coated gold core-shell nanoparticles

The surface-enhanced Raman spectrum (A) caused by different concentrations of Salmonella in Figure 3, the linear relationship between the relative Raman intensity and the concentration of Salmonella (B)

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

Example 1

1) Synthesis of Raman-enhanced substrates

First take 0.5mL of 1% chloroauric acid solution in a 100mL round bottom flask, add 49.5mL of ultrapure water, heat and stir until boiling; then quickly add 1.5mL of 1% trisodium citrate solution After continuing to boil for 15 minutes, the color of the solution turned into wine red, the reaction was stopped, and it was naturally cooled to room temperature. Then take 2 mL of prepared nano gold in a 50 mL round bottom flask, add 1 mL of ultrapure water, 0.4 mL of L-ascorbic acid (0.1 mol/L), and keep stirring at room temperature for 5 min, then add 1.2 mL of silver nitrate (1×10 ^-3 mol/L), and continued to stir for 1 hour, the solution changed from wine red to orange, and the reaction was terminated, and the resulting solution was a Raman-enhanced substrate-silver-coated gold core-shell nanoparticles. Figure 2 is a transmission electron microscope image (TEM) of silver-coated gold core-shell nanoparticles. It can be seen from the figure that the particle size of the nanoparticles is about 20nm, and the thickness of the silver shell is about 4nm.

2) Immobilization of aptamers

Take 190 μL of silver-coated gold core-shell nanoparticles, add 10 μL of aptamer (10 μmol/L), so that the final concentration of aptamer is 500 nmol/L, place in a shaker at 4 °C, 75 r/min, and incubate for 12 h. Centrifuge at 12000r/min for 15min, discard the supernatant, wash twice with PBS buffer, and resuspend in 100 μL PBS buffer for later use.

3) Detection of Salmonella in buffer

Obtain the Salmonella bacterial solution with a concentration of 1.7×10 ⁷ cfu/mL by plate counting method, and then dilute the bacterial solution to 1.7×10 ⁶ cfu/mL, 1.7×10 ⁵ cfu/mL, and 1.7×10 ⁴ cfu/mL , 1.7×10 ³ cfu/mL, 1.7×10 ² cfu/mL; use the aptamer-modified silver-coated gold composite material synthesized in step 2) as the Raman enhancing reagent and capture probe, and take 175 μL of the composite Mix with 10 μL of the sample to be tested and incubate at 37°C for 45min. Then 15 μL (10 μmol/L) of aptamers modified with ROX signal molecules were added, and incubated at 37° C. for 45 minutes. Then centrifuge at 3000r/min for 5min and wash twice. Samples were resuspended and detected by Raman spectrometer in buffer. Fig. 3A shows the Raman spectra caused by Salmonella in the concentration range of 17-1.7×10 ⁵ cfu/mL. It can be seen from the figure that as the concentration of Salmonella increases, the Raman intensity also increases correspondingly. With 1628cm ^-1 as the quantitative characteristic peak, Fig. 3B shows the linear curve of Salmonella. Within the concentration range of 17～1.7×10 ⁵ cfu/mL, Salmonella has a good linear relationship with the relative Raman intensity at 1628cm ^-1 , the linear equation is y=580.29x-737.59 (R=0.9925), and the minimum detection limit is 17cfu/mL.

Example 2

1) Synthesis of Raman-enhanced substrates

First take 0.5mL of 1% chloroauric acid solution in a 100mL round bottom flask, add 49.5mL of ultrapure water, heat and stir until boiling; then quickly add 1.5mL of 1% trisodium citrate solution After continuing to boil for 15 minutes, the color of the solution turned into wine red, the reaction was stopped, and it was naturally cooled to room temperature. Then take 2 mL of prepared nano gold in a 50 mL round bottom flask, add 1 mL of ultrapure water, 0.4 mL of L-ascorbic acid (0.1 mol/L), and keep stirring at room temperature for 5 min, then add 1.2 mL of silver nitrate (1×10 ^-3 mol/L), and continued to stir for 1 hour, the solution changed from wine red to orange, and the reaction was terminated, and the resulting solution was a Raman-enhanced substrate-silver-coated gold core-shell nanoparticles. Figure 1 is a transmission electron microscope image (TEM) of silver-coated gold core-shell nanoparticles. It can be seen from the figure that the particle size of the nanoparticles is about 20nm, and the thickness of the silver is about 4nm.

2) Immobilization of aptamers

Take 190 μL of silver-coated gold core-shell nanoparticles, add 10 μL of aptamer (10 μmol/L), so that the final concentration of aptamer is 500 nmol/L, place in a shaker at 4 °C, 75 r/min, and incubate for 12 h. Centrifuge at 12000r/min for 15min, discard the supernatant, wash twice with PBS buffer, and resuspend in 100 μL PBS buffer.

3) Detection of Salmonella in milk

Milk was used as the actual sample; 5 mL of milk was taken and centrifuged at 7000 r/min for 10 min at 10°C to completely remove the upper fat layer. The obtained sample was diluted 1:20 with deionized water, and then filtered through a disposable syringe filter to finally obtain the test solution, and different concentrations of Salmonella were prepared and added to the test solution. Detect with the method of the present invention, and calculate recovery rate, the result is shown in Table 1.

Table 1 The inventive method detects the result of Salmonella in milk

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the 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.