Method for rapid broad-spectrum detection of salmonella

Abstract

The invention discloses a method for rapid broad-spectrum detection of salmonella, and relates to the technical field of bacterial detection. Comprising the following steps: (1) preparing a labeled SERS probe; (2) preparing a Fe3O4 (at) Au (at) Ag magnetic composite material; (3) combining Fe3O4 (at) Au (at) AgNPs with Ag (at) 4MBA (at) AuNPs to construct an aptamer sensor; and (4) carrying out SERS detection on the salmonella. According to the novel surface enhanced Raman scattering (SERS) aptamer sensor constructed on the basis of noble metal nanoparticles and a magnetic composite material, according to the inverse proportion relation that the higher the salmonella concentration is, the weaker the Raman signal is generated according to the principle of a competition law, the ultrasensitive rapid field detection is carried out on different salmonella.

Description

Technical Field

[0001] The invention relates to the technical field of bacteria detection, and more particularly to a method for rapid and broad-spectrum detection of Salmonella. Background Art

[0002] Salmonella belongs to the Enterobacteriaceae family, including Salmonella enterica (S.enterica) and Salmonella bongori (S.bongori). It is a large class of non-spore, non-capsule Gram-negative facultative anaerobic bacteria. Salmonella is the main pathogen that causes bacterial foodborne diseases worldwide, and is also a pathogen that is generally controlled by countries and international organizations. It is widely distributed in nature and often lives in humans and animals, especially in the intestines of poultry, livestock and pets. Salmonella is often associated with food poisoning incidents around the world, and the main contaminated foods are: dairy products, meat products, egg products, etc. Salmonella does not decompose protein, and the appearance and taste of food infected by it are difficult to detect, and the infection dose of Salmonella is relatively low. Salmonella is a serious hazard to human health. After surviving the acidic environment of the stomach, the ingested pathogens will enter the small intestine, cross the mucus layer of the small intestine and adhere to the intestinal epithelial cells, inducing a series of intestinal inflammations and even systemic infections, manifested as vomiting, diarrhea, and abdominal pain. In severe cases, it can cause dehydration and shock. Some patients may develop sepsis, which is more common in children and people with low immunity. For people with low immune function, Salmonella infection may even be life-threatening.

[0003] Traditional detection methods such as standard plate count method, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have high accuracy and specificity, but they have some corresponding disadvantages. The plate count method takes a long time to detect, usually requiring several days of culture to obtain the test results, which is time-consuming and labor-intensive, and is not suitable for multi-sample detection and rapid on-site detection; although PCR has high sensitivity, it often produces false negative or false positive identification results. At the same time, PCR can only detect the presence of DNA or RNA and cannot directly reflect the activity of microorganisms; although ELISA is widely used in antigen detection, it has high requirements for experimental conditions (such as temperature and pH), resulting in limited sensitivity in some cases, especially in the detection of lower concentrations, cross-reactions may occur, leading to false positive results, and standard samples are required for quantification, which may be difficult to obtain and preserve. In addition, fluorescent labeling, liposome analysis, chip technology, bioconjugated nanoprobes and other technologies have also been developed, but all of these methods have some disadvantages, such as many pretreatment steps, complex instruments, low sensitivity, the need for skilled personnel, and cumbersome detection procedures.

[0004] Therefore, it is an urgent problem for technicians in this field to provide a new technology that is rapid, highly selective and ultra-sensitive to detect Salmonella pathogens in a timely and accurate manner and prevent foodborne diseases. Summary of the invention

[0005] In view of this, the present invention provides a method for rapid and broad-spectrum detection of Salmonella based on a SERS aptamer biosensor.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for rapid broad-spectrum detection of Salmonella, comprising the following steps:

[0008] (1) Preparation of labeled SERS (surface enhanced Raman scattering) probes

[0009] 1) 17 mg AgNO 3 Dissolve in 100 mL ultrapure water, heat to boiling under vigorous stirring, quickly inject 2 mL 1% trisodium citrate, boil for 40 min, and cool naturally to room temperature to obtain Ag NPs (nanoparticle) solution; the final solution containing silver nanoparticles is yellow-green;

[0010] 2) Take 10 mL of the Ag NPs solution prepared in step 1) and add 200 μL of 0.1 mM 4MBA (4-mercaptobenzoic acid) solution. After vigorous stirring for 5 h, centrifuge the mixture and redisperse the precipitate in 2 mL of ultrapure water to obtain the Ag@MBA NPs solution, which is stored at 4°C for later use;

[0011] 3) Add 222 μL 2% HAuCl 4 solution, 240 μL 0.2M NaOH solution, 3 mL 0.01M Na 2 SO 3 The solution was added into 4.538 mL ultrapure water to obtain Au growth solution, which was stored at 4°C for later use;

[0012] 4) 2 mL of the Ag@4MBA NPs solution prepared in step 2), 2.55 mL of ultrapure water, 1 mL of 5% PVP (polyvinyl pyrrolidone) solution, 200 μL of 0.5 M AA (ascorbic acid) solution, 200 μL of 0.5 M NaOH solution, 50 μL of 0.1 M Na 2 SO 3 The solution and 4 mL of the Au growth solution prepared in step 3) were added to a 25 mL glass vial. After magnetic stirring for 30 min, the solution was centrifuged and the precipitate was redispersed in 2 mL of ultrapure water to obtain the Ag@4MBA@AuNPs solution for later use.

[0013] Surface-enhanced Raman spectroscopy (SERS) is a combination of Raman spectroscopy and nanotechnology. In recent years, surface-enhanced Raman scattering (SERS) has been considered a promising method for rapid bacterial detection due to its remarkable sensitivity and specificity, strong fingerprint recognition ability, non-destructive analysis, and fast spectral acquisition speed. It has been widely used in food safety, pharmaceutical research, and environmental control. Precious metal nanoparticles are of great significance in the preparation of SERS substrates, which have a significant impact on the stability, repeatability, and enhancement of the Raman signal of SERS sensors. The small gaps between nanoparticles or the sharp outward branching tips are called "hot spots", which help to enhance the Raman signal of SERS active substrates (such as nanoparticles, nanostars, tetrahedrons, and satellite structures, etc.). In addition, precious metal nanoparticles, after being functionalized with Raman dye molecules or biorecognition molecules (such as antibodies, aptamers, lectins, antibiotics, etc.), produce characteristic SERS signals and can specifically bind to target bacteria.

[0014] (2)Fe 3 O 4 Preparation of @Au@Ag magnetic composite materials

[0015] 5) 1.35 g FeCl 3 6H 2 O and 1g PEG (polyethylene glycol) were added to 40mL ethylene glycol and stirred until a clear solution was obtained. 3.6g NaAc was slowly added and vigorously stirred for 30min. The mixed solution was transferred to a Teflon-lined stainless steel autoclave and reacted at 200℃ for 10h. After cooling to room temperature, ultrapure water and anhydrous ethanol were used alternately for magnetic separation and washing. The washing product was placed in a vacuum drying oven at 60℃ and dried for 10h for standby use to obtain Fe 3 O 4 MNPs (magnetic nanoparticles);

[0016] 6) Add 100 mL 0.01% HAuCl 4 The solution was heated to boiling in an oil bath, and 1 mL of 1.0% trisodium citrate solution was quickly added under vigorous stirring. The solution first changed from light yellow to gray-black, and finally to wine red and remained unchanged. At this time, the solution was kept boiling for 15 minutes and then cooled at room temperature to obtain AuNPs solution, which was stored at 4°C for later use. 1.5 mL of 0.1MAA solution and 0.5 mL of 1 mM AgNO 3 The solution was mixed into 10 mL of Au NPs solution and stirred vigorously for 30 min until the wine red color changed to orange yellow. The stirred solution was centrifuged and the precipitate was redispersed in 10 mL of ultrapure water to obtain the Au@AgNPs solution for use.

[0017] 7) Take 40 mg of Fe prepared in step 5) 3 O 4 MNPs were added to 40 mL of 10 mg / mL PEI solution and mixed with ultrasound for 2 h to obtain Fe 3 O 4 @PEI solution was washed 3 times with ultrapure water magnetic separation to remove excess PEI and obtain Fe 3 O 4 @PEI; 10mgFe 3 O 4 @PEI was added to 100 mL of the Au@Ag NPs solution prepared in step 6), and ultrasonicated for 30 min. The product was separated by a magnet, washed four times with deionized water, and redispersed in 4 mL of ultrapure water to obtain Fe 3 O 4 @Au@Ag MNPs solution is ready for use;

[0018] Magnetic nanoparticles have attracted extensive attention due to their unique properties, such as small size effect, surface effect, special magnetism and good biocompatibility. The shell configuration of magnetic nanoparticles is a more stable structure, and the shell provides a platform for surface modification and functionalization. 3 O 4 @Au@Ag) is particularly interesting because the gold-silver core-shell structure can not only provide binding sites for antibodies or aptamers, but also can be surface derivatized to help reduce particle aggregation and improve biocompatibility through steric or electronic repulsion.

[0019] (3)Fe 3 O 4 @Au@Ag NPs combined with Ag@4MBA@AuNPs to construct aptamer sensors

[0020] 8) Take 1 mL of the Ag@4MBA@Au NPs solution prepared in step 4) and add 20 μL of 10 μM SH-Apt solution, incubate at room temperature for 12 h, remove excess SH-Apt by centrifugation, wash the product three times with PBS buffer, and redisperse in 1 mL of PBS buffer to obtain Ag@4MBA@Au-Apt NPs solution;

[0021] 9) Take 1 mL of Fe prepared in step 7) 3 O 4 40 μL of 5 μM SH-cDNA solution was added to the @Au@Ag MNPs solution and incubated with a digital oscillator at room temperature for 12 h. The solution was then treated with 2 μM MCH for 1 h to block the unbound sites. The product was collected after washing with ethanol magnetic separation and 1 mL of PBS buffer was added to obtain Fe 3 O4 @Au@Ag-cDNA MNPs solution;

[0022] Aptamers are single-stranded DNA or RNA oligonucleotides with defined sequences. Compared with antibodies, aptamers as chemical antibodies have better target affinity, chemical stability and lower antioxidant properties. Due to their high purity, flexible modification, low production cost, small molecular weight, easy synthesis and good storage stability, they have demonstrated their superiority and practicality for pathogen determination;

[0023] 10) Take 900 μL of the Ag@4MBA@Au-AptNPs solution prepared in step 8) and 600 μL of the Fe 3 O 4 The solution of @Au@Ag-cDNA MNPs was mixed and gently incubated on a digital oscillator for 1 h. The mixture was magnetically separated and washed three times and redispersed in the original volume of PBS buffer to obtain Ag@4MBA@Au-Fe 3 O 4 @Au@Ag magnetic composite nanoparticle solution is the SERS aptamer sensor;

[0024] The SERS biosensor prepared by combining aptamer-modified noble metal nanoprobes with aptamer complementary chain-modified magnetic composite nanomaterials can improve detection sensitivity and specificity and achieve rapid on-site detection of target bacteria in complex samples;

[0025] (4) SERS detection of Salmonella

[0026] Add 50 μL of sample to 100 μL of Ag@4MBA@Au-Fe 3 O 4 @Au@Ag magnetic composite nanoparticle solution was vortexed for 2 minutes. Due to the preferential binding between the aptamer and Salmonella, part of the Ag@4MBA@Au-Apt was removed from the Fe 3 O 4 @Au@Ag-cDNA was separated from the Ag@4MBA@Au-Apt in the supernatant, and then the Ag@4MBA@Au-Apt was removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer and dried on a clean silicon chip, and detected and analyzed using a portable Raman spectrometer.

[0027] Furthermore, the centrifugation condition is 8000 rpm, 10 min.

[0028] Furthermore, the concentration of the PBS buffer is 10 mM and the pH is 7.4.

[0029] Furthermore, in step 8), the sequence of the SH-Apt is:

[0030] 5′-SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACA G-3′, as shown in SEQ ID NO.1.

[0031] Furthermore, the sequence of the SH-cDNA in step 8) is:

[0032] 5′-SH-C6-CTGTCATAATGTCAAGTC-3′, as shown in SEQ ID NO.2.

[0033] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0034] The present invention discloses a novel surface enhanced Raman scattering (SERS) aptamer sensor constructed based on noble metal nanoparticles and magnetic composite materials. The sensor uses the characteristic peaks of Raman signal molecules to perform rapid, sensitive and broad-spectrum detection of different subtypes of Salmonella (including Enteritidis Salmonella, Typhimurium Salmonella, etc.), has good specificity and accuracy, and the detection limit is as low as 1 cfu / mL. In addition, the method does not need to pre-treat the sample, can be directly detected, and the detection time only takes 2 minutes, which reduces the test complexity and greatly shortens the detection time compared with the current detection method, and has good time superiority and detection broad spectrum. At the same time, the method uses a portable (or small) Raman spectrometer to directly detect the sample on site, does not require skilled professionals, saves costs to a large extent, solves direct economic problems such as expensive and cumbersome detection procedures, has the advantages of being simple and fast, intuitive results, strong specificity, high sensitivity, good repeatability, good stability, etc., and provides an efficient and sensitive detection method in food safety monitoring and bacterial infection diagnosis.

[0035] Fe 3 O 4 The Au@Ag nanoparticles on the Ag@Au@Ag MNPs not only enhance the SERS signal, but also provide sufficient aptamer binding sites. Raman reporter molecules (4MBA) are embedded in Ag@Au NPs. This embedded structure can prevent external interference and greatly enhance and stabilize the SERS signal. SH-cDNA and SH-Apt bind to Fe via Ag-S covalent bonds and Au-S covalent bonds, respectively. 3 O 4 @Au@Ag MNPs and Ag@4MBA@Au NPs were combined and then, the cDNA-Fe 3 O 4The SERS aptamer sensor of Salmonella was constructed by nucleic acid hybridization of @Au@Ag MNPs and Apt-Ag@4MBA@Au. At this time, due to the multiple SERS effects, the SERS aptamer sensor of Salmonella has the strongest SERS signal. Finally, with the addition of Salmonella, the aptamer will specifically and preferentially bind to Salmonella, causing Ag@4MBA@Au to be released from the aptamer sensor, resulting in a decrease in the SERS intensity after magnetic separation. According to the inverse relationship between the concentration of Salmonella and the SERS intensity, different concentrations of Salmonella can be quantitatively determined.

[0036] This detection method can be widely used in food safety monitoring, environmental monitoring, public health monitoring and other fields. During food processing, transportation and storage, Salmonella may cause serious foodborne diseases. This sensor can quickly and accurately detect different subtypes of Salmonella in food on-site, improving the level of food safety; for the detection of water and soil samples, this sensor can assess the contamination of bacteria in the environment and help formulate environmental protection measures; in addition, in hospitals or public places, through rapid detection of samples, the spread of Salmonella can be discovered and controlled in a timely manner, reducing the risk of epidemic infection and ensuring public safety.

[0037] 1. SERS sensors based on bacteria detection can be divided into two types: label-free and label-based SERS methods. The label-free SERS method can identify and quantify bacteria by directly detecting the SERS of bacteria adsorbed on the surface of the active substrate, but the information obtained by this method is directly related to the type of bacteria. The source of bacterial SERS signals has not yet been determined, and there is a lack of standard bacterial SERS spectrum databases, and the sensitivity is low. The label-based SERS method uses sharp Raman spectral peaks and highly sensitive substances as optical markers. The antibodies or aptamers on the SERS tags specifically bind to bacteria and have good biocompatibility. At the same time, the SERS label probes assembled with specific organic Raman labeling molecules, target recognition molecules and plasma nanoparticles can form an amplified characteristic Raman signal. The optical properties of labeled SERS probes are similar to those of fluorescent probes using organic dyes and quantum dots, but labeled SERS probes have incomparable qualities that other probes cannot match: the peak width of the Raman spectrum is only about 1nm, and Raman spectroscopy can achieve online simultaneous detection of multiple components; the ultra-high sensitivity provided by the SERS probe can be used for trace analysis of biological samples; in addition, the SERS probe has strong photostability and can effectively avoid photobleaching and signal quenching; the Raman shift is not affected by the frequency of the excitation light, so the interference of spontaneous fluorescence in the biological system can be avoided by selecting a near-infrared light source.

[0038] 2. The present invention uses a precious metal core-shell structure with embedded internal standard molecules to prepare the substrate. The advantages of this method are: 1) The internal standard molecule layer is sandwiched between the core and the shell layer, and is protected by the shell layer to avoid the influence of the external environment; 2) The shell surface is completely free and can be accessed by the target molecule without worrying about dynamic replacement; 3) The nanoparticles act as both internal standards and SERS enhancement substrates, and will not interfere with the spatial distribution of the target molecules; 4) The signal comes from the target molecules adsorbed on the surface of the shell layer. The flexibility of this method in the selection of Raman probes, core and shell materials (size, shape) makes it suitable for quantitative SERS detection of various targets. At the same time, Au and Ag are heterogeneous metals, and the two layers of dense heterogeneous metals generate a large number of hot spots under the effective inter-particle coupling, induce electromagnetic field enhancement, and can adjust the intensity of SERS, thereby further obtaining a better SERS enhancement effect. Au@Ag with embedded Raman signal molecules has excellent performance in terms of stability, sensitivity and repeatability of detection results.

[0039] 3. The magnetic nanocomposite material has efficient separation ability and good biocompatibility due to its superparamagnetism and Au@Ag nanoparticle surface. This property enables the magnetic nanoparticles to be easily separated and aggregated under the action of an external magnetic field. When the external magnetic field disappears, the magnetic nanoparticles can be evenly dispersed in the sample to be tested, and the aptamer and Salmonella can be specifically bound in a directional manner, which improves the separation and enrichment efficiency of Salmonella under the interference of complex sample matrices with high protein, high salt and high sugar, and reduces the time and complexity of sample processing.

[0040] 4. Utilizing aptamer-modified Fe 3 O 4 @Au@Ag can easily separate the target bacteria from complex samples into clean PBS solution, avoiding the interference of environmental factors (e.g., pH value, salt ion strength) and ensuring the stability of the proposed SERS platform.

[0041] 5. This project successfully developed a rapid and sensitive aptamer biosensor for detecting Salmonella based on the highly conserved sequence gene of Salmonella aptamers. The detection of Salmonella in actual samples of milk, shrimp and lake water achieved satisfactory results with a detection range of 10-10 8 cfu / mL, the detection limit reaches 1 cfu / mL, and the detection time is shortened to 2 minutes, which provides a more rapid, sensitive, broad-spectrum, and specific portable detection method for the detection of Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0043] Figure 1 This is a diagram of the research ideas of the present invention;

[0044] Figure 2 Design principle diagram for the present invention;

[0045] Figure 3 A schematic diagram of the nanoparticles prepared by the present invention; Figure 3 a is a schematic diagram of Ag@4MBA@Au nanoparticles. Figure 3 b is Fe 3 O 4 Schematic diagram of magnetic nanoparticles. Figure 3 c is Fe 3 O 4 Schematic diagram of @Au@Ag magnetic nanoparticles;

[0046] Figure 4 This is a sensitivity test diagram of five types of Salmonella in the present invention;

[0047] Figure 5 It is a schematic diagram of the detection of five subtypes of Salmonella, Escherichia coli, Staphylococcus aureus and Vibrio parahaemolyticus of the present invention;

[0048] Figure 6 Schematic diagram of the detection of three actual samples (lake water, milk, shrimp) of the present invention. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] Example 1

[0051] A rapid and broad-spectrum detection method for Salmonella Figure 1 This is a diagram of the research ideas of the present invention; Figure 2 The present invention is designed as a schematic diagram), comprising the following steps:

[0052] 1. A method for rapid broad-spectrum detection of Salmonella, characterized in that it comprises the following steps:

[0053] (1) Preparation of labeled SERS probes

[0054] 1) 17 mg AgNO 3 Dissolve in 100 mL ultrapure water, heat to boiling under vigorous stirring, quickly inject 2 mL 1% trisodium citrate, boil for 40 min, and cool naturally to room temperature to obtain AgNPs solution; the final solution containing silver nanoparticles is yellow-green;

[0055] 2) Take 10 mL of the AgNPs solution prepared in step 1) and add 200 μL of 0.1 mM 4MBA solution. After vigorous stirring for 5 h, centrifuge the mixture at 8000 rpm for 10 min, and redisperse the precipitate in 2 mL of ultrapure water to obtain an Ag@MBANPs solution, which is stored at 4°C for later use;

[0056] 3) Add 222 μL 2% HAuCl 4 solution, 240 μL 0.2M NaOH solution, 3 mL 0.01M Na 2 SO 3 The solution was added into 4.538 mL ultrapure water to obtain Au growth solution, which was stored at 4°C for later use;

[0057] 4) 2 mL of the Ag@4MBANPs solution prepared in step 2), 2.55 mL of ultrapure water, 1 mL of 5% PVP, 200 μL of 0.5 MAA, 200 μL of 0.5 M NaOH solution, 50 μL of 0.1 M Na 2 SO 3 The solution and 4 mL of the Au growth solution prepared in step 3) were added to a 25 mL glass vial. After magnetic stirring for 30 min, the solution was centrifuged at 8000 rpm for 10 min, and the precipitate was redispersed in 2 mL of ultrapure water to obtain the Ag@4MBA@AuNPs solution for later use (see Figure 3 a);

[0058] (2)Fe 3 O 4 Preparation of @Au@Ag magnetic composite materials

[0059] 5) 1.35 g FeCl 3 6H 2 O and 1g PEG were added to 40mL ethylene glycol and stirred until a clear solution was obtained. 3.6g NaAc was slowly added and vigorously stirred for 30min. The mixed solution was transferred to a Teflon-lined stainless steel autoclave and reacted at 200℃ for 10h. After cooling to room temperature, ultrapure water and anhydrous ethanol were used alternately for magnetic separation and washing. This was repeated 3 times. The washed product was placed in a vacuum drying oven and dried at 60℃ for 10h for standby use. Fe 3 O 4MNPs (see Figure 3 b);

[0060] 6) Add 100 mL 0.01% HAuCl 4 The solution was heated to boiling in an oil bath, and 1 mL of 1.0% trisodium citrate solution was quickly added under vigorous stirring. The solution first changed from light yellow to gray-black, and finally to wine red and remained unchanged. At this time, the solution was kept boiling for 15 minutes and then cooled at room temperature to obtain AuNPs solution, which was stored at 4°C for later use. 1.5 mL of 0.1 M ascorbic acid solution and 0.5 mL of 1 mM AgNO 3 The solution was mixed into 10 mL of AuNPs solution and stirred vigorously for 30 min until the wine red color changed to orange yellow. The stirred solution was centrifuged at 8000 rpm for 10 min, and the precipitate was redispersed in 10 mL of ultrapure water to obtain the Au@AgNPs solution for use.

[0061] 7) Take 40 mg of Fe prepared in step 5) 3 O 4 MNPs were added to 40 mL of 10 mg / mL PEI solution and mixed with ultrasound for 2 h to obtain Fe 3 O 4 @PEI solution was washed 3 times with ultrapure water magnetic separation to remove excess PEI and obtain Fe 3 O 4 @PEI; 10mgFe 3 O 4 @PEI was added to 100 mL of the Au@AgNPs solution prepared in step 6), and ultrasonicated for 30 min. The product was separated by a magnet, washed four times with deionized water, and redispersed in 4 mL of ultrapure water to obtain Fe 3 O 4 @Au@Ag MNPs solution is available (see Figure 3 c);

[0062] (3)Fe 3 O 4 @Au@AgNPs combined with Ag@4MBA@AuNPs to construct aptamer sensor

[0063] 8) Take 1 mL of the Ag@4MBA@Au NPs solution prepared in step 4) and add 20 μL of 10 μM SH-Apt solution;

[0064] The sequence of SH-Apt is:

[0065] 5′-SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACA G-3′, as shown in SEQ ID NO.1;

[0066] After incubation at room temperature for 12 h, the excess SH-Apt was removed by centrifugation at 8000 rpm for 10 min, and the product was washed three times with PBS buffer (10 mM, pH 7.4) and redispersed in 1 mL of PBS buffer (10 mM, pH 7.4) to obtain Ag@4MBA@Au-AptNPs solution;

[0067] 9) Take 1 mL of Fe prepared in step 7) 3 O 4 @Au@Ag MNPs solution was added with 40 μL of 5 μM SH-cDNA solution;

[0068] The sequence of SH-cDNA is:

[0069] 5′-SH-C6-CTGTCATAATGTCAAGTC-3′, as shown in SEQ ID NO.2.

[0070] The mixture was incubated at room temperature with a digital oscillator for 12 h, and then treated with 2 μM MCH for 1 h to block the unbound sites. The product was collected after washing with ethanol magnetic separation, and 1 mL of PBS buffer (10 mM, pH 7.4) was added to obtain Fe 3 O 4 @Au@Ag-cDNA MNPs solution;

[0071] 10) Take 900 μL of the Ag@4MBA@Au-AptNPs solution prepared in step 8) and 600 μL of the Fe 3 O 4 The Ag@4MBA@Au-Fe@Ag-cDNA MNPs solution was mixed and gently incubated on a digital oscillator for 1 h. The mixture was magnetically separated and washed three times and redispersed in the original volume of PBS buffer (10 mM, pH 7.4) to obtain Ag@4MBA@Au-Fe 3 O 4 @Au@Ag magnetic composite nanoparticle solution is the SERS aptamer sensor;

[0072] (4) SERS detection of Salmonella

[0073] Add 50 μL of sample to 100 μL of Ag@4MBA@Au-Fe 3 O 4 @Au@Ag magnetic composite nanoparticle solution was vortexed for 2 minutes. Due to the preferred binding between the aptamer and Salmonella, part of the Ag@4MBA@Au-Apt was removed from the Fe 3 O 4@Au@Ag-cDNA was separated from the Ag@4MBA@Au-Apt in the supernatant, and then the Ag@4MBA@Au-Apt was removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer (10 mM, PH 7.4), dried on a clean silicon chip, and detected and analyzed using a portable Raman spectrometer.

[0074] Specificity analysis

[0075] In the SERS detection of Salmonella, 50 μL of different serotypes of Salmonella (S. Typhimurium, S. Enteritidis, S. Kentucky, S. Indiana, S. NO) and three non-Salmonella (S. aureus, E. coil, V. Parahemolyticus) were added to 100 μL of Ag@4MBA@Au-Fe 3 O 4 In the @Au@Ag magnetic composite nanoparticle solution, due to the preferred binding between the aptamer and Salmonella, part of the Ag@4MBA@Au-Apt from Fe 3 O 4 @Au@Ag-cDNA was separated from the supernatant, and then the Ag@4MBA@Au-Apt was removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer and dried on a clean silicon chip, and detected and analyzed using a portable Raman spectrometer.

[0076] The corresponding results are as follows:

[0077] Sensitive detection of five Salmonella species Figure 4 As shown. With the increase of Salmonella concentration (S.Typhimurium, S.Enteritidis, S.Kentucky, S.IndiaNa, S.NO) (10-10 8 cfu / mL), the Raman signal weakened, 4MBA at 1076cm -1 The SERS intensity at 400 nm showed a good linear relationship with the Salmonella concentration (logarithm), and the linear regression equations were Y = -982.395Log(X) + 12834, Y = -865.029Log(X) + 16129.432, Y = -1006.329Log(X) + 16129.432, Y = -979.627Log(X) + 11325.494, Y = -1038.691Log(X) + 13473.466, R 2The values ​​were as high as 0.997, 0.997, 0.994, 0.9997, and 0.997, and the corresponding detection limits were as low as 1.782, 1.637, 1.941, 1.632, and 1.875 cfu / mL, respectively, indicating that the sensor has good sensitivity and extremely low detection limit.

[0078] Specific analysis of eight bacterial species Figure 5 It is obvious from the SERS spectra that the SERS intensity of the five Salmonella species is significantly lower than that of the blank sample and the other three analytes (S. aureus, E. coil, V. Parahemolyticus), indicating that the SERS-based aptamer sensor has a high selectivity for the detection of Salmonella, which is also the result of the specific selection of the Salmonella aptamer.

[0079] SERS analysis in real samples (lake water, milk, shrimp)

[0080] Lake water and milk do not need to be pretreated. For shrimp samples, fresh shrimp meat can be used after extraction and grinding. Take appropriate amount of lake water, milk and shrimp samples and add them to the standard sample of Salmonella Enteritidis to prepare the concentration of Salmonella Enteritidis (2×10 3 -2×10 7 cfu / mL) was used to test the practicality and sensitivity of the sensor. 50 μL of lake water standard solution was added to 100 μL of Ag@4MBA@Au-Fe 3 O 4 @Au@Ag solution for 1 hour (the same as for milk and shrimp samples), and the supernatant was removed after magnetic separation. The precipitate was redispersed in 10μL PBS buffer and then dried on a clean silicon chip. It can then be detected and analyzed using a portable Raman spectrometer.

[0081] The SERS detection results of the three substances are as follows Figure 6 shown.

[0082] In view of the excellent performance of the sensor of the present invention, its applicability was evaluated. Since the samples did not require pretreatment, the detection time of three actual samples (lake water, milk and shrimp) was only 2 minutes, and their SERS spectra at 1076 cm -1The Raman intensities at 100 and 100 μg / mL were all acceptable linear, and the corresponding spiked recoveries were 99.35%-102.38%, 95.82%-102.398%, 98.64%-102.08%, and the LODs were 1.884 cfu / mL, 2.228 cfu / mL, and 1.858 cfu / mL, respectively. These results indicate that the aptamer sensor has satisfactory accuracy and anti-interference performance in the analysis of real samples, and compared with other methods, this method also shows significant reliability even at trace concentration levels. Therefore, the method of the present invention is ultrasensitive, rapid, and non-interference, and has high potential in detecting Salmonella in real samples.

[0083] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined in the present invention may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown in the present invention, but will conform to the widest scope consistent with the principles and novel features disclosed in the present invention.

Claims

1. A method for rapid broad-spectrum detection of Salmonella, characterized in that: The following steps are involved: (1) Preparation of labeled SERS probes 1) 17 mg of AgNO3 was dissolved in 100 mL of ultrapure water, heated to boiling under vigorous stirring, and 2 mL of 1% trisodium citrate was quickly injected, boiled for 40 min, and naturally cooled to room temperature to obtain AgNPs solution; 2) Take 10 mL of the AgNPs solution prepared in step 1) and add 200 μL of 0.1 mM 4MBA solution. After vigorous stirring for 5 h, centrifuge the mixture and redisperse the precipitate in 2 mL of ultrapure water to obtain an Ag@MBANPs solution, which is stored at 4°C for later use; 3) Add 222 μL of 2% HAuCl4 solution, 240 μL of 0.2M NaOH solution, and 3 mL of 0.01M Na2SO3 solution into 4.538 mL of ultrapure water to obtain an Au growth solution, which is then stored at 4°C for later use; 4) 2 mL of the Ag@4MBANPs solution prepared in step 2), 2.55 mL of ultrapure water, 1 mL of 5% PVP, 200 μL of 0.5 MAA, 200 μL of 0.5 M NaOH solution, 50 μL of 0.1 M Na2SO3 solution, and 4 mL of the Au growth solution prepared in step 3) were added to a 25 mL glass vial. After magnetic stirring for 30 min, the solution was centrifuged and the precipitate was redispersed in 2 mL of ultrapure water to obtain the Ag@4MBA@AuNPs solution for later use; (2) Preparation of Fe3O4@Au@Ag magnetic composite materials 5) Add 1.35 g FeCl3·6H2O and 1 g PEG to 40 mL ethylene glycol and stir until a clear solution is obtained. Slowly add 3.6 g NaAc and vigorously stir for 30 min. Transfer the mixed solution to a Teflon-lined stainless steel autoclave and react at 200 °C for 10 h. Cool to room temperature and alternately use ultrapure water and anhydrous ethanol for magnetic separation and washing. Repeat this process three times. Place the washed product in a vacuum drying oven and dry it at 60 °C for 10 h to obtain Fe3O4 MNPs. 6) Heat 100 mL of 0.01% HAuCl4 solution to boiling in an oil bath, and quickly add 1 mL of 1.0% trisodium citrate solution under vigorous stirring. The solution first changes from light yellow to gray-black, and finally to wine red and remains unchanged. At this time, keep the solution boiling for 15 minutes and then cool it at room temperature to obtain an AuNPs solution, which is stored at 4°C for later use; 1.5 mL of 0.1MAA solution and 0.5 mL of 1mMAgNO3 solution are mixed into 10 mL of AuNPs solution. After vigorous stirring for 30 minutes, the wine red turns to orange-yellow. The stirred solution is centrifuged, and the precipitate is redispersed in 10 mL of ultrapure water to obtain an Au@AgNPs solution for later use; 7) 40 mg of Fe3O4 MNPs prepared in step 5) was added to 40 mL of 10 mg / mL PEI solution and ultrasonically mixed for 2 hours to obtain Fe3O4@PEI solution, which was then magnetically separated and washed with ultrapure water for 3 times to remove excess PEI to obtain Fe3O4@PEI; 10 mg of Fe3O4@PEI was added to 100 mL of Au@AgNPs solution prepared in step 6), ultrasonically separated for 30 min, the product was separated with a magnet, washed with deionized water four times, and redispersed in 4 mL of ultrapure water to obtain Fe3O4@Au@AgMNPs solution for later use; (3) Construction of aptamer sensor by combining Fe3O4@Au@AgNPs with Ag@4MBA@AuNPs 8) Take 1 mL of the Ag@4MBA@Au NPs solution prepared in step 4) and add 20 μL of 10 μM SH-Apt solution, incubate at room temperature for 12 h, remove excess SH-Apt by centrifugation, wash the product three times with PBS buffer, and redisperse in 1 mL of PBS buffer to obtain Ag@4MBA@Au-Apt NPs solution; 9) Take 1 mL of the Fe3O4@Au@Ag MNPs solution prepared in step 7) and add 40 μL of 5 μM SH-cDNA solution, incubate with a digital oscillator at room temperature for 12 h, and then treat with 2 μM MCH for 1 h to block the unbound sites. Collect the product after washing with ethanol magnetic separation, and add 1 mL of PBS buffer to prepare a Fe3O4@Au@Ag-cDNA MNPs solution; 10) 900 μL of the Ag@4MBA@Au-AptNPs solution prepared in step 8) and 600 μL of the Fe3O4@Au@Ag-cDNA MNPs solution prepared in step 9) were mixed, gently shaken and incubated on a digital oscillator for 1 hour, the mixture was magnetically separated and washed 3 times, and redispersed in the original volume of PBS buffer to obtain the Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution, which is the SERS aptamer sensor; (4) SERS detection of Salmonella Add 50 μL of sample to 100 μL of Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution prepared in step 10) and vortex for 2 minutes. Due to the preferential binding between the aptamer and Salmonella, part of the Ag@4MBA@Au-Apt is separated from the Fe3O4@Au@Ag-cDNA in the competitive binding system. Then, the Ag@4MBA@Au-Apt separated from the supernatant is removed by magnetic separation. Finally, the precipitate is redispersed in 10 μL of PBS buffer, placed on a clean silicon chip for drying, and detected and analyzed using a portable Raman spectrometer.

2. The method according to claim 1, characterized in that The centrifugal condition is 8000 rpm, 10 min.

3. The method according to claim 1, characterized in that The concentration of the PBS buffer is 10 mM and the pH is 7.

4.

4. The method according to claim 1, characterized in that: Step 8) The sequence of the SH-Apt is: 5′-SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACA G-3′, as shown in SEQ ID NO.

1.

5. The method according to claim 1, characterized in that Step 8) The sequence of the SH-cDNA is: 5′-SH-C6-CTGTCATAATGTCAAGTC-3′, as shown in SEQ ID NO.2.

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