A rapid and broad-spectrum method for detecting Salmonella
The SERS aptamer sensor constructed by precious metal nanoparticles and magnetic composite materials solves the problems of long detection time and low sensitivity of Salmonella, and achieves fast, accurate and highly selective detection effects.
Patent Information
- Application Number
- CN202411788483.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-09-23
- Estimated Expiration
- 2044-12-06
AI Technical Summary
Existing technologies for Salmonella detection have problems such as long detection time, low sensitivity, high false positive rate, strict requirements on experimental conditions and cumbersome detection procedures, making it difficult to achieve rapid, accurate and highly selective detection.
A SERS aptamer biosensor method was adopted, and a surface-enhanced Raman scattering sensor was constructed using noble metal nanoparticles and magnetic composite materials. Ag@4MBA@Au and Fe3O4@Au@Ag magnetic composite nanoparticles were prepared and combined with aptamers for rapid detection.
It achieves rapid, sensitive and broad-spectrum detection of Salmonella, with a detection limit as low as 1 cfu/mL, a detection time of only 2 minutes, and no sample pretreatment is required, reducing detection cost and complexity, and has good specificity and accuracy.
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Figure CN119555662B_ABST
Abstract
Description
Technical Field
[0001] The present 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, which includes Salmonella enterica (S. enterica) and Salmonella bongori (S. bongori). It is a large group of non-spore-forming, non-capsulated, Gram-negative, facultative anaerobic bacteria. Salmonella is the main pathogen causing bacterial foodborne illnesses worldwide and is widely regulated by countries and international organizations. It is widely distributed in nature and often resides in humans and animals, especially in the intestines of poultry, livestock, and pets. Salmonella is often associated with food poisoning incidents worldwide, and the main contaminated foods include dairy products, meat products, and egg products. Salmonella does not break down proteins, so contaminated food is difficult to detect in appearance and taste, and the infectious dose of Salmonella is relatively low. Salmonella poses a serious threat 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 counts, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA), while highly accurate and specific, also have several drawbacks. Plate counts are time-consuming, often requiring several days of incubation to obtain results. This is labor-intensive and time-consuming, making them unsuitable for multi-sample testing and rapid on-site testing. While PCR offers high sensitivity, it often produces false-negative or false-positive identification results. Furthermore, PCR can only detect the presence of DNA or RNA and cannot directly reflect microbial activity. Although widely used for antigen detection, ELISA has high requirements for experimental conditions (such as temperature and pH), resulting in limited sensitivity in some cases. Cross-reactions can occur, leading to false-positive results, especially at lower concentrations. Furthermore, standards are required for quantification, which can be difficult to obtain and preserve. Furthermore, fluorescent labeling, liposome analysis, chip technology, and bioconjugated nanoprobes have also been developed. However, all of these methods have drawbacks, such as multiple pretreatment steps, complex instrumentation, low sensitivity, the need for skilled personnel, and cumbersome detection procedures.
[0004] Therefore, it is an urgent problem for those skilled in the art to provide a new technology that is rapid, highly selective and ultrasensitive 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 solutions:
[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] 17 mg of AgNO₃ was dissolved in 100 mL of ultrapure water and heated to boiling with vigorous stirring. 2 mL of 1% trisodium citrate was rapidly added and the mixture was boiled for 40 minutes. The mixture was then cooled to room temperature to obtain a Ag NPs (nanoparticle) solution. The final solution containing silver nanoparticles was yellow-green.
[0010] 2) Add 200 μL of 0.1 mM 4-mercaptobenzoic acid (4-MBA) solution to 10 mL of the Ag NPs solution prepared in step 1). After vigorous stirring for 5 h, the mixture was centrifuged and the precipitate was redispersed in 2 mL of ultrapure water to obtain the Ag@MBA NPs solution, which was stored at 4°C until use.
[0011] 3) Add 222 μL of 2% HAuCl4 solution, 240 μL of 0.2 M NaOH solution, and 3 mL of 0.01 M Na2SO3 solution to 4.538 mL of ultrapure water to obtain an Au growth solution, which was stored at 4°C until 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 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 minutes, 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 fusion of Raman spectroscopy and nanotechnology. In recent years, SERS has been recognized as a promising method for rapid bacterial detection due to its remarkable sensitivity and specificity, strong fingerprint recognition capabilities, non-destructive analysis, and rapid spectral acquisition. It has been widely used in fields such as food safety, pharmaceutical research, and environmental control. Noble metal nanoparticles are crucial in the preparation of SERS substrates, significantly impacting the stability, reproducibility, and enhancement of the Raman signal from SERS sensors. Small gaps between nanoparticles or sharp, outward-pointing branches, known as "hotspots," enhance the Raman signal of SERS-active substrates (such as nanoparticles, nanostars, tetrahedrons, and satellite structures). Furthermore, when functionalized with Raman dyes or biorecognition molecules (such as antibodies, aptamers, lectins, and antibiotics), noble metal nanoparticles produce characteristic SERS signals and can specifically bind to target bacteria.
[0014] (2) Preparation of Fe3O4@Au@Ag magnetic composite materials
[0015] 5) 1.35 g of FeCl3·6H2O and 1 g of PEG (polyethylene glycol) were added to 40 mL of ethylene glycol and stirred until a clear solution was obtained. 3.6 g of NaAc was slowly added and vigorously stirred for 30 min. The mixed solution was transferred to a Teflon-lined stainless steel autoclave and reacted at 200°C for 10 h. After cooling to room temperature, the mixture was subjected to magnetic separation and washing using ultrapure water and anhydrous ethanol alternately, and this was repeated three times. The washed product was placed in a vacuum drying oven at 60°C for 10 h before use to obtain Fe3O4 MNPs (magnetic nanoparticles).
[0016] 6) Heat 100 mL of 0.01% HAuCl4 solution to boiling in an oil bath. Rapidly 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 the AuNPs solution, which is stored at 4°C for later use. Mix 1.5 mL of 0.1MAA solution and 0.5 mL of 1 mM AgNO3 solution into 10 mL of AuNPs solution. After vigorous stirring for 30 minutes, the wine red turns to orange-yellow. Centrifuge the stirred solution, and redisperse the precipitate in 10 mL of ultrapure water to obtain the Au@AgNPs solution for later use.
[0017] 7) 40 mg of the Fe3O4 MNPs prepared in step 5) were added to 40 mL of a 10 mg / mL PEI solution and ultrasonically mixed for 2 hours to obtain a Fe3O4@PEI solution. The Fe3O4@PEI solution was then magnetically washed three times with ultrapure water to remove excess PEI, thereby obtaining Fe3O4@PEI. 10 mg of the Fe3O4@PEI solution was added to 100 mL of the Au@Ag NPs solution prepared in step 6) and ultrasonically mixed for 30 minutes. The product was separated using a magnet, washed four times with deionized water, and redispersed in 4 mL of ultrapure water to obtain a Fe3O4@Au@Ag MNPs solution for later use.
[0018] Magnetic nanoparticles have attracted widespread attention due to their unique properties, such as small size effects, surface effects, exceptional magnetic properties, and excellent biocompatibility. Magnetic nanoparticle shells provide a more stable structure, and the shell provides a platform for surface modification and functionalization. The synthesis of magnetic composite nanomaterials (Fe3O4@Au@Ag) is particularly interesting because the gold-silver core-shell structure not only provides binding sites for antibodies or aptamers, but also allows for surface derivatization to reduce particle aggregation through steric or electronic repulsion, thereby improving biocompatibility.
[0019] (3) Construction of aptamer sensors by combining Fe3O4@Au@Ag NPs with Ag@4MBA@AuNPs
[0020] 8) Add 20 μL of 10 μM SH-Apt solution to 1 mL of the Ag@4MBA@Au NPs solution prepared in step 4) and incubate at room temperature for 12 h. Excess SH-Apt was removed by centrifugation, and the product was washed three times with PBS buffer and redispersed in 1 mL of PBS buffer to obtain an Ag@4MBA@Au-Apt NPs solution.
[0021] 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. Then treat with 2 μM MCH for 1 h to block unbound sites. After washing with ethanol magnetic separation, collect the product and add 1 mL of PBS buffer to prepare the Fe3O4@Au@Ag-cDNA MNPs solution.
[0022] Aptamers are single-stranded DNA or RNA oligonucleotides with defined sequences. Compared to 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 detection.
[0023] 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 and gently shaken on a digital oscillator for 1 hour. The mixture was magnetically separated and washed three times and redispersed in the original volume of PBS buffer to obtain an Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution, which 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, enabling rapid on-site detection of target bacteria in complex samples.
[0025] (4) SERS detection of Salmonella
[0026] 50 μL of sample was added to 100 μL of the Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution prepared in step 10) and vortexed for 2 minutes. Due to the preferential binding between the aptamer and Salmonella, some Ag@4MBA@Au-Apt was separated from the Fe3O4@Au@Ag-cDNA in the competitive binding system. The Ag@4MBA@Au-Apt detached from the supernatant was then removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer and placed on a clean silicon chip for drying. It was then detected and analyzed using a portable Raman spectrometer.
[0027] Furthermore, the centrifugal 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, the sequence of the SH-Apt in step 8) 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 Salmonella Enteritidis, Salmonella Typhimurium, etc.), with good specificity and accuracy, and a detection limit as low as 1 cfu / mL. In addition, the method does not require pretreatment of the sample and can be directly detected. The detection time is only 2 minutes, which reduces the test complexity and greatly shortens the detection time compared to the current detection methods, and has good time advantages and broad-spectrum detection. At the same time, the method uses a portable (or small) Raman spectrometer to perform direct on-site detection of the sample, does not require skilled professionals, saves costs to a large extent, and solves direct economic problems such as expensive and cumbersome detection procedures. It has the advantages of simplicity and rapidity, intuitive results, strong specificity, high sensitivity, good repeatability, and good stability, providing an efficient and sensitive detection method in food safety monitoring and bacterial infection diagnosis.
[0035] The Au@Ag nanoparticles on the Fe3O4@Au@Ag MNPs not only enhance the SERS signal but also provide sufficient aptamer binding sites. A Raman reporter molecule (4MBA) is embedded in the Ag@Au NPs. This embedded structure prevents external interference, greatly enhancing and stabilizing the SERS signal. SH-cDNA and SH-Apt bind to the Fe3O4@Au@Ag MNPs and Ag@4MBA@Au NPs via Ag-S covalent bonds and Au-S covalent bonds, respectively. A Salmonella SERS aptasensor is then constructed through nucleic acid hybridization between cDNA-Fe3O4@Au@Ag MNPs and Apt-Ag@4MBA@Au. This aptasensor exhibits the strongest SERS signal due to the multiple SERS effects. Finally, with the addition of Salmonella, the aptamer will specifically and preferentially bind to Salmonella, causing the 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 Salmonella concentration and 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. Salmonella can cause serious foodborne illnesses during food processing, transportation, and storage. This sensor can quickly and accurately detect different subtypes of Salmonella in food on-site, improving food safety. By testing water and soil samples, the sensor can assess bacterial contamination in the environment and assist in developing environmental protection measures. Furthermore, rapid sample testing in hospitals and public places can promptly detect and control the spread of Salmonella, reducing the risk of infection and safeguarding public safety.
[0037] 1. SERS sensors based on bacterial 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. However, the information obtained by this method is directly related to the bacterial species. The source of the bacterial SERS signal has not yet been determined. There is also a lack of a standard bacterial SERS spectrum database, and the sensitivity is low. The label-based SERS method uses sharp Raman spectral peaks and highly sensitive substances as optical labels. The antibodies or aptamers on the SERS label specifically bind to bacteria and have good biocompatibility. At the same time, the SERS label probe 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 they 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 and 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 autofluorescence in biological systems can be avoided by selecting a near-infrared light source.
[0038] 2. The present invention uses a precious metal core-shell structure with an embedded internal standard molecule to prepare the substrate. The advantages of this method are: 1) The internal standard molecule layer is sandwiched between the core and shell layers, protected by the shell layer and shielded from the influence of the external environment; 2) The shell surface is completely free and accessible to the target molecules without the need for dynamic replacement; 3) The nanoparticles serve as both internal standards and SERS-enhancing substrates without interfering with the spatial distribution of the target molecules; and 4) The signal comes from the target molecules adsorbed on the shell surface. The flexibility of this method in selecting Raman probes, core, and shell materials (size and shape) makes it suitable for quantitative SERS detection of various targets. Furthermore, Au and Ag, as heterogeneous metals, create a large number of hotspots due to effective interparticle coupling, inducing electromagnetic field enhancement and modulating the SERS intensity, thereby further achieving a better SERS enhancement effect. The Au@Ag with embedded Raman signal molecules exhibits excellent performance in terms of detection stability, sensitivity, and repeatability.
[0039] 3. Due to its superparamagnetic properties and Au@Ag nanoparticle surface, the magnetic nanocomposite material has efficient separation capabilities and good biocompatibility. This property enables the magnetic nanoparticles to be easily separated and aggregated in a directional manner under the action of an external magnetic field. When the external magnetic field disappears, the magnetic nanoparticles can be evenly dispersed into the sample to be tested, achieving directional and specific binding between the aptamer and Salmonella, improving the separation and enrichment efficiency of Salmonella under the interference of complex sample matrices with high protein, high salt, and high sugar, and reducing the time and complexity of sample processing.
[0040] 4. The aptamer-modified Fe3O4@Au@Ag can easily separate the target bacteria from complex samples into clean PBS solution, avoiding the interference of environmental factors (such as pH value and 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.
[0043] Figure 1 This is a diagram of the research ideas of the present invention;
[0044] Figure 2 Design principle diagram of the present invention;
[0045] Figure 3 Schematic diagram of the nanoparticles prepared in the present invention; Figure 3 a is a schematic diagram of Ag@4MBA@Au nanoparticles. Figure 3 b is a schematic diagram of Fe3O4 magnetic nanoparticles, Figure 3 c is a schematic diagram of Fe3O4@Au@Ag magnetic nanoparticles;
[0046] Figure 4 This is a sensitivity test chart for five types of Salmonella in the present invention;
[0047] Figure 5 Schematic diagram of the detection of five subtypes of Salmonella, Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus according to the present invention;
[0048] Figure 6 Schematic diagram of the detection of three actual samples (lake water, milk, and shrimp) of the present invention. DETAILED DESCRIPTION
[0049] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.
[0050] Example 1
[0051] A rapid and broad-spectrum method for detecting 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, comprising the following steps:
[0053] (1) Preparation of labeled SERS probes
[0054] 17 mg of AgNO₃ was dissolved in 100 mL of ultrapure water and heated to boiling with vigorous stirring. 2 mL of 1% trisodium citrate was rapidly added and the mixture was boiled for 40 min. The mixture was then cooled to room temperature to obtain a AgNPs solution. The final solution containing silver nanoparticles was yellow-green.
[0055] 2) Add 200 μL of 0.1 mM 4MBA solution to 10 mL of the AgNPs solution prepared in step 1) and vigorously stir for 5 h. Centrifuge the mixture at 8000 rpm for 10 min, and redisperse the precipitate in 2 mL of ultrapure water to obtain the Ag@MBANPs solution, which is stored at 4°C until use.
[0056] 3) Add 222 μL of 2% HAuCl4 solution, 240 μL of 0.2 M NaOH solution, and 3 mL of 0.01 M Na2SO3 solution to 4.538 mL of ultrapure water to obtain an Au growth solution, which was stored at 4°C until 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 M NaOH solution, 200 μL of 0.5 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 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) Preparation of Fe3O4@Au@Ag magnetic composite materials
[0059] 5) 1.35 g of FeCl3·6H2O and 1 g of PEG were added to 40 mL of ethylene glycol and stirred until a clear solution was obtained. 3.6 g of NaAc was slowly added and vigorously stirred for 30 min. The mixed solution was transferred to a Teflon-lined stainless steel autoclave and reacted at 200°C for 10 h. After cooling to room temperature, the mixed solution was washed by magnetic separation using ultrapure water and anhydrous ethanol alternately. This was repeated three times. The washed product was placed in a vacuum drying oven and dried at 60°C for 10 h before use to obtain Fe3O4 MNPs (see Figure 3 b);
[0060] 6) Heat 100 mL of 0.01% HAuCl4 solution to boiling in an oil bath. Rapidly 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. Mix 1.5 mL of 0.1 M ascorbic acid solution and 0.5 mL of 1 mM AgNO3 solution into 10 mL of AuNPs solution. After vigorous stirring for 30 minutes, the wine red turns to orange-yellow. Centrifuge the stirred solution at 8000 rpm for 10 minutes, and redisperse the precipitate in 10 mL of ultrapure water to obtain an Au@AgNPs solution for later use.
[0061] 7) 40 mg of Fe3O4 MNPs prepared in step 5) were 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 three times with ultrapure water 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 minutes, and the product was separated with a magnet, washed four times with deionized water, and redispersed in 4 mL of ultrapure water to obtain Fe3O4@Au@Ag MNPs solution for use (see Figure 3 c);
[0062] (3) Construction of aptamer sensors by combining Fe3O4@Au@AgNPs with Ag@4MBA@AuNPs
[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] The mixture was incubated at room temperature for 12 h, centrifuged at 8000 rpm for 10 min to remove excess SH-Apt, 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 the Fe3O4@Au@Ag MNPs solution prepared in step 7) and add 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 with a digital oscillator at room temperature 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 prepare the Fe3O4@Au@Ag-cDNA MNPs solution;
[0071] 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 and gently shaken 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 an Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution, which is the SERS aptamer sensor.
[0072] (4) SERS detection of Salmonella
[0073] 50 μL of sample was added to 100 μL of the Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution prepared in step 10) and vortexed for 2 minutes. Due to the preferred binding between the aptamer and Salmonella, part of the Ag@4MBA@Au-Apt was separated from the Fe3O4@Au@Ag-cDNA in the competitive binding system. The Ag@4MBA@Au-Apt detached from the supernatant was then removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer (10 mM, pH 7.4), placed on a clean silicon chip for drying, 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 species (S. aureus, E. coil, and V. Parahemolyticus) were added to a 100 μL solution of Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticles. Due to the preferential binding between the aptamer and Salmonella, a portion of the Ag@4MBA@Au-Apt was separated from the Fe3O4@Au@Ag-cDNA in a competitive binding system. The detached Ag@4MBA@Au-Apt was then removed from the supernatant by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer, dried on a clean silicon chip, 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 2 The sensitivity was 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 5It is obvious from the SERS spectra that the SERS intensities of the five Salmonella species are significantly lower than those of the blank sample and the other three analytes (S. aureus, E. coil, and V. Parahemolyticus), indicating that the SERS-based aptamer sensor has 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 extracted and ground before use. Take appropriate amounts of lake water, milk, and shrimp samples and add them to the S. Enteritidis standard to prepare a S. Enteritidis concentration of (2×10 3 -2×10 7 A standard solution (100 μL, 0.1 μg / mL) was used to test the sensor's practicality and sensitivity. 50 μL of the lake water standard solution was added to 100 μL of the Ag@4MBA@Au-Fe3O4@Au@Ag solution and incubated for 1 hour (the same procedure was used for milk and shrimp samples). After magnetic separation, the supernatant was removed, and the precipitate was redispersed in 10 μL of PBS buffer. The precipitate was then dried on a clean silicon chip 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 do not require pretreatment, the detection time of three actual samples (lake water, milk and shrimp) is only 2 minutes, and their SERS spectra at 1076 cm -1 Acceptable linearity was observed across all Raman intensities at 400 nm and 800 nm, with corresponding spike recoveries ranging from 99.35% to 102.38%, 95.82% to 102.398%, and 98.64% to 102.08%, respectively. LODs were 1.884 cfu / mL, 2.228 cfu / mL, and 1.858 cfu / mL, respectively. These results demonstrate that the aptasensor exhibits satisfactory accuracy and anti-interference performance in real sample analysis. Compared to other methods, this method exhibits significant reliability even at trace concentration levels. Therefore, the proposed method is ultrasensitive, rapid, and interference-free, and has great potential for detecting Salmonella in real samples.
[0083] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein, but is intended to be embodied in the widest possible manner consistent with the principles and novel features disclosed herein.
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 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 rapidly injected. The mixture was boiled for 40 min and allowed to cool naturally to room temperature to obtain an AgNPs solution. 2) Add 200 μL of 0.1 mM 4MBA solution to 10 mL of the AgNPs solution prepared in step 1) and vigorously stir for 5 h. Centrifuge the mixture and redisperse the precipitate in 2 mL of ultrapure water to obtain the Ag@MBANPs solution, which is stored at 4°C until use. 3) Add 222 μL of 2% HAuCl4 solution, 240 μL of 0.2 M NaOH solution, and 3 mL of 0.01 M Na2SO3 solution to 4.538 mL of ultrapure water to obtain an Au growth solution, which was stored at 4°C until 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 M NaOH solution, 200 μL of 0.5 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) 1.35 g of FeCl3·6H2O and 1 g of PEG were added to 40 mL of ethylene glycol and stirred until a clear solution was obtained. 3.6 g of NaAc was slowly added and vigorously stirred for 30 min. The mixed solution was transferred to a Teflon-lined stainless steel autoclave and reacted at 200°C for 10 h. After cooling to room temperature, the mixture was washed by magnetic separation using ultrapure water and anhydrous ethanol alternately, and the washing was repeated three times. The washed product was placed in a vacuum drying oven at 60°C for 10 h before use to obtain Fe3O4 MNPs. 6) Heat 100 mL of 0.01% HAuCl4 solution to boiling in an oil bath. Rapidly add 1 mL of 1.0% trisodium citrate solution while vigorously stirring. The solution first changes from light yellow to gray-black and finally to wine red and remains unchanged. Keep boiling the solution for 15 minutes and then cool it at room temperature to obtain the AuNPs solution, which is stored at 4°C for later use. Mix 1.5 mL of 0.1mA A solution and 0.5 mL of 1mM AgNO3 solution into 10 mL of AuNPs solution. After vigorously stirring for 30 minutes, the wine red turns to orange-yellow. Centrifuge the stirred solution, and redisperse the precipitate in 10 mL of ultrapure water to obtain the Au@AgNPs solution for later use. 7) 40 mg of the Fe3O4 MNPs prepared in step 5) were added to 40 mL of a 10 mg / mL PEI solution and ultrasonically mixed for 2 hours to obtain a Fe3O4@PEI solution. The Fe3O4@PEI solution was then magnetically washed three times with ultrapure water to remove excess PEI, thereby obtaining Fe3O4@PEI. 10 mg of the Fe3O4@PEI solution was added to 100 mL of the Au@AgNPs solution prepared in step 6), ultrasonically mixed for 30 minutes, and the product was separated using a magnet, washed four times with deionized water, and redispersed in 4 mL of ultrapure water to obtain a Fe3O4@Au@AgMNPs solution for later use. (3) Construction of aptamer sensors by combining Fe3O4@Au@AgNPs with Ag@4MBA@AuNPs 8) Add 20 μL of 10 μM SH-Apt solution to 1 mL of the Ag@4MBA@Au NPs solution prepared in step 4) and incubate at room temperature for 12 h. Excess SH-Apt was removed by centrifugation, and the product was washed three times with PBS buffer and redispersed in 1 mL of PBS buffer to obtain an 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. Then treat with 2 μM MCH for 1 h to block unbound sites. After washing with ethanol magnetic separation, collect the product and add 1 mL of PBS buffer to prepare the 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 and gently shaken on a digital oscillator for 1 hour. The mixture was magnetically separated and washed three times and redispersed in the original volume of PBS buffer to obtain an Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution, which is the SERS aptamer sensor. (4) SERS detection of Salmonella 50 μL of sample was added to 100 μL of the Ag@4MBA@Au-Fe3O4@Au@Ag magnetic composite nanoparticle solution prepared in step 10) and vortexed for 2 minutes. Due to the preferential binding between the aptamer and Salmonella, some Ag@4MBA@Au-Apt was separated from the Fe3O4@Au@Ag-cDNA in the competitive binding system. The Ag@4MBA@Au-Apt detached from the supernatant was then removed by magnetic separation. Finally, the precipitate was redispersed in 10 μL of PBS buffer and placed on a clean silicon chip for drying. It was then 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 solution is 10 mM and the pH is 7.
4.
4. The method according to claim 1, wherein 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, wherein Step 8) The sequence of the SH-cDNA is: 5′-SH-C6-CTGTCATAATGTCAAGTC-3′, as shown in SEQ ID NO.2.