A multiple sers signal enhanced nano "sandwich" bacterial detection system and a preparation method thereof

Abstract

This invention discloses a nano-"sandwich" bacterial detection system with multiple SERS signal enhancement and its preparation method, belonging to the fields of functional material preparation and Raman detection. The nano-"sandwich" bacterial detection system includes highly adhesive labeled nanoparticles D-Au@Ag-C, whose structure consists of Au@Ag core-shell nanoparticles grown in situ on the surface of dendritic mesoporous silica DMSN, simultaneously modified with concanavalin A and a SERS tag; and a capture substrate Fe3O4@Au-Ab, whose structure consists of Fe3O4 nanoparticles with a gold coating on the surface, simultaneously modified with bacterial targeting antibodies. The nano-"sandwich" bacterial detection system of this invention can highly identify Staphylococcus aureus in aqueous solutions or blood, with extremely high detection accuracy and a very low detection limit, showing great promise for on-site detection of clinical bacterial infection-related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of functional material preparation and Raman detection, and particularly relates to a nano "sandwich" bacterial detection system with multiple SERS signal enhancement and a preparation method thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Bacterial infectious diseases are a major public health threat worldwide, and it is essential to develop a rapid, sensitive and effective diagnostic method. Traditional blood bacterial detection methods involve three main techniques, including standard plate colony counting, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). However, the standard plate colony counting method usually requires complex and time-consuming biochemical processes (usually several days) for separation, incubation and counting. The PCR method requires expensive professional equipment, complex processing procedures (cell lysis, nucleic acid extraction, etc.). The ELASA method is cumbersome, labor-intensive, time-consuming, and has many external interference factors and poor repeatability. In order to overcome these shortcomings, it is urgent to develop a technology that can detect blood bacteria in bacterial infectious diseases in a shorter time and with simple operation.

[0004] With the deepening of research, more and more means and techniques are applied to the detection of blood bacteria. Based on the need for rapid sensing, surface-enhanced Raman scattering (SERS), which benefits from the localized surface plasmon resonance (LSPR) of noble metals, has become a promising means of non-destructive detection of bacteria. SERS is a powerful analytical technique, and its portable detection equipment makes it simple and fast to operate. The main advantage of detecting biomarkers is its inherent ability to provide specific high signal "fingerprint" spectra that can selectively detect pathogenic bacteria in complex environments.

[0005] In recent years, by constructing excellent SERS sensors, the detection of a variety of pathogenic bacteria has been successfully achieved. However, the preparation of high-sensitivity SERS sensors is still a serious challenge, and the detection sensitivity of the current SERS sensor still does not reach the ideal effect. SUMMARY

[0006] In order to solve the problems of the prior art, the present application aims to provide a nano "sandwich" bacterial detection system with multiple SERS signal enhancement and a preparation method thereof. The nano "sandwich" bacterial detection system provided by the present application has good hemolytic performance, good SERS enhancement effect, and can well capture Staphylococcus aureus in an aqueous solution or blood, with a very low detection lower limit.

[0007] To achieve the above object, the technical scheme of the present application is:

[0008] In the first aspect of the present application, a nano "sandwich" bacterial detection system with multiple SERS signal enhancement is provided, comprising a labeled nanoparticle D-Au@Ag-C with high bacterial adhesion, and a capture substrate Fe3O4@Au-Ab.

[0009] The D-Au@Ag-C structure is: dendritic mesoporous silica DMSN surface in situ growth of Au@Ag core-shell nanoparticles, while modified with concanavalin A (Con A) and SERS labels.

[0010] The Fe3O4@Au-Ab is: Fe3O4 nanoparticles coated with a gold coating on the surface, while modified with a bacterial targeting antibody.

[0011] Preferably, the SERS label is 4-mercaptobenzoic acid (4-MBA).

[0012] Preferably, the dendritic mesoporous silica DMSN has a diameter of 160-180 nm.

[0013] Preferably, the bacteria are Staphylococcus aureus.

[0014] The nano "sandwich" bacterial detection system of the present application can form a sandwich structure of D-Au@Ag-C / bacteria / Fe3O4@Au-Ab when detecting bacteria, with multiple enhancement effects. Especially for the detection of Staphylococcus aureus, the lower limit of detection is extremely low, the selectivity is good, and the detection sensitivity is high.

[0015] In the second aspect of the present application, a preparation method of the nano "sandwich" bacterial detection system with multiple SERS signal enhancement is provided, comprising the following steps:

[0016] 1) Preparation of D-Au@Ag-C:

[0017] DMSN is modified with amino groups to make the surface positively charged to obtain DMSN-NH2; DMSN-NH2 is used as a raw material to grow gold on the surface of DMSN by seed solution growth method to obtain DMSN-Au; DMSN-Au is modified with SERS labels to obtain DMSN-Au 4-MBA ; silver Ag is grown on the surface of DMSN-Au 4-MBA to obtain DMSN-Au@Ag; DMSN-Au@Ag is modified with SERS labels to obtain DMSN-Au@Ag 4-MBA ; DMSN-Au@Ag 4-MBA is modified with concanavalin A (Con A) to obtain D-Au@Ag-C;

[0018] 2) Preparation of Fe3O4@Au-Ab:

[0019] Preparation of gold seed solution; surface modification of Fe3O4 nanoparticles to make them positive potential, then mixed with gold seed solution, using seed solution growth method to grow gold on the surface of Fe3O4 nanoparticles, to obtain Fe3O4@Au; surface modification of Fe3O4@Au with bacterial targeting antibody Ab to obtain Fe3O4@Au-Ab.

[0020] Preferably, the preparation method of dendritic mesoporous silica DMSN comprises the following steps: triethanolamine is fully dissolved into ultrapure water, the obtained solution is heated and stirred; sodium salicylate and cetyltrimethylammonium bromide are added to the solution and stirred; under stirring, tetraethyl orthosilicate is added dropwise to the above solution, and after the dropwise addition is completed, the stirring is continued; after the solution is cooled to room temperature, the product is collected by centrifugation; washed; remove cetyltrimethylammonium bromide; dry, to obtain dendritic mesoporous silica DMSN.

[0021] Preferably, in step 1), the seed solution growth method is specifically as follows: DMSN-NH2 is dispersed into deionized water, tetrachloroauric acid solution is added, and ultrasonic mixing is performed; under vigorous stirring, sodium borohydride solution is quickly injected into the mixed solution, stirring reaction is performed, centrifugation is performed and ultrapure water is used for multiple washing, to obtain DMSN-m-Au seed nanoparticles; the DMSN-m-Au seed solution is resuspended into hydroxylamine hydrochloride solution, vigorous stirring is performed at room temperature, and gold chloride solution is quickly added; stirring is performed, and centrifugation is performed to collect DMSN-Au.

[0022] Preferably, the surface growth of silver Ag on DMSN-Au is specifically as follows: DMSN-Au is dispersed into polyvinylpyrrolidone aqueous solution and stirred; silver nitrate and ascorbic acid aqueous solution are added to the mixed solution for incubation, to reduce silver ions into metallic silver; centrifugation is performed, and washing is performed to remove excess reactants, to obtain DMSN-Au@Ag. 4-MBA 4-MBA Preferably, the surface growth of silver Ag on DMSN-Au is specifically as follows: DMSN-Au is dispersed into polyvinylpyrrolidone aqueous solution and stirred; silver nitrate and ascorbic acid aqueous solution are added to the mixed solution for incubation, to reduce silver ions into metallic silver; centrifugation is performed, and washing is performed to remove excess reactants, to obtain DMSN-Au@Ag.

[0023] Preferably, the Con A modification of DMSN-Au@Ag is specifically as follows: the carboxyl group on 4-MBA in DMSN-Au@Ag is activated by using a mixed solution of tetramethylammonium hydroxide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt; Con A solution is mixed with the activated DMSN-Au@Ag, and stirring is performed at room temperature, to obtain D-Au@Ag-C. 4-MBA 4-MBA Preferably, the Con A modification of DMSN-Au@Ag is specifically as follows: the carboxyl group on 4-MBA in DMSN-Au@Ag is activated by using a mixed solution of tetramethylammonium hydroxide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt; Con A solution is mixed with the activated DMSN-Au@Ag, and stirring is performed at room temperature, to obtain D-Au@Ag-C. 4-MBA

[0024] ​​​Preferably, in step 2), the preparation method of the iron oxide nanoparticles is as follows: ferric chloride hexahydrate and trisodium citrate (TSC) are dissolved in ethylene glycol, and ethylene glycol containing sodium acetate is added under magnetic stirring; the mixture is stirred vigorously at room temperature; then, the mixture is transferred to an autoclave, and the autoclave is placed in an oven at 200°C for heating to obtain a black precipitate; the precipitate is washed, collected with a magnet, and dried to obtain the iron oxide nanoparticles.

[0025] Preferably, the surface modification of the Fe3O4 nanoparticles specifically involves: dispersing the Fe3O4 nanoparticles in a polyethyleneimine (PEI) aqueous solution, mixing thoroughly, and then modifying the surface of the Fe3O4 nanoparticles with a layer of PEI through self-assembly, thereby modifying the Fe3O4 nanoparticles to a positive potential.

[0026] Preferably, in step 2), the modification of the Fe3O4@Au surface with bacterial targeting antibody Ab to obtain Fe3O4@Au-Ab specifically involves: adding polyethylene glycol (PEG) to Fe3O4@Au, ultrasonically mixing, centrifuging to remove excess PEG, and obtaining Fe3O4@Au-PEG; activating the carboxyl groups on the surface of Fe3O4@Au-PEG; ultrasonicating at room temperature, resuspending the activated nanoparticles in phosphate-buffered saline containing bacterial targeting antibody Ab, and stirring to obtain Fe3O4@Au-Ab.

[0027] A third aspect of the present invention provides a bacterial detection method employing the aforementioned multiple SERS signal enhancement nano-"sandwich" bacterial detection system, the specific steps of which are as follows:

[0028] The Fe3O4@Au-Ab capture substrate was added to the sample for testing, incubated, washed, and then D-Au@Ag-C labeled nanoparticles with high bacterial adhesion were added, mixed, washed using magnetic separation, and transferred to a silicon wafer. Its temperature at 1583 cm⁻¹ was measured. -1 Raman signal.

[0029] Preferably, the bacteria is Staphylococcus aureus.

[0030] The beneficial effects of this invention are as follows:

[0031] Inspired by the natural phenomenon of fruit-laden branches growing close together, the inventors of this invention introduced dendritic mesoporous silica (DMSN) nanoparticles with pollen morphology into SERS-labeled nanoparticles. Due to multivalent interactions, the nanoscale topology enhances the interaction between the nanoparticles and bacteria, thereby enhancing bacterial capture. Au@Ag core-shell nanomaterials with excellent SERS performance were loaded onto the DMSN surface via in-situ growth, and simultaneously modified with concanavalin A and the SERS tag 4-MBA to obtain labeled nanoparticles D-Au@Ag-C with high bacterial adhesion. Furthermore, to enhance the intensity of bacterial detection signals, an innovation was made based on traditional magnetic Fe3O4 nanomaterials. A magnetic substrate Fe3O4@Au-Ab MNPs with SERS enhancement function was synthesized using a noble metal shell coating method, and simultaneously modified with bacterial-targeting antibodies. This gave the magnetic substrate Fe3O4@Au-Ab not only magnetic separation properties, SERS enhancement properties, and stability.

[0032] The multi-SERS signal-enhanced nano-"sandwich" bacterial detection system of this invention, when performing bacterial detection, ultimately forms a sandwich structure of D-Au@Ag-C / bacteria / Fe3O4@Au-Ab, exhibiting multiple enhancement effects:

[0033] (1) The Au@Ag core-shell structure in D-Au@Ag-C material can provide a stronger coupled SERS signal than pure Au nanoparticles; the introduction of DMSN plays a role in spatial enrichment of Au@Ag, and a high coupling field can be generated between two adjacent Au@Ag nanoparticles.

[0034] (2) The Au shell on the outer layer of the magnetic Fe3O4@Au MNPs has high SERS activity, which can enhance the signal of nearby Raman molecules; the inner Fe3O4 core has good magnetic reaction, realizing the separation and enrichment of the target substance from other substances in the mixture.

[0035] The present invention provides a nano-"sandwich" bacterial detection system with enhanced multiple SERS signals, which successfully identifies Staphylococcus aureus in aqueous solution or blood with extremely high accuracy. This sandwich detection strategy has great potential for on-site detection of clinical bacterial infection-related diseases. Attached Figure Description

[0036] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0037] Figure 1The diagram shows the synthesis and principle mechanism of the nano-"sandwich" bacterial detection system with multiple SERS signal enhancement of the present invention; a) is a schematic diagram of D-Au@Ag synthesis; b) is a schematic diagram of Fe3O4@Au synthesis; c) is a principle mechanism diagram of the nano-"sandwich" bacterial detection system with multiple SERS signal enhancement during bacterial detection, and shows three typical types of enhancement "hot spots" generated in the sandwich structure, where 1 is a weak "hot spot" generated by the surface of the plasma nanoparticles (Au@Ag); 2 is a strong "hot spot" formed by the nano-gap formed by the close proximity of Au@Ag nanoparticles under the constraint of DMSN; and 3 is a coupling "hot spot" between Fe3O4@Au (part of the enrichment module) and Au@Ag (part of the signal module).

[0038] Figure 2 TEM images of DMSN-Au@Ag and Fe3O4@Au prepared in the embodiments of the present invention; wherein, a) is a representative TEM image of DMSN nanoparticles, b) is a representative TEM image of DMSN-Au@Ag labeled nanoparticles with high bacterial adhesion, and c) is a representative TEM image of Fe3O4@Au trapping substrate;

[0039] Figure 3 The following are the ultraviolet-visible absorption spectra of different nanoparticles prepared in the embodiments of the present invention; wherein, a) is the ultraviolet-visible absorption spectrum of DMSN, DMSN-m-Au, DMSN-Au and DMSN-Au@Ag nanoparticles, and b) is the ultraviolet-visible absorption spectrum of Fe3O4, Fe3O4-PEI, Fe3O4-Au and Fe3O4@Au nanoparticles;

[0040] Figure 4 Zeta potential diagrams of different nanoparticles prepared in the embodiments of the present invention; wherein, a) is the zeta potential of DMSN, DMSN-m-Au, DMSN-Au, DMSN-Au@Ag and D-Au@Ag-C nanoparticles, and b) is the zeta potential of Fe3O4, Fe3O4-PEI, Fe3O4-Au, Fe3O4@Au and Fe3O4@Au-Ab nanoparticles;

[0041] Figure 5 The hemolytic properties of D-Au@Ag-C and Fe3O4@Au-Ab nanoparticles prepared in the embodiments of the present invention;

[0042] Figure 6 SEM images of SERS tags and capture substrates capturing Staphylococcus aureus, where 2 is the SERS tag with a size of approximately 170 nm; 3 is the capture substrate with a size of approximately 80 nm; and 1 is Staphylococcus aureus with a size of approximately 1 micrometer.

[0043] Figure 7 Raman spectra of DMSN-Au, DMSN-Au@Ag, and D-Au@Ag-C prepared for embodiments of the present invention;

[0044] Figure 8 The surface spectroscopy of different nanoparticles prepared for embodiments of the present invention and their wavelengths at 1583 cm⁻¹ -1 The Raman peak intensities are shown at 1583 cm⁻¹; where a) is the Raman spectrum of Au@Ag and DMSN-Au@Ag, and b) is the Raman spectrum of Au@Ag and DMSN-Au@Ag at 1583 cm⁻¹. -1 c) shows the Raman peak intensity at 1583 cm⁻¹, representing the Raman spectra of the D-Au@Ag-C / bacteria and D-Au@Ag-C / bacteria / Fe3O4@Au-Ab “sandwich” bacterial detection systems. d) shows the corresponding Raman peak intensity at 1583 cm⁻¹ for both the D-Au@Ag-C / bacteria and D-Au@Ag-C / bacteria / Fe3O4@Au-Ab “sandwich” bacterial detection systems. -1 The Raman peak at that location is strong;

[0045] Figure 9 The diagram shows the detection of different bacteria by the "sandwich" bacterial detection system of the present invention; where a) represents the specificity of the detection of Staphylococcus aureus, and b) represents the repeatability of the detection of Staphylococcus aureus.

[0046] Figure 10 The graph shows the detection results of the "sandwich" bacterial detection system of the present invention for different concentrations of Staphylococcus aureus in aqueous solution; wherein, a) is the detection performance of Staphylococcus aureus in aqueous solution, and b) is the result of the detection at 1583 cm⁻¹ in graph a). -1 Raman peak intensity fitting diagram;

[0047] Figure 11 The graph shows the detection results of the "sandwich" bacterial detection system of this invention on different concentrations of Staphylococcus aureus in 1% blood solution; where a) is the detection performance of Staphylococcus aureus in 1% blood solution, and b) is the result at 1583 cm⁻¹ in graph a). -1 Raman peak intensity fitting diagram;

[0048] Figure 12 The "sandwich" bacterial detection system of this invention displays the detection spectrum of blood bacterial samples from infected mice; wherein, a) is the Raman spectrum of the blood bacterial sample from infected mice, and b) is the spectrum based on the 1583 cm⁻¹ in figure a). -1 The comparison between the bacterial count calculated from the Raman peak intensity and the results obtained from plate culture, with the inset in b) showing the recovery rate of the labeled nanoparticles with high bacterial adhesion and the capture substrate. Detailed Implementation

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] Example 1

[0051] A method for preparing a nano-"sandwich" bacterial detection system with enhanced multiple SERS signals includes the following steps:

[0052] I. Materials: Tetraethyl orthosilicate (TEOS), hydrochloric acid (HCl), acetone, cetyltrimethylammonium bromide (CTAB), sodium salicylate (NaSal), triethanolamine (TEA), sodium borohydride (NaBH4), phosphate buffered saline (PBS), tetramethylammonium hydroxide 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide sodium salt (NHS) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ferric chloride hexahydrate (FeCl3·6H2O), anhydrous sodium acetate (NaAc), sodium citrate, ethylene glycol (EG), polyvinylpyrrolidone K30 (PVP-K30), silver nitrate (AgNO3), ammonia (28%), 4-mercaptobenzoic acid (4-MBA), (3-aminopropyl)triethoxysilane (APTES), hydroxyammonium chloride (NH2OH·HCl), ascorbic acid (AA), gold(III) chloride trihydrate (HAuCl4·3H2O), ethanol, bovine serum albumin (BSA), and concanavalin A (Con A) were purchased from Yuanye Reagent Co., Ltd. (Shanghai, China). All other chemicals were obtained from Adamas beta and were ready for use without further purification. All experiments used deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ).

[0053] II. Experimental Scheme

[0054] 1. Preparation and amino modification of dendritic mesoporous silica (DMSN) nanoparticles:

[0055] First, 0.068 g of TEA was completely dissolved in 25 mL of ultrapure water. The resulting solution was heated to 80 °C and stirred continuously for 0.5 h. Then, 168 mg of sodium salicylate (NaSal) and 380 mg of CTAB were added to the reaction solution. After stirring for 1 h, 4 mL of TEOS solution was added dropwise to the solution at a gentle stirring speed (300 rpm), and stirring was continued gently for 2 h. After cooling the solution to room temperature, the product was collected by high-speed centrifugation (20,000 rpm, 15 min). The product was washed three times each with water and ethanol to remove excess reactants. Finally, the material was resuspended in a mixture of hydrochloric acid and ethanol (HCl:ethanol = 1:9) and refluxed at 80 °C for 6 h. This extraction was repeated three times to remove the surfactant (CTAB). The finally purified DMSN was vacuum dried overnight at 50 °C.

[0056] To impart a positive charge to the DMSN surface, amino groups were modified onto it. 100 μL of APTES was added to 10 mL of DMSN solution (10 mg / mL), and the mixture was heated to 80 °C and stirred for 12 h to obtain DMSN-NH2 nanoparticles. After washing several times with ultrapure water, the nanoparticles were stored at 4 °C for later use.

[0057] 2. Preparation of DMSN-Au@Ag nanoparticles:

[0058] 50 mg of the prepared DMSN-NH2 NPs were dispersed in 5 mL of deionized water. Then, 500 μL of HAuCl4 solution (20 mM) was added, and the mixture was sonicated for 0.5 h. Under vigorous stirring, 5 mL of freshly prepared NaBH4 solution (0.1 M) was rapidly injected into the mixture. After stirring for 1 h, the mixture was centrifuged and washed repeatedly with ultrapure water. Au nanoparticles with a diameter of approximately 3 nm were grown on the surface of the DMSN-NH2 NPs, yielding DMSN-m-Au seed nanoparticles. 4 mL of the DMSN-m-Au NPs seed solution (5 mg / mL) was resuspended in hydroxylamine hydrochloride solution (12.5 mL, 2.8 mM), and the mixture was stirred vigorously at room temperature. 150 μL of HAuCl4 solution (50 mM) was rapidly added. After stirring for 0.3 h, the DMSN-Au NPs were collected by centrifugation and resuspended in ethanol. Subsequently, 50 μL of 4-MBA (10 mM) solution was added. After stirring for two hours, several centrifugations and washings were performed to obtain DMSN-Au. 4-MBA Nanoparticles were collected and stored at 4°C. Additionally, to enhance the SERS activity of the nanoparticles, DMSN-Au... 4-MBA Ag shells are grown on the surface of nanoparticles. 6 mg DMSN-Au 4-MBANPs were dispersed in 60 mL of PVP-K30 aqueous solution (2 mg / mL) and stirred thoroughly for 30 min. Then, 600 μL of silver nitrate (10 mM) and 600 μL of ascorbic acid (10 mM) aqueous solution were added to the mixture, and the mixture was incubated for 1 h to reduce silver ions to metallic silver. The mixture was centrifuged at 9000 rpm for 15 min and washed multiple times with ultrapure water to remove excess reactants, yielding purified DMSN-Au@Ag nanoparticles.

[0059] 3. Preparation of labeled nanoparticles with high bacterial adhesion (D-Au@Ag-C nanoparticles):

[0060] 6 mL of DMSN-Au@Ag ethanol solution was thoroughly mixed with 60 μL of 4-MBA (10 mM) solution and stirred at room temperature for 2 h. 4-MBA was modified onto the nanoparticles by forming Ag-S bonds with thiol groups. Excess 4-MBA was then removed by centrifugation, and the resulting sample was resuspended in 2 mL of PBS solution to obtain monodisperse DMSN-Au@Ag. 4-MBA Nanoparticle solution. DMSN-Au@Ag was activated with an EDC / NHS mixed solution. 4-MBA Carboxyl groups on 4-MBA in nanoparticles. 200 μL of Con A solution was mixed with the activated nanoparticles and stirred at room temperature for 12 h. Subsequently, 200 μL of 10 wt% BSA solution was added, and stirring continued for 1 h to block excess amino groups and reduce non-specific binding of the nanoparticles. The prepared D-Au@Ag-C nanoparticles were resuspended in PBS buffer (10 mM) for later use.

[0061] 4. Preparation of 3nm Au nanoparticles:

[0062] Trisodium citrate (1.5 mL, 1 wt%) and tetrachloroauric acid aqueous solution (148.5 mL, 0.01 wt%) were thoroughly mixed and vigorously stirred at room temperature for 15 min. Then, freshly prepared sodium borohydride ice-water solution (4.5 mL, 0.1 M) was rapidly injected into the mixture. Stirring continued for 4 h to obtain a colloidal gold solution with a particle size of 3 nm.

[0063] 5. Preparation of Fe3O4 nanoparticles:

[0064] 1.62 g of ferric chloride hexahydrate and 0.5 g of trisodium citrate (TSC) were dissolved in 40 mL of ethylene glycol. 40 mL of ethylene glycol containing 6.64 g of sodium acetate (NaAc) was added under magnetic stirring. The mixture was stirred vigorously at room temperature for 0.5 h. The mixture was then transferred to a 100 mL autoclave lined with polytetrafluoroethylene (PTFE). The autoclave was heated in a 200 °C oven for 10 h to obtain a black precipitate. The precipitate was then washed four times each with ethanol and water under manual shaking, collected magnetically, and vacuum dried at 60 °C for 6 h for further use.

[0065] 6. Preparation of Fe3O4@Au core-shell nanoparticles:

[0066] 10 mg of Fe3O4 nanoparticles were dispersed in 20 mL of PEI aqueous solution (5 mg / mL), thoroughly mixed, and sonicated for 2 h. A layer of PEI was then applied to the surface of the Fe3O4 nanoparticles via self-assembly, modifying the material to a positive potential, resulting in Fe3O4-PEI nanoparticles. The obtained Fe3O4-PEI nanoparticles were mixed with 3 nm Au seeds and sonicated for 30 min to prepare Fe3O4-Au seed nanoparticles. The product was magnetically collected and washed four times with deionized water under manual shaking to remove excess Au seeds. The prepared Fe3O4-Au nanoparticles were redispersed in 50 mL of HAuCl4 solution (0.4 mM), and 0.5 mL of NH2OH·HCl aqueous solution and 0.15 g of PVP-K30 were rapidly added under an ultrasonic atmosphere. After reacting for 15 min, the final product Fe3O4@Au was magnetically collected, washed multiple times, and resuspended in 10 mL of deionized water for later use.

[0067] 7. Preparation of the trapping substrate (Fe3O4@Au-Ab nanoparticles):

[0068] 1 mg of polyethylene glycol (PEG) was added to Fe3O4@Au (5 mL). The solution was sonicated for 3 hours, then centrifuged at 9000 rpm for 10 minutes to remove excess PEG. Subsequently, the modified nanoparticles were resuspended in PBS buffer (5 mL; pH 7.4) containing EDC (200 μg / mL) and NHS (200 μg / mL) to activate the surface carboxyl groups. After sonication at room temperature for 30 min, the activated nanoparticles were resuspended in PBS (1 mL) containing 20 μg of bacterial targeting antibody (Ab), and stirring was continued for 2 h. Finally, 500 μL of 10 wt% BSA aqueous solution was added to block excess binding sites. The resulting Fe3O4@Au-Ab nanoparticles were stored at 4 °C for later use.

[0069] 8. Morphology of labeled nanoparticles with high bacterial adhesion and the trapping substrate:

[0070] Take 100 μL of DMSN, DMSN-Au@Ag nanoparticles and Fe3O4@Au nanoparticles, dilute them to 1 mL with deionized water, and drop 10 μL of the diluted nanoparticles onto a copper grid. Dry them overnight in an electronic dehumidifier and observe the morphology of the nanoparticles using a transmission electron microscope (TEM).

[0071] 9. UV-Vis absorption spectra of labeled nanoparticles with high bacterial adhesion and the trapping substrate:

[0072] DMSN, DMSN-Au, and DMSN-Au@Ag nanoparticles were diluted with deionized water to the same concentration. The solutions were placed in quartz cuvettes, and the UV-Vis absorption spectra of each solution were detected using a UV-Vis spectrophotometer in the wavelength range of 380 nm to 700 nm. A control treatment was performed to verify the synthesis of SERS-tagged D-Au@Ag-C nanoparticles.

[0073] Fe3O4, Fe3O4-PEI, Fe3O4-Au, and Fe3O4@Au nanoparticles were diluted with deionized water to the same concentration. The solutions were placed in quartz cuvettes, and the UV-Vis absorption spectra of each solution were detected using a UV-Vis spectrophotometer in the wavelength range of 300 nm to 700 nm. A control group was established to verify the synthesis of the Fe3O4@Au-Ab nanoparticles used for capturing the substrate.

[0074] 10. Characterization of the zeta potential of labeled nanoparticles with high bacterial adhesion and the trapping substrate:

[0075] Take 100 μL of DMSN, DMSN-NH2, DMSN-m-Au, DMSN-Au, DMSN-Au@Ag and D-Au@Ag-C nanoparticles, dilute to 2 mL with deionized water, and measure the zeta potential of the nanoparticles in an aqueous environment using a nanoparticle size analyzer. Repeat the process three times and record the data.

[0076] Take 100 μL of Fe3O4, Fe3O4-PEI, Fe3O4-Au, Fe3O4@Au and Fe3O4@Au-Ab nanoparticles, dilute them to 2 mL with deionized water, and use a nanoparticle size analyzer to detect the zeta potential of the nanoparticles in an aqueous environment. Repeat the process three times and record the data.

[0077] 11. Hemolysis experiment of labeled nanoparticles with high bacterial adhesion and capturing substrate:

[0078] 1.2 mL of blood was drawn from the eyeballs of healthy 7-week-old mice and thoroughly mixed with EDTA solution. The mixture was centrifuged at 2000 rpm for 10 minutes, the supernatant was removed, and the particles were washed with PBS to obtain pure red blood cells. The resulting 20% ​​red blood cell suspension was then diluted with PBS buffer to a series of different concentrations (containing 25, 50, 100, and 200 μg / mL). -1 A mixture of D-Au@Ag-C / Fe3O4@Au-Ab materials (DMSN and Fe3O4 nanoparticles) was prepared. Blood samples in PBS and deionized water served as negative and positive controls, respectively. All samples incubated at 37°C for 2 hours were centrifuged, and the absorbance of the supernatant was measured.

[0079] 12. Bacterial culture:

[0080] Staphylococcus aureus, Escherichia coli, Salmonella plague, Bacillus subtilis, MRSA, and Pseudomonas aeruginosa were used in the experiment. These bacteria were grown overnight in LB medium at 37°C, and the growth was determined by centrifugation. The bacterial concentration in PBS solution was obtained by measuring the optical density at 600 nm. The OD values ​​of Staphylococcus aureus and Escherichia coli were 1, corresponding to concentrations of 4 × 10⁻⁶. 9 and 2×10 9 .

[0081] 13. The ability of labeled nanoparticles with high bacterial adhesion and the trapping substrate to capture bacteria:

[0082] To investigate the excellent bacterial capture ability of SERS tags, the adhesion characteristics of surface morphology to bacteria were studied using scanning electron microscopy. D-Au@Ag-C and Fe3O4@Au-Ab NPs were mixed with bacteria and incubated in a shaker for 30 minutes. The mixture was then fixed with 2.5% glutaraldehyde for 2 hours, followed by dehydration with a series of ethanol-water gradients (50%, 70%, 90%, and 100%) for 10 minutes each. Finally, the sample was dropped onto a silicon wafer, dried, and then sprayed with platinum before SEM imaging.

[0083] 14. Investigation into the SERS performance of labeled nanoparticles with high bacterial adhesion:

[0084] DMSN-Au, DMSN-Au@Ag, and D-Au@Ag-C nanoparticles were diluted to the same concentration with deionized water. 10 μL of the diluted nanoparticles were dropped onto a silicon wafer, and their Raman spectra were measured using a micro Raman spectrometer. The experiment was repeated three times.

[0085] 15. Study on the enhancement performance of SERS coupling by DMSN and the effect of Fe3O4@Au-Ab on SERS coupling performance:

[0086] DMSN-Au@Ag and Au@Ag nanoparticles were diluted to the same concentration, and 10 μL was dropped onto a silicon wafer. Measurements were taken from 1000 to 1700 cm⁻¹. -1 Raman spectra within the range.

[0087] D-Au@Ag-C and 10 6 Incubate with CFU / mL Staphylococcus aureus for 30 min, then divide into two portions. One portion is left to stand, while the other portion is incubated with 100 μL of Fe3O4@Au-Ab nanoparticles for 30 min. After incubation, resuspend the nanoparticles in 100 μL of PBS using centrifugation and magnetic enrichment. Test at 1000–1700 cm⁻¹. -1 Raman spectra within the range.

[0088] 16. Investigation into the specific detection of Staphylococcus aureus using a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate:

[0089] Prepare 1 mL of deionized water at different concentrations (20, 50, 10). 2 10 3 and 10 4 CFU mL -1 Bacterial samples were collected. 50 μL of the Fe3O4@Au-Ab NPs nanosystem was added to each sample. Incubation was performed for 30 minutes. After multiple washes with PBS, D-Au@Ag-C NPs were added, and the mixture was gently shaken and incubated for another 30 minutes. Finally, the resulting D-Au@Ag-C / S. aureus / Fe3O4@Au-Ab complex was washed three times using magnetic separation and transferred to a silicon wafer, where its temperature at 1583 cm⁻¹ was measured. -1 The Raman signal was obtained. All the above experiments were repeated three times.

[0090] 17. Investigation on the specificity and repeatability of the sandwich system of labeled nanoparticles with high bacterial adhesion for the detection of Staphylococcus aureus:

[0091] To perform selective analysis, 1 mL of a 10... 3 Staphylococcus aureus at a concentration of 10 cells / mL and 5 other bacteria at a concentration of 10 5 Interfering bacteria (Escherichia coli, Salmonella typhimurium, Bacillus subtilis, MRSA, and Pseudomonas aeruginosa) at cell / mL concentrations were incubated with the detection nanosystem for 30 min with gentle shaking (250 rpm, 37°C). Finally, the mixture was washed three times and magnetically enriched. SERS signals of the six different bacterial species were measured on the finally enriched trace solutions and plotted on a 1583 cm⁻¹ graph. -1 Raman intensity histogram.

[0092] To investigate the repeatability of Staphylococcus aureus detection, 10 batches of nanomaterials with a repeatable measurement concentration of 10 were prepared. 5 Staphylococcus aureus at cell / mL, plotted on a 1583 cm⁻¹ plot. -1 Raman intensity histograms were plotted. The SERS testing experiments for each batch of materials were repeated three times.

[0093] 18. Detection of bacteria in aqueous solution using a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate:

[0094] Staphylococcus aureus samples of different concentrations (0, 20, 50, 10 mL) were prepared using deionized water. 2 10 3 and 10 4 (CFU / ml). Then, 50 μL of Fe3O4@Au-Ab NPs was added to each sample. Incubation for 30 minutes. After washing with PBS, D-Au@Ag-C NPs were added and the mixture was gently shaken. Finally, the resulting D-Au@Ag-C / S. aureus / Fe3O4@Au-Ab complex was washed three times using magnetic separation and transferred to a silicon wafer, where its temperature was measured at 1583 cm⁻¹. -1 The Raman signal was obtained. All the above experiments were repeated three times.

[0095] 19. Detection of bacteria in 1% blood using a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate:

[0096] Fresh mouse blood was diluted with deionized water to a 1% blood concentration solution for later use. Different concentrations of Staphylococcus aureus (0, 10, ...) were prepared in 1 mL of the 1% blood solution. 2 10 3 10 4 and 10 5 Samples were prepared in CFU / ml. Then, 50 μL of Fe3O4@Au-Ab NPs were added to each sample. Incubation was performed for 30 minutes. After washing with PBS, D-Au@Ag-CNPs were added and the mixture was gently shaken. Finally, the resulting D-Au@Ag-C / S. aureus / Fe3O4@Au-Ab complex was washed three times using magnetic separation and transferred to a silicon wafer, where its temperature was measured at 1583 cm⁻¹. -1 The Raman signal was obtained. All the above experiments were repeated three times.

[0097] 20. Detection of bacteria in blood samples from infected mice using a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate:

[0098] To detect bacteria in mouse blood, 100 μL of bacterial PBS suspension (5 × 10⁻⁶) was prepared.9 A mouse model of bacterial infection was established by injecting CFU (conjugated Staphylococcus aureus quinquefolius) into the bloodstream of Balb / c mice via tail vein. One hour post-infection, 10 μL of blood was drawn from the orbital cavity of the mice and diluted to 1% with PBS. Subsequently, 50 μL of Fe3O4@Au-Ab NPs was incubated with the blood sample at 37°C for 30 minutes, followed by the addition of 50 μL of D-Au@Ag-C. Finally, magnetic enrichment was performed, followed by multiple washes to remove unbound bacteria and blood cells. The complex was transferred to a silicon wafer, and the corresponding Raman signal was detected. The bacterial count was calculated based on the intensity in the standard curve and compared with the results of bacterial electroplating.

[0099] In the experiment, 6-week-old female Balb / c mice, weighing 20 grams, were obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd. They underwent a one-week acclimatization period in the laboratory before the experiment. All animal experiments complied with the regulations of the Experimental Animal Center of Shandong University.

[0100] III. Experimental Results

[0101] 1. Morphological characterization of the sandwich system of labeled nanoparticles with high bacterial adhesion and trapping substrate:

[0102] like Figure 2 As shown in a), the synthesized DMSN nanoparticles have a diameter of approximately 170 nm, are uniform in size, and have good dispersibility. Obvious dendritic mesoporous morphology can be observed on the surface, proving that DMSN nanoparticles were successfully synthesized.

[0103] DMSN-Au@Ag nanoparticles morphology as Figure 2 As shown in b), Au@Ag nanoparticles grown in situ can be clearly observed on the surface of DMSN nanoparticles, proving that Au@Ag core-shell nanoparticles with SERS properties have been successfully loaded onto the surface of DMSN nanoparticles to obtain DMSN-Au@Ag nanoparticles. These nanoparticles have stable morphology, good dispersibility, and their size is not significantly different from that of DMSN nanoparticles, approximately 175 nm.

[0104] like Figure 2 As shown in c), the synthesized Fe3O4@Au nanoparticles have a diameter of approximately 80 nm, are uniform in size, have good dispersibility, and show a relatively obvious Au shell on the surface, proving that Fe3O4@Au core-shell nanoparticles for capturing substrates have been successfully synthesized.

[0105] 2. UV-Vis absorption spectrum of the sandwich system of labeled nanoparticles with high bacterial adhesion and trapping substrate:

[0106] like Figure 3As shown in a), the DMSN-Au@Ag nanoparticles exhibit characteristic peaks of Au and Ag nanoparticles at 520 nm and 450 nm, proving that the Au@Ag nanoparticles were successfully loaded into the DMSN nanoparticles, thus obtaining the DMSN-Au@Ag nanoparticles.

[0107] like Figure 3 As shown in b), the characteristic peak of Fe3O4@Au nanoparticles at around 600 nm shows a significant blue shift compared to Fe3O4-Au nanoparticles, proving that an Au shell was synthesized on the surface of Fe3O4, and Fe3O4@Au core-shell nanoparticles were successfully prepared.

[0108] 3. Characterization of the potential of the sandwich system of labeled nanoparticles with high bacterial adhesion and trapping substrate:

[0109] like Figure 4 As shown in a), the zeta potential of D-Au@Ag-C nanoparticles changes continuously through gradual modification and coating, proving the successful preparation of D-Au@Ag-C nanoparticles.

[0110] like Figure 4 As shown in b), the zeta potential of Fe3O4@Au-Ab nanoparticles changes continuously through gradual modification and coating, proving the successful preparation of Fe3O4@Au-Ab nanoparticles.

[0111] 4. Hemolytic activity of the labeled nanoparticles with high bacterial adhesion and the sandwich system for capturing substrates:

[0112] like Figure 5 As shown, except for the deionized water positive control sample, all samples exhibited good hemolytic properties, demonstrating that the sandwich strategy has good blood compatibility.

[0113] 5. Evaluation of the bacterial capture ability of labeled nanoparticles and capture substrates with high bacterial adhesion:

[0114] like Figure 6 As shown in the SEM image, D-Au@Ag-C nanoparticles and Fe3O4@Au-C nanoparticles are clearly adsorbed on the surface of Staphylococcus aureus, proving that the sandwich strategy has a good performance in capturing Staphylococcus aureus.

[0115] 6. Evaluation of SERS performance of labeled nanoparticles with high bacterial adhesion:

[0116] like Figure 7 As shown, with the synthesis of D-Au@Ag-C nanoparticles, the nanoparticles located at 1073 cm⁻¹... -1 and 1583cm -1The Raman peak intensity gradually increased, proving that the synthesized D-Au@Ag-C nanoparticles have superior SERS performance compared to simple DMSN-Au nanoparticles.

[0117] 7. Study on the SERS coupling enhancement performance of DMSN and the SERS coupling performance of Fe3O4@Au-Ab:

[0118] like Figure 8 a) Figure 8 As shown in b), the introduction of DMSN effectively improves the SERS performance of Au@Ag nanomaterials.

[0119] like Figure 8 c) Figure 8 As shown in d), the final sandwich structure shows that Fe3O4@Au-Ab has a good SERS enhancement effect on the D-Au@Ag-C tag nanomaterials enriched on the bacterial surface, proving that the present invention has successfully prepared a nano "sandwich" bacterial detection system with multiple SERS signal enhancement.

[0120] 8. Evaluation of the specificity and repeatability of the sandwich system of highly adhesive labeled nanoparticles and trapping substrate for the detection of Staphylococcus aureus:

[0121] like Figure 9 As shown in a), the 1583 cm⁻¹ of the Staphylococcus aureus sample... -1 The peak intensity has a significant ability to distinguish between different bacteria, proving that the SERS "sandwich" system has good selectivity for Staphylococcus aureus.

[0122] like Figure 9 As shown in b), the SERS sandwich detection system prepared at different times in 10 batches showed uniform results for the detection of Staphylococcus aureus, with an RSD value of 4.5%, proving that the SERS "sandwich" structure has good repeatability.

[0123] 9. Evaluation of the application of a sandwich system consisting of highly adhesive labeled nanoparticles and a trapping substrate for the detection of Staphylococcus aureus in aqueous solution:

[0124] like Figure 10 As shown in a), the Raman peak intensity shows a significant increasing trend with increasing Staphylococcus aureus concentration. Figure 10 As shown in b), 1583cm -1The peak intensity at a given point exhibits a strong linear relationship with bacterial concentration, with an R-squared value of 0.982 and a linear equation of y = -3598 + 3046.4x. Using the formula LOD = 3σ / S, the LOD value is calculated to be 7 CFU. This demonstrates that the "sandwich" detection strategy of this invention has great application potential in bacterial detection in aqueous solutions, exhibiting an extremely low detection limit.

[0125] 10. Evaluation of the detection results of a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate in 1% blood:

[0126] like Figure 11 As shown in a), the Raman peak intensity shows a significant increasing trend with increasing concentration of Staphylococcus aureus in 1% blood solution. Figure 11 As shown in b), 1583cm -1 The peak intensity at a given point exhibits a strong linear relationship with bacterial concentration, with an R-squared value of 0.964 and a limit of detection (LOD) of 97 CFU. This demonstrates that the "sandwich" detection strategy of this invention has great application potential in the detection of bacteria in blood.

[0127] 11. Detection of bacteria in blood samples from infected mice using a sandwich system of labeled nanoparticles with high bacterial adhesion and a trapping substrate:

[0128] like Figure 12 As shown in a), the nano-"sandwich" bacterial detection system with multiple SERS signal enhancement proposed in this invention was used to quantify bacteria in blood collected from mice infected with bacteria. The SERS signals of blood bacterial samples from the three groups of mice were basically uniform. Furthermore, as... Figure 12 As shown in b), the nano-"sandwich" bacterial detection system based on multiple SERS signal enhancement was used to quantitatively analyze the corresponding bacterial concentrations. The results were then compared with those obtained using the standard plate culture method, showing a high degree of consistency. The recoveries of the highly adhesive labeled nanoparticles and the capture substrate were also calculated to determine the accuracy of the results obtained from detecting bacteria together. The recoveries ranged from 91 to 102. Figure 12 Illustration in b).

[0129] These results strongly demonstrate that the nano-"sandwich" bacterial detection system with enhanced multiple SERS signals developed in this invention has extremely high accuracy in blood sample analysis and has broad application prospects in the clinical application of ultrasensitive bacterial detection.

[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nano-"sandwich" bacterial detection system with multiple SERS signal enhancement, characterized in that, Including highly bacterially adhesive labeled nanoparticles D-Au@Ag-C and a trapping substrate Fe3O4@Au-Ab; The D-Au@Ag-C structure is: Au@Ag core-shell nanoparticles are grown in situ on the surface of dendritic mesoporous silica DMSN, and are modified with concanavalin A and SERS tags. The Fe3O4@Au-Ab is: Fe3O4 nanoparticles coated with a gold coating and modified with bacterial targeting antibodies.

2. The nano-"sandwich" bacterial detection system with multiple SERS signal enhancement as described in claim 1, characterized in that, The SERS label is 4-mercaptobenzoic acid (4-MBA).

3. The nano-"sandwich" bacterial detection system with multiple SERS signal enhancement as described in claim 1, characterized in that, The dendritic mesoporous silica DMSN has a diameter of 160-180 nm.

4. The nano-"sandwich" bacterial detection system with multiple SERS signal enhancement as described in claim 1, characterized in that, The bacteria in question is Staphylococcus aureus.

5. A method for preparing the nano-"sandwich" bacterial detection system with multiple SERS signal enhancement as described in any one of claims 1-4, characterized in that, Includes the following steps: 1) Preparation of D-Au@Ag-C: Amino groups were modified on the surface of DMSN to make it positively charged, yielding DMSN-NH2. Using DMSN-NH2 as a raw material, gold was grown on the surface of DMSN via a seed solution growth method to obtain DMSN-Au. DMSN-Au was then modified with a SERS tag to obtain DMSN-Au. 4-MBA ; in DMSN-Au 4-MBA Silver Ag was grown on the surface to obtain DMSN-Au@Ag; DMSN-Au@Ag was then modified with a SERS tag to obtain DMSN-Au@Ag. 4-MBA ; For DMSN-Au@Ag 4-MBA Con A modification with concanavalin A yielded D-Au@Ag-C; 2) Preparation of Fe3O4@Au-Ab: A gold seed solution was prepared; Fe3O4 nanoparticles were surface-modified to a positive potential, then mixed with the gold seed solution, and gold was grown on the surface of the Fe3O4 nanoparticles using a seed solution growth method to obtain Fe3O4@Au; Fe3O4@Au was modified with bacterial targeting antibody Ab to obtain Fe3O4@Au-Ab.

6. The preparation method according to claim 5, characterized in that, The preparation method of dendritic mesoporous silica (DMSN) includes the following steps: Triethanolamine was fully dissolved in ultrapure water, and the resulting solution was heated and stirred. Sodium salicylate and hexadecyltrimethylammonium bromide were added to the solution and stirred. While stirring, tetraethyl orthosilicate was added dropwise to the above solution, and stirring was continued after the addition was completed. After the solution was cooled to room temperature, the product was collected by centrifugation. The product was washed to remove hexadecyltrimethylammonium bromide and dried to obtain dendritic mesoporous silica DMSN.

7. The preparation method according to claim 5, characterized in that, In step 1), the seed solution growth method specifically involves: dispersing DMSN-NH2 in deionized water, adding tetrachloroauric acid solution, and ultrasonically mixing; rapidly injecting sodium borohydride solution into the mixed solution under vigorous stirring, stirring the reaction, centrifuging, and washing multiple times with ultrapure water to obtain DMSN-m-Au seed nanoparticles; resuspending the DMSN-m-Au seed solution in hydroxylamine hydrochloride solution, stirring vigorously at room temperature, and rapidly adding gold chloride solution; stirring, centrifuging, and collecting DMSN-Au; Or, the one mentioned in DMSN-Au 4-MBA The specific process for growing silver (Ag) on ​​the surface is as follows: DMSN-Au 4-MBA Disperse the mixture in an aqueous solution of polyvinylpyrrolidone and stir; add silver nitrate and ascorbic acid aqueous solution to the mixed solution and incubate to reduce silver ions to metallic silver; Centrifuge and wash to remove excess reactants to obtain DMSN-Au@Ag; Or, the above refers to DMSN-Au@Ag 4-MBA The Con A modification of concanavalin A involved activating DMSN-Au@Ag with a mixed solution of tetramethylammonium hydroxide 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt. 4-MBA The carboxyl group on 4-MBA; Con A solution and activated DMSN-Au@Ag 4-MBA Mix and stir at room temperature to obtain D-Au@Ag-C.

8. The preparation method according to claim 5, characterized in that, In step 2), the preparation method of iron oxide nanoparticles is as follows: ferric chloride hexahydrate and trisodium citrate are dissolved in ethylene glycol, and ethylene glycol containing sodium acetate is added under magnetic stirring; the mixture is stirred vigorously at room temperature; then, the mixture is transferred to an autoclave, and the autoclave is placed in an oven at 200°C for heating to obtain a black precipitate; the precipitate is washed, collected with a magnet, and dried to obtain iron oxide nanoparticles. Alternatively, the surface modification of the Fe3O4 nanoparticles specifically involves dispersing the Fe3O4 nanoparticles in a polyethyleneimine (PEI) aqueous solution, mixing them thoroughly, and then modifying the surface of the Fe3O4 nanoparticles with a layer of PEI through self-assembly, thereby modifying the Fe3O4 nanoparticles to a positive potential.

9. The preparation method according to claim 5, characterized in that, In step 2), the modification of the Fe3O4@Au surface with bacterial targeting antibody Ab to obtain Fe3O4@Au-Ab specifically involves: adding polyethylene glycol (PEG) to Fe3O4@Au, ultrasonically mixing, centrifuging to remove excess PEG, and obtaining Fe3O4@Au-PEG; activating the carboxyl groups on the surface of Fe3O4@Au-PEG; ultrasonicating at room temperature, resuspending the activated nanoparticles in phosphate-buffered saline containing bacterial targeting antibody Ab, and stirring to obtain Fe3O4@Au-Ab.

10. A method for detecting bacteria, characterized in that, The specific steps of using the nano-"sandwich" bacterial detection system with multiple SERS signal enhancement as described in any one of claims 1-4 are as follows: The Fe3O4@Au-Ab capture substrate was added to the sample for testing, incubated, washed, and then D-Au@Ag-C labeled nanoparticles with high bacterial adhesion were added, mixed, washed using magnetic separation, and transferred to a silicon wafer. Its temperature at 1583 cm⁻¹ was measured. -1 Raman signal.

11. The bacterial detection method as described in claim 10, characterized in that, The bacteria in question is Staphylococcus aureus.

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