Staphylococcus aureus capturing system and application thereof

Abstract

The invention relates to a staphylococcus aureus capturing system and application thereof. The staphylococcus aureus capture system is obtained by assembling a sulfydryl-modified staphylococcus aureus aptamer on the surface of a nano microsphere / AuPt. Due to the high specificity of the aptamer to the staphylococcus aureus, the aptamer is preferentially combined with target bacteria, and enzyme active sites on the surface of the nano microsphere / AuPt are shielded, so that the catalytic performance is reduced, the absorbance of oxTMB at 652nm is reduced, and the detection of the staphylococcus aureus is realized. The detection method is simple and quick, and can be completed by only one step. And meanwhile, when the nano microspheres are polydopamine nano microspheres (PDA), the PDA / AuPt has excellent photothermal conversion performance. After the apt-PDA / AuPt and the target bacteria are incubated, the apt-PDA / AuPt and the target bacteria are tightly combined through a specific aptamer, and after the apt-PDA / AuPt and the target bacteria are irradiated by a 808nm near-infrared laser, the temperature of a solution system is sharply increased, cell structures of the bacteria are damaged by high temperature, and photo-thermal killing of the bacteria is completed.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, in particular to a Staphylococcus aureus capture system and application thereof. Background Art

[0002] Staphylococcus aureus (S. aureus) is the third largest microbial pathogen after Salmonella and Vibrio parahaemolyticus, and is widely present in nature. Studies have shown that food poisoning caused by Staphylococcus aureus accounts for about 25% of food poisoning pathologies caused by foodborne pathogens. Traditional methods for detecting pathogens include plate counts, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction assay (PCR), but these methods have the defects of cumbersome operation steps, long analysis time, and low sensitivity, and can no longer meet the requirements of rapid and accurate detection. At the same time, in order to prevent the contamination and re-transmission of Staphylococcus aureus, it is necessary not only to focus on the detection of pathogens, but also to implement effective elimination of pathogens. Photothermal sterilization is increasingly being used by people because of its advantages of broad-spectrum sterilization, no drug resistance, short time, and few side effects. Sensitive detection and timely killing of pathogens help to ensure environmental hygiene and safety, but existing technologies rarely achieve detection and killing of pathogens at the same time.

[0003] Nanomaterials have become a research hotspot for the construction of new biosensors and photothermal sterilizers due to their advantages such as simple synthesis, stable properties, diverse enzyme-mimicking activities and excellent functions. Among them, AuPt nanoparticles have good peroxide-mimicking enzyme activity, but their particle size is small, they are easy to aggregate in the solution, and the chemical reaction sites are blocked, which limits the further application of AuPt in the detection field. At the same time, polydopamine nanoparticles (PDA) are a kind of nanoparticles with good water solubility, good dispersibility, high loading rate and excellent photothermal conversion performance. They are often used as photothermal agents in photothermal sterilization. However, the disadvantage is that PDA has a single function. Although the above nanoparticles have been studied in the field of food detection or control, their own defects limit their further application in the field of bacterial detection and control. Therefore, it is urgent to construct a new nanomaterial that has both enzyme-mimicking activity and photothermal conversion performance, and at the same time has stable properties, so as to achieve sensitive detection and photothermal killing of foodborne pathogens. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the lack of a Staphylococcus aureus capturing system that can simultaneously detect and kill Staphylococcus aureus in the prior art.

[0005] To solve the above technical problems, the present invention constructs a Staphylococcus aureus capture system based on the stable support ability of nano-microspheres and the good peroxidase activity of gold-platinum nanoparticles (AuPt), which can realize the colorimetric detection of Staphylococcus aureus and the photothermal killing of Staphylococcus aureus in vitro. The present invention enriches gold and platinum ions on the surface of nano-microspheres, and sodium citrate is used as a reducing agent to assist the in-situ growth of AuPt nanoparticles, and the nano-microspheres / AuPt with the best enzyme activity are obtained by optimizing the addition amount and addition ratio of AuPt. Then, the thiol-modified Staphylococcus aureus aptamer is assembled on the surface of nano-microspheres / AuPt. Due to the high specificity of the aptamer to Staphylococcus aureus, it preferentially binds to the target bacteria, and the enzyme active sites on the surface of the nano-microspheres / AuPt are shielded, resulting in reduced catalytic performance, and the absorbance of the catalytic product oxTMB at 652nm decreases, thereby realizing the detection of Staphylococcus aureus. The detection means is simple and fast, and only one step is required. At the same time, when the nanospheres are polydopamine nanospheres (PDA), PDA / AuPt has excellent photothermal conversion performance. After Apt-PDA / AuPt is incubated with the target bacteria, the two are tightly bound through specific aptamers, and the temperature rises sharply under the irradiation of 808nm near-infrared light. The high temperature destroys the cell structure of the bacteria and completes the photothermal killing of the bacteria.

[0006] The first object of the present invention is to provide a Staphylococcus aureus capture system, the Staphylococcus aureus capture system comprising a nano-microsphere carrier, AuPt loaded on the surface of the nano-microsphere carrier, and an aptamer connected to the AuPt;

[0007] Wherein, the aptamer recognizes and binds to the Staphylococcus aureus, and the sequence of the aptamer is shown as SEQ ID NO.1.

[0008] Furthermore, the aptamer is covalently linked to the AuPt.

[0009] Furthermore, the aptamer is modified by thiol group.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned Staphylococcus aureus capture system, comprising the following steps:

[0011] S1, heating and incubating the nanosphere carrier in a system containing chloroauric acid, chloroplatinic acid and a reducing agent to obtain a nanosphere / gold platinum nanoparticle solution;

[0012] S2. Co-incubate the aptamer with the nano-microsphere / gold-platinum nanoparticle solution to connect the aptamer with the gold-platinum nanoparticles, thereby obtaining the Staphylococcus aureus capture system.

[0013] Furthermore, the reducing agent includes sodium citrate.

[0014] Furthermore, the mass ratio of chloroauric acid to chloroplatinic acid is (0-3):(0-3), such as 1:0, 3:1, 1:1, 1:3, 0:1, including but not limited to the above-listed values.

[0015] Furthermore, the added concentrations of the chloroauric acid and chloroplatinic acid solutions are both (10-30) mM.

[0016] The third object of the present invention is to provide an application of the above-mentioned Staphylococcus aureus capture system in the preparation of a Staphylococcus aureus detection product.

[0017] Furthermore, the application comprises the following steps:

[0018] S1, incubating the Staphylococcus aureus capture system with a sample to be tested containing Staphylococcus aureus;

[0019] S2. Add enzyme-catalyzed substrate and colorimetric substrate to the co-incubated solution for reaction and perform colorimetric detection.

[0020] Furthermore, the enzyme catalytic substrate includes H2O2, and the color development substrate includes 3,3',5,5'-tetramethylbenzidine (TMB). The nanospheres / AuPt have peroxidase activity. When H2O2 is present, the nanospheres / AuPt catalyzes the H2O2 reaction and produces reactive oxygen species, which can oxidize the colorless TMB into blue ox TMB, thereby causing a color development reaction. When Staphylococcus aureus binds to the aptamer, Staphylococcus aureus shields the enzyme active sites on the nanospheres / AuPt, causing the enzyme activity of the nanospheres / AuPt to decrease, thereby causing the ability of TMB to catalyze oxidation into oxTMB to decrease, and the absorbance of ox TMB at 652nm to decrease.

[0021] Furthermore, in the Staphylococcus aureus detection product, the nano-microsphere carrier includes a polydopamine nano-microsphere carrier.

[0022] The fourth object of the present invention is to provide an application of the above Staphylococcus aureus capture system in the preparation of a Staphylococcus aureus sterilization product.

[0023] Furthermore, in the Staphylococcus aureus sterilization product, the nano-microsphere carrier includes a polydopamine nano-microsphere carrier.

[0024] Furthermore, the Staphylococcus aureus sterilization product is achieved by contacting the Staphylococcus aureus capture system with Staphylococcus aureus and performing near-infrared light irradiation, wherein the power of the near-infrared light irradiation is 1.5W-2W.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention uses aptamers as specific recognition molecules. Compared with antibodies in traditional immunoassays, aptamers have the advantages of low cost, stable performance and easy labeling, and can achieve high affinity and high specificity for Staphylococcus aureus.

[0027] (2) The present invention attaches AuPt nanoparticles on the surface of polydopamine nanospheres. The surface of the nanospheres has a large number of catechol, amine and imine groups, which have a strong affinity for metal ions, can bind to metal ions and form catalytic sites in situ. Using them as a supporting framework, AuPt is evenly distributed on the surface of the nanospheres, which effectively improves the dispersibility of the AuPt nanoparticles, prevents the aggregation of the AuPt nanoparticles in the solution, and ensures the peroxidase catalytic activity of the nanocomposite material.

[0028] (3) The present invention conjugates the thiol-modified Staphylococcus aureus aptamer with the nanospheres / AuPt in large quantities to achieve the construction of a specific binding probe.

[0029] (4) The present invention utilizes the catalytic performance of apt-nanocarrier / AuPt, and the aptamer preferentially binds to the target bacteria, the target bacteria shield the catalytic site of apt-nanocarrier / AuPt, the enzyme catalytic ability of the nanomaterial is weakened, the colorimetric signal is reduced, and the absorbance change of oxTMB at 652nm is proportional to the concentration of Staphylococcus aureus, thereby achieving quantitative detection of bacteria. This detection mode is simple and fast, can be completed in the same system, shortens the detection time, and has broad application prospects.

[0030] (5) The present invention utilizes the excellent photothermal conversion performance of PDA nanomaterials. After apt-PDA / AuPt is incubated with the target bacteria, the solution is irradiated with an 808nm near-infrared laser, the temperature of the system rises sharply, and the high temperature destroys the Staphylococcus aureus tissue, thereby achieving rapid elimination of the bacteria and preventing re-contamination by pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0032] Figure 1 It is a schematic diagram of the Staphylococcus aureus capture system of the present invention and its application in detection products and photothermal therapy products;

[0033] Figure 2 Transmission electron microscopy (TEM) images of PDA (a) and PDA / AuPt (b), EDS elemental spectra of PDA / AuPt (c, d);

[0034] Figure 3It is the PDA / AuPt synthesis optimization diagram: AuPt addition concentration (a), addition ratio (b);

[0035] Figure 4 is the storage stability diagram of PDA / AuPt;

[0036] Figure 5 The zeta potential graphs of PDA, PDA / AuPt, and apt-PDA / AuPt (a) and the UV-visible absorption graph of the supernatant of the PDA / AuPt surface before and after modification with apt (b);

[0037] Figure 6 The UV-visible absorption graph of the apt-PDA / AuPt colorimetric system in the presence or absence of target bacteria (a), the corresponding detection standard curve of the sensor (b) and the specificity analysis graph (c);

[0038] Figure 7 The photothermal temperature distribution diagram of PDA / AuPt after 5 on / off cycles (a); the number of colonies on the plate after different sterilization treatments (b, c, d). DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Example 1: Preparation of PDA / AuPt

[0041] PDA: Pour 2 mL of ammonia water, 40 mL of ethanol and 90 mL of deionized water into a beaker, stir in a 30°C water bath for 30 min, then dissolve 0.5 g of dopamine hydrochloride in 10 mL of deionized water, add it to the above solution, and continue to react for 24 h. Centrifuge and wash three times to obtain PDA nanoparticles. Then resuspend in deionized water to prepare a 2 mg / mL PDA solution, and store at 4°C.

[0042] PDA / AuPt: Add 2.5mL PDA solution to 6.5mL water and sonicate for 30min. Then, at room temperature, add 750uL chloroauric acid trihydrate (25mM) solution and 250uL chloroplatinic acid hexahydrate (25mM) solution and stir for 5min to mix evenly. Heat the solution to boiling and immediately add 1mL sodium citrate solution (20mg / mL). Keep boiling for 10min and then stop heating and continue stirring for 20min. After the end, cool naturally to room temperature, centrifuge the reaction product at 8000rpm for 10min, wash it three times with deionized water, resuspend it in water, and obtain a 1mg / mL PDA / AuPt solution, which is stored at 4°C for use. In order to further obtain PDA / AuPt with the best catalytic activity, the addition concentration and ratio of AuPt were optimized.

[0043] Through transmission electron microscopy ( Figure 2 a, 2b), EDS element spectrum ( Figure 2 c, 2d), the prepared PDA nanospheres have uniform size, and AuPt is distributed on the surface of the PDA nanospheres. The optimization results of synthesizing PDA / AuPt are shown in Figure 3 As shown, AuPt addition concentration = 25 mM, AuPt addition ratio = 3:1. The changes in the enzyme activity of PDA / AuPt during the storage of aqueous solution were analyzed by measuring the absorbance of oxTMB at 652 nm ( Figure 4 ), the enzyme activity of PDA / AuPt was relatively stable within 24 days of storage, and the enzyme activity decreased to 67% after 24 days, indicating that PDA / AuPt had good storage stability.

[0044] Example 2: Preparation of apt-PDA / AuPt

[0045] SH-apt (10 μM, 100 μL) was added to 1 mL of PDA / AuPt solution, incubated at 25°C for 4 h, centrifuged at 8000 rpm for 6 min, washed with deionized water, and stored at 4°C for use.

[0046] Thiol-modified Staphylococcus aureus aptamer chain (SEQ ID NO: 1): GCAATG GTA CGG TAC TTCCTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CACGCT ACT TTG CTA A

[0047] The zeta potential of PDA, PDA / AuPt and apt-PDA / AuPt nanocomposites is shown in Figure 2 Figure 5As shown in a, they are 12.83±0.70, -23.65±1.64, and -36.60±0.53 mV, respectively. The PDA nanospheres are positively charged, and the attachment of AuPt nanoparticles causes PDA / AuPt to be negatively charged. At the same time, due to the electronegativity of naked DNA, the potential of apt-PDA / AuPt is further reduced. In addition, after the aptamer modification, the absorbance of the supernatant at 260 nm is significantly reduced ( Figure 5 b). The above results prove that the synthesis results of apt-PDA / AuPt are consistent with expectations.

[0048] Example 3: Preparation for experimental feasibility verification

[0049] In this example, ATCC 29213 Staphylococcus aureus standard strain was used, and Staphylococcus aureus solution (final concentration of 1.4×10 6 CFU / mL) was added to a mixed solution containing apt-PDA / AuPt nanocomposite (1 μg) and acetate buffer (pH = 3.8) to make a total volume of 800 μL, and sterile water was used as a negative control. The mixture was incubated at room temperature for 20 min, and 100 μL of TMB (5 mM) solution and 100 μL of H2O2 (100 mM) were added to catalyze the reaction at room temperature for 25 min, and then the absorbance change at 652 nm was detected.

[0050] like Figure 6 As shown in (a), when the bacteria were captured by apt-PDA / AuPt, the absorbance at 652 nm further decreased, confirming that the bacteria could shield the catalytic active sites of PDA / AuPt.

[0051] Example 4: Establishment of a standard curve for Staphylococcus aureus detection

[0052] The Staphylococcus aureus stock solution was diluted with sterile water to different concentrations (1.4×10 2 -1.4×10 7 CFU / mL). Staphylococcus aureus solution was added to a mixed solution containing apt-PDA / AuPt nanocomposite (1μg) and acetic acid buffer (pH=3.8) to make a total volume of 800μL. Sterile water was used as a negative control. The mixture was incubated at room temperature for 20 minutes. 100μL TMB (5mM) solution and 100μL H2O2 (100mM) were added. The reaction was catalyzed at room temperature for 25 minutes, and then the absorbance at 652nm was detected. The logarithmic value of the concentration of Staphylococcus aureus was used as the horizontal axis, and the absorbance difference of oxTMB at 652nm was used as the vertical axis to determine the standard curve. Experimental results ( Figure 6b) It can be obtained that the A0-A value (A0 and A are the absorbance of oxTMB at 652nm before and after incubation with Staphylococcus aureus, respectively) and the logarithmic value of the concentration of Staphylococcus aureus show a good linear relationship, and the linear regression equation is Y=0.0752X-0.08548 (where Y is the absorbance difference of oxTMB at 652nm, and X is the logarithmic value of the concentration of Staphylococcus aureus), and the determination coefficient (R 2 =0.98633), according to the formula LOD = 3б / S, the detection limit of this test is calculated to be 44CFU / mL (б is the relative standard deviation of the absorbance of 11 parallel blank tests, S is the slope of the standard curve), which proves that the colorimetric sensor can realize the detection of Staphylococcus aureus.

[0053] Example 5: Selectivity Analysis

[0054] Escherichia coli O157:H7 (ATCC 03820), Salmonella typhimurium (ATCC 10420), Listeria monocytogenes (ATCC 19115), Shigella dysenteriae (ATCC 13313), Staphylococcus xylosus (CICC 21445) and Pediococcus pentosaceus (ATCC 33316) were selected (OD of all strains 600 The interfering bacteria were 0.3, and the sterile water sample was used as the blank control. Figure 6 As can be seen in c, the Staphylococcus aureus sample shows an obvious absorbance difference, while the colorimetric signals of other samples are weak. The results show that the colorimetric sensor has good selectivity.

[0055] Example 6: Spike recovery experiment

[0056] Pork samples were purchased from a local vegetable market. 1 g of pork was added to 9 mL of sterile water and ground into pieces. The mixture was centrifuged at 5000 rpm for 5 min. The supernatant was diluted 10 times with sterile water and sterilized by ultraviolet irradiation. The plate count method was used to determine whether the sample was completely sterilized. A known concentration of Staphylococcus aureus (1.4 × 10 3 -1.4×10 5 CFU / mL) for further testing and verification by conventional plate count method, all analyses were performed in triplicate. As shown in Table 1, the recovery rate in pork samples was 99.4-110.8%, and the relative standard deviation was between 1.93-7.66%. The results confirmed that the colorimetric strategy can achieve effective detection of Staphylococcus aureus in spiked samples, and at the same time, the detection results of the colorimetric sensor of the present invention and the plate count method are not much different.

[0057] Table 1 Recovery and RSD of Staphylococcus aureus in spiked pork (x±s, n=3)

[0058]

[0059] Example 7: Photothermal conversion performance of PDA / AuPt

[0060] 200 μL of PDA / AuPt solution (150 μg / mL) was transferred to a 96-well plate and irradiated with a near-infrared laser (808 nm, 2 W) for 5 min, followed by natural cooling, for 5 consecutive cycles. The infrared thermal imager was used to collect data every 30 s. Figure 7 As shown in a, under 808 nm laser irradiation, the temperature of PDA / AuPt increased rapidly within 5 min, and there was no obvious temperature change during 5 cycles, and it still maintained good photothermal conversion ability.

[0061] Example 8: In vitro photothermal killing of Staphylococcus aureus

[0062] apt-PDA / AuPt (final concentration 150 μg / mL) was mixed with Staphylococcus aureus (10 6 CFU / mL) for 20 minutes, then irradiated with a near-infrared laser (808nm, 2W) for 5 minutes, 100uL of the solution was diluted 10 times to the appropriate concentration, evenly spread on the LB agar plate, placed in an incubator at 37℃ for 24 hours, and the colony growth on the LB agar plate was observed. The laser-irradiated Staphylococcus aureus solution and the apt-PDA / AuPt+Staphylococcus aureus solution that was not treated with laser irradiation were used as negative controls. The results showed that ( Figure 7 b, c, d), after 5 min of near-infrared laser irradiation, the photothermal killing effect of bacteria reached 95%, proving the broad prospects of apt-PDA / AuPt in the field of food sterilization.

[0063] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A Staphylococcus aureus capture system, characterized in that: The Staphylococcus aureus capture system comprises a nano-microsphere carrier, gold-platinum nanoparticles loaded on the surface of the nano-microsphere carrier, and an aptamer connected to the gold-platinum nanoparticles; Wherein, the sequence of the aptamer is shown as SEQ ID NO.

1.

2. The Staphylococcus aureus capture system according to claim 1, characterized in that: The aptamer is covalently linked to the gold platinum nanoparticles.

3. A method for preparing the Staphylococcus aureus capture system according to claim 1 or 2, characterized in that: The following steps are involved: S1, heating and incubating the nanosphere carrier in a system containing chloroauric acid, chloroplatinic acid and a reducing agent to obtain a nanosphere / gold platinum nanoparticle solution; S2. Co-incubate the aptamer with the nano-microsphere / gold-platinum nanoparticle solution to connect the aptamer with the gold-platinum nanoparticles, thereby obtaining the Staphylococcus aureus capture system.

4. The preparation method according to claim 3, characterized in that: The mass ratio of the chloroauric acid to the chloroplatinic acid is (0-3):(0-3).

5. Use of the Staphylococcus aureus capture system according to claim 1 or 2 in the preparation of a Staphylococcus aureus detection product.

6. The use according to claim 5, characterized in that: The following steps are involved: S1, incubating the Staphylococcus aureus capture system with a sample to be tested containing Staphylococcus aureus; S2. Add enzyme-catalyzed substrate and colorimetric substrate to the co-incubated solution for reaction and perform colorimetric detection.

7. The use according to claim 6, characterized in that: The enzyme catalytic substrate includes hydrogen peroxide, and the color developing substrate includes 3,3',5,5'-tetramethylbenzidine.

8. Use of the Staphylococcus aureus capture system according to claim 1 or 2 in the preparation of a Staphylococcus aureus sterilization product.

9. The use according to claim 8, characterized in that: In the Staphylococcus aureus sterilization product, the nano-microsphere carrier comprises a polydopamine nano-microsphere carrier.

10. The use according to claim 9, characterized in that: The Staphylococcus aureus sterilization product is achieved by contacting the Staphylococcus aureus capture system with Staphylococcus aureus and performing near-infrared light irradiation.

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