An aptamer fluorescence sensing method for bacterial recognition, capture and sterilization

Abstract

The present invention discloses an aptamer fluorescence sensing method for bacterial identification, capture and sterilization, including: preparing an AIE CuNCs solution, and performing polymyxin E modification on the AIE CuNCs solution to obtain polymyxin E (PME)-modified AIE CuNCs; culturing bacteria, and preparing aptamer-functionalized magnetic beads; linking streptavidin-labeled Escherichia coli aptamers to PME@AIE Cu NCs to identify and capture Escherichia coli, and obtaining an IMB@Apt‑E / E.coli / PME@AIE CuNCs sandwich structure; breaking the photocontrol group PC‑linker in the aptamer by ultraviolet light irradiation, taking the supernatant after magnetic separation, and measuring the fluorescence intensity by a fluorescence spectrometer to achieve identification and capture of bacteria. The present invention improves the sensitivity and selectivity of bacterial detection and achieves instant killing of bacteria.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent nano-biotechnology, and in particular relates to an aptamer fluorescent sensing method for bacterial identification, capture and sterilization. Background Art

[0002] Over the past few decades, bacterial infections have been considered one of the biggest challenges facing public health around the world, and rapid detection and treatment of bacterial infections are crucial. Bacterial infections usually include cellulitis, abscesses, and postoperative infections. The most common bacteria in bacterial infections are mainly Gram-negative and Gram-positive bacteria, such as Escherichia coli and Staphylococcus aureus. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are ubiquitous and can sometimes cause various complications and chronic infections, and in severe cases may induce sepsis, pneumonia, meningitis and other diseases. Therefore, it is urgent to achieve rapid detection, diagnosis and treatment of bacterial pathogen infections.

[0003] Traditional bacterial identification methods are often time-consuming and have limited sensitivity. With the development of nanotechnology, the combination of nanomaterials and bacterial identification has provided a new way to quickly and accurately identify bacteria. In particular, the combination of biorecognition molecules such as aptamers and nanoclusters can achieve high sensitivity and high specificity for specific bacteria. The emergence of this technology not only improves the efficiency of bacterial identification, but also provides a precise target for subsequent bactericidal treatment.

[0004] In recent years, nanoclusters, as an emerging nanomaterial, have shown unique advantages in the fields of biomedicine, optoelectronics, etc. In particular, copper nanoclusters (Cu NCs) with aggregation-induced emission (AIE) have become a hot topic of research due to their excellent optical properties and biocompatibility. AIE nanoclusters exhibit strong fluorescence emission in the aggregated state, which makes them have great application potential in biomarkers, bioimaging and sensing. In addition to being an identification tool, nanoclusters themselves also have bactericidal effects. Studies have shown that some nanoclusters can kill bacteria by destroying the cell wall of bacteria or inhibiting the growth and metabolism of bacteria. Combining antibiotics with nanoclusters can further enhance their bactericidal effect. This synergistic effect can not only achieve efficient killing of bacteria, but also reduce the use of antibiotics and reduce the risk of drug resistance. In view of this, it is urgent to propose an aptamer fluorescence sensing technology that can simultaneously achieve bacterial recognition, capture and sterilization. Summary of the invention

[0005] To achieve the above objectives, the present invention provides an aptamer fluorescence sensing method for bacterial identification, capture and sterilization, aiming to achieve efficient identification, capture and sterilization of bacteria. It includes:

[0006] preparing an AIE CuNCs solution, and performing polymyxin E modification on the AIE CuNCs solution to obtain polymyxin E modified AIE CuNCs;

[0007] Cultivating bacteria and preparing aptamer-functionalized magnetic beads;

[0008] The streptavidin-labeled magnetic beads with E. coli aptamers were linked to PME@AIE Cu NCs to recognize and capture E. coli, and the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure was obtained;

[0009] The photocontrollable group PC-linker in the aptamer is broken by ultraviolet light irradiation. After magnetic separation, the supernatant is taken and the fluorescence intensity is measured by a fluorescence spectrometer to achieve the recognition and capture of bacteria.

[0010] Preferably, the process of preparing the AIE CuNCs solution comprises:

[0011] Under magnetic stirring at room temperature, anhydrous CuSO4 solution was added to 2.4 mL of ultrapure water, mixed thoroughly, and then GSH solution was added;

[0012] The mixture was transferred to a 37°C water bath and magnetically stirred for 30 min, and then the pH of the system was adjusted to 4.0-5.5 with 1 M NaOH solution;

[0013] The reaction solution was vigorously stirred at 37°C for 1 h to prepare a CuNCs solution;

[0014] Add Ce(NO3)3·6H2O solution to the CuNCs solution, make up to volume, and stir at room temperature for 10 min;

[0015] The AIE CuNCs solution was obtained by dialyzing with a dialysis bag of MW = 500Da for 1 h.

[0016] Preferably, the molar ratio of the anhydrous CuSO4 solution to the GSH solution is 8:1;

[0017] The pH range is 4.6 to 4.7;

[0018] The amount of the Ce(NO3)3·6H2O solution used is 50 μL of a 20 mM solution.

[0019] Preferably, the process of modifying the AIE CuNCs solution with polymyxin E to obtain polymyxin E-modified AIE CuNCs comprises:

[0020] Polymyxin E and cerium nitrate solution were added to CuNCs, and the volume was adjusted to 8 mL with water and then magnetically stirred to prepare polymyxin E-modified AIE CuNCs.

[0021] Preferably, the amount of CuNCs used is 1.6 mL;

[0022] The dosage of polymyxin E was 2.4 mL 4 mg / mL, and the volume was adjusted to 8 mL with water, and then vigorously stirred for 1 hour.

[0023] Preferably, the process of culturing bacteria comprises:

[0024] The bacterial colony was placed in 10 mL of LB liquid medium, and then the bacteria were placed in a constant temperature shaker at 37°C and 180 rpm for overnight cultivation.

[0025] Preferably, the bacteria include Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes.

[0026] Preferably, the process of preparing aptamer-functionalized magnetic beads comprises:

[0027] The Escherichia coli aptamer Apt-E labeled with biotin and photocontrollable group PC-linker was reacted with streptavidin-labeled magnetic beads at 27°C for 1 hour. Subsequently, the beads were washed twice with PBS buffer solution and magnetically separated to obtain aptamer-functionalized magnetic beads IMB@Apt-E.

[0028] Preferably, the process of linking the streptavidin-tagged magnetic beads with the E. coli aptamer to PME@AIE CuNCs to recognize and capture E. coli to obtain the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure comprises:

[0029] The magnetic beads IMB@Apt-E were mixed with bacterial solutions of different concentrations and diluted to 1 mL with 1×PBS. The solution was incubated with shaking and washed twice with Tri-HCl after magnetic separation. The supernatant was discarded to wash away the free bacterial solution.

[0030] Polymyxin E-modified AIE CuNCs were added to the solution respectively, and incubated with shaking at 27°C for 15 h to form an IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure.

[0031] Preferably, the concentration of the bacterial solution is: 10 8 ~10 2 CFU / mL;

[0032] The temperature and time of the shaking incubation are 27° C. and 1 h, respectively;

[0033] The Tri-HCl buffer contains NaCl and MgCl2 and has a pH of 7.20;

[0034] The ultraviolet lamp is a 365nm ultraviolet lamp, and the irradiation time is 4 minutes.

[0035] Compared with the prior art, the present invention has the following advantages and technical effects:

[0036] The aptamer fluorescence sensing method of the present invention prepares PME@AIE Cu NCs nanomaterials, which have excellent luminescence performance and stability. Through the interaction between the material and IMB@Apt-E and the recognition and capture of Escherichia coli, a "sandwich structure" of IMB@Apt-E / E.coli / PME@AIE CuNCs is formed between the immunomagnetic beads, the target bacteria and the metal nanoclusters modified by antibiotics, and a biosensor that can efficiently identify bacteria is constructed and obtained, which can be used for the study of integrated identification, capture and antibacterial of bacteria. After magnetic separation, after the bacteria and the metal nanoclusters modified by antibiotics act together for a period of time, some bacteria are killed under the synergistic effect of the metal nanoclusters and the antibiotics. The aptamer fluorescence sensing technology proposed in the present invention realizes the integration of bacterial identification, capture and sterilization by constructing a "sandwich structure", improves the antibacterial efficiency, reduces the risk of drug resistance, and provides a new strategy for the treatment of bacterial infections.

[0037] The metal nanoclusters prepared in the present invention have antibacterial properties, and in vitro antibacterial studies of the "sandwich structure" show that the antibacterial rate of the metal nanoclusters in combination with antibiotics against E. coli is 91.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0039] Figure 1 Schematic diagram of the synthesis conditions of optimized AIE Cu NCs according to the embodiment of the present invention; wherein (a) is a graph showing the molar ratio of GSH and CuSO4; (b) is a graph showing the pH of the reaction system; (c) is a graph showing the dosage of Cu NCs; (d) is a graph showing the Ce 3+ (e) is the volume relationship diagram; (f) is the fluorescence spectrum diagram;

[0040] Figure 2Schematic diagram of the results of the optimized bacterial liquid detection of the embodiment of the present invention; wherein, (a) is a diagram showing the relationship between the dosage of optimized AIE Cu NCs; (b) is a diagram showing the relationship between the concentration of PME for optimizing the detection of E. coli; (c) is a diagram showing the relationship between the time of co-incubation of optimized PME and AIE Cu NCs;

[0041] Figure 3 Schematic diagram of the stability of PME and PME@AIE Cu NCs of the embodiment of the present invention; wherein (a) is a stability relationship diagram under different pH; (b) is a stability relationship diagram under continuous excitation of a xenon lamp within 1 hour; (c) is a stability relationship diagram in the presence of different concentrations of NaNO3;

[0042] Figure 4 Schematic diagram of AIE Cu NCs of an embodiment of the present invention at different excitation wavelengths; wherein (a) is a diagram of the maximum excitation wavelength of AIE Cu NCs; (b) is a diagram of the emission wavelength and fluorescence spectrum of AIE Cu NCs;

[0043] Figure 5 The Uv-vis absorption spectra of PME, AIE Cu NCs and PME@AIE Cu NCs of the embodiments of the present invention;

[0044] Figure 6 FT-IR spectra of PME, AIE Cu NCs and PME@AIE Cu NCs of the embodiments of the present invention;

[0045] Figure 7 Zeta potential diagrams of PME, AIE Cu NCs and PME@AIE Cu NCs of the embodiments of the present invention;

[0046] Figure 8 330nm excitation fluorescence intensity diagram of the embodiment of the present invention; wherein, (a) is a linear relationship diagram between the logarithm of E. coli and the fluorescence intensity of PME@AIE Cu NCs; (b) is a corresponding fluorescence spectrum diagram;

[0047] Fig. 9 Graphs showing conditions for optimizing detection of bacterial solution in an embodiment of the present invention; wherein (a) is a graph showing the concentration relationship of the E. coli aptamer; (b) is a graph showing the time relationship of capturing E. coli; and (c) is a graph showing the time relationship of optimizing the irradiation of ultraviolet light;

[0048] Fig.10 Specific relationship diagram of PME@AIE Cu NCs of the embodiment of the present invention;

[0049] Fig.11 Schematic diagram of the antibacterial performance of AIE Cu NCs according to an embodiment of the present invention. DETAILED DESCRIPTION

[0050] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0051] It should be noted that the steps shown in the flowcharts of the accompanying drawings can be executed in a computer system such as a set of computer executable instructions, and that, although a logical order is shown in the flowcharts, in some cases, the steps shown or described can be executed in an order different from that shown here.

[0052] like Figure 1-2 As shown, this embodiment provides an aptamer fluorescence sensing method for bacterial identification, capture and sterilization, including:

[0053] preparing an AIE Cu NCs solution, and modifying the AIE CuNCs solution with polymyxin E (PME) to obtain polymyxin E (PME)-modified AIE CuNCs;

[0054] Cultivating bacteria and preparing aptamer-functionalized magnetic beads;

[0055] The streptavidin-labeled magnetic beads with E. coli aptamers were linked to PME@AIE Cu NCs to recognize and capture E. coli, and the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure was obtained;

[0056] The photocontrollable group PC-linker in the aptamer is broken by ultraviolet light irradiation. After magnetic separation, the supernatant is taken and the fluorescence intensity is measured by a fluorescence spectrometer to achieve the recognition and capture of bacteria.

[0057] Furthermore, the process of preparing the AIE CuNCs solution includes:

[0058] Under magnetic stirring at room temperature, anhydrous CuSO4 solution was added to 2.4 mL of ultrapure water, mixed thoroughly, and then GSH solution was added;

[0059] The mixture was transferred to a 37°C water bath and magnetically stirred for 30 min, and then the pH of the system was adjusted to 4.0-5.5 with 1 M NaOH solution;

[0060] The reaction solution was vigorously stirred at 37°C for 1 h to prepare a CuNCs solution;

[0061] Ce(NO3)3·6H2O solution was added to the Cu NCs solution, and the solution was stirred at room temperature for 10 min after the volume was fixed.

[0062] The AIE CuNCs solution was obtained by dialyzing with a dialysis bag of MW = 500Da for 1 h.

[0063] To further optimize the scheme, the molar ratio of anhydrous CuSO4 solution to GSH solution was 8:1;

[0064] The pH of the system was specifically adjusted to 4.6-4.7 with NaOH solution;

[0065] The amount of Ce(NO3)3·6H2O solution used was 50 μL of a 20 mM solution.

[0066] Furthermore, the AIE CuNCs solution is modified with polymyxin E (PME) to obtain the AIE CuNCs modified with polymyxin E (PME), which includes:

[0067] Polymyxin E and cerium nitrate solution were added to CuNCs, and the volume was adjusted to 8 mL with water and then magnetically stirred to prepare polymyxin E (PME)-modified AIE CuNCs.

[0068] The scheme was further optimized, and the amount of CuNCs used was 1.6 mL;

[0069] The dosage of polymyxin E (PME) was 2.4 mL 4 mg / mL. After diluting to 8 mL with water, it was vigorously stirred for 1 h.

[0070] Further, the process of culturing bacteria includes:

[0071] The bacterial colony was placed in 10 mL of LB liquid medium, and then the bacteria were placed in a constant temperature shaker at 37°C and 180 rpm for overnight cultivation.

[0072] The scheme was further optimized, and the bacteria included Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Listeria monocytogenes.

[0073] Furthermore, the process of preparing aptamer-functionalized magnetic beads includes:

[0074] The E. coli aptamer Apt-E labeled with biotin and photo-controllable group PC-linker was reacted with streptavidin-labeled magnetic beads at 27°C for 1 hour, then washed twice with PBS buffer solution and magnetically separated to obtain aptamer-functionalized magnetic beads IMB@Apt-E. PC-linker (photoleavable) is a non-nucleoside molecule with unique photo-controllable properties. When ultraviolet light irradiates PC-linker, photolysis occurs and the DNA chain breaks.

[0075] Furthermore, the process of linking the streptavidin-tagged magnetic beads with E. coli aptamers to PME@AIE Cu NCs to recognize and capture E. coli to obtain the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure includes:

[0076] The magnetic beads IMB@Apt-E were mixed with bacterial solutions of different concentrations and diluted to 1 mL with 1×PBS. The solution was incubated with shaking and washed twice with Tri-HCl after magnetic separation. The supernatant was discarded to wash away the free bacterial solution.

[0077] Polymyxin E-modified AIE CuNCs were added to the solution respectively, and incubated with shaking at 27°C for 2 h to form an IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure.

[0078] To further optimize the solution, the concentration of the bacterial solution is: 10 8 ~10 2 CFU / mL;

[0079] Furthermore, the concentration of the bacterial solution is: 1×10 5 CFU / mL.

[0080] The temperature and time of shaking incubation were 27°C and 1 h, respectively;

[0081] Tri-HCl buffer contains NaCl and MgCl2 and has a pH of 7.20;

[0082] The photocontrol group PC-linker in the aptamer was broken by irradiation with ultraviolet light, the ultraviolet light was 365nm, and the irradiation time was 4 minutes.

[0083] Furthermore, it also includes in vitro antibacterial research on the "sandwich structure", and the specific steps are as follows:

[0084] After magnetic separation, 100 μL of the "sandwich structure" solution was spread on LB solid culture medium and cultured at 37°C overnight.

[0085] The reaction system in a was compared with a blank control group cultured at 37°C.

[0086] Example

[0087] Reagents:

[0088] 98% glutathione (reduced) was purchased from Aladdin Reagent Co., Ltd. Anhydrous CuSO4 was purchased from Tianjin Damao Chemical Reagent Factory Sodium hydroxide was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Sodium chloride was purchased from Tianjin Fengchuan Chemical Reagent Factory Technology Co., Ltd. 1×PBS was purchased from Beijing Solebao Reagent Streptavidin magnetic beads and Apt-E were purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.

[0089] instrument:

[0090] F-7000 Fluorescence Spectrophotometer; TU -1 901 double beam UV-visible spectrophotometer; Fourier transform infrared spectroscopy (FT-IR); NanoBrook 90Plus PALSZeta potential and particle size analyzer; TG16-W high-speed centrifuge; B13-3 magnetic stirrer; XH-C vortex mixer.

[0091] Example 1

[0092] Preparation of antibiotic-modified metal nanoclusters;

[0093] Under magnetic stirring at room temperature, 10 mM anhydrous CuSO4 solution (3 mL) was added to ultrapure water (2.4 mL). After thorough mixing, 0.2 M GSH solution (1.2 mL) was slowly added to the above solution. The solution gradually turned gray-black, and continued to add dropwise to present a light yellow color. It was then transferred to a 37 ° C water bath and magnetically stirred for 30 min. It can be seen that the solution gradually changed from light yellow to a white turbid liquid. Subsequently, the pH of the system was adjusted to the range of 4.6-4.7 with 1 M NaOH solution until the white turbid liquid gradually became clear and transparent, and finally presented a light yellow color. Next, the reaction solution was vigorously stirred at 37 ° C for 1 h to prepare Cu NCs. 20 mM Ce (NO3) 3·6H2O solution (50 μL) was added to the previously prepared Cu NCs solution (400 μL), and its volume was fixed to 1 mL and stirred at room temperature for 10 min. The above solution was dialyzed for 1 h using a dialysis bag with MW = 500Da to obtain an AIE Cu NCs solution. A 10 mL glass bottle was placed on a magnetic stirrer at room temperature, 1.6 mL of the prepared Cu NCs were added, followed by 2.4 mL of 4 mg / mL polymyxin E, and then 200 μL of 20 mM Ce (NO3) 3 solution was added, followed by dilution to 8 mL with water, and PME-modified AIE Cu NCs were obtained after vigorous stirring for 1 h.

[0094] Optimization of experimental conditions;

[0095] The preparation of AIE Cu NCs was optimized by (1) the molar ratio of GSH to Cu SO4; (2) the pH of the system; (3) the amount of CuNCs; (4) the amount of Ce 3+ volume; (5) reaction time. The results show that the molar ratio of anhydrous CuSO4 and GSH is 8:1. Figure 1 As shown in (a), when the pH of the system is in the range of 4.6-4.7, the fluorescence intensity of Cu NCs is the strongest. Figure 1 (b) Rare earth ion Ce 3+ can induce the aggregation of Cu NCs and have an 3+ The volume of Cu NCs produced fluorescence dependence. The optimal dose of Cu NCs was 400 μL. Figure 1 (c) and then optimized Ce 3+ The reaction time with Cu NCs, such as Figure 1 As shown in (d), the reaction is complete and reaches equilibrium in 10 min. 3+ As the volume increases, the fluorescence intensity of Cu NCs gradually increases and reaches equilibrium after 50 μL. Figure 1 (e) In the range of 5-50 μL, Ce 3+ The volume and fluorescence intensity showed a good linear relationship (y = 39.42x-302.49, R 2 =0.9921), the fluorescence spectrum is shown in Figure 1 (f) Therefore, adding Ce 3+ The optimal volume is 50 μL for subsequent experiments.

[0096] In order to obtain the best detection results in the process of detecting E. coli in bacterial liquid, the dosage of metal nanoclusters was first optimized. The optimal dosage of AIE Cu NCs was 200 μL, such as Figure 2 As shown in (a). Since antibiotics have a certain bactericidal effect and play a key role in the detection, capture and antibacterial process of bacteria, the effects of different concentrations of PME on the fluorescence intensity were compared during the synthesis of PME@AIECu NCs. Figure 2 As shown in (b), 4 mg / mL PME is the optimal concentration. Secondly, the optimal incubation time of PME and AIE Cu NCs was optimized to be 1.5 h. Figure 2 (c) and can remain stable within 12 hours.

[0097] Investigation of the stability of AIE Cu NCs before and after adding antibiotics;

[0098] like Figure 3As shown in (a), the pH stability of the material was investigated, indicating that the pH stability was improved to a certain extent after the metal nanocluster surface was modified with antibiotics. Figure 3 As shown in (b), the AIE Cu NCs surface was modified with PME before and after multiple excitations by a xenon lamp, and it could maintain resistance to white light bleaching within 1 hour. Figure 3 As shown in (c), before and after the AIE Cu NCs bind to the antibiotic, when the concentration of NaNO3 is ≤500mM, its fluorescence intensity remains basically unchanged. In summary, after the AIE Cu NCs surface is modified with antibiotics, in addition to a slight enhancement in fluorescence intensity, it still has good pH stability, resistance to white light bleaching, and salt tolerance, and has no effect on the properties of the metal nanoclusters themselves.

[0099] Characterization of PME@AIE Cu NCs materials;

[0100] First, if Figure 4 As shown in (a), the excitation wavelength and emission wavelength of AIE Cu NCs are 330 nm and 580 nm, respectively. Figure 4 (b) is the fluorescence spectra of AIE Cu NCs at different excitation wavelengths. The optical absorption of antibiotics, metal nanoclusters and the combination of the two was then analyzed using UV-visible absorption spectra. Figure 5 As shown, the UV-visible absorption spectrum of AIE Cu NCs has weak absorption bands near 280nm and 340nm, which is mainly due to the π-π* transition of sp2 C=C and the n-π* transition of C=O, while PME has no obvious absorption peak, and PME@AIE Cu NCs only has a weak absorption peak of AIE Cu NCs. Therefore, we speculate that PME@AIE Cu NCs may be successfully synthesized.

[0101] Figure 6 FTIR spectra of GSH, PME, AIE Cu NCs and PME@AIE Cu NCs, respectively. The figure shows that 2520cm -1 is the stretching vibration peak of -SH on GSH, while the AIE Cu NCs is located at 2520 cm -1 The stretching vibration peaks near 3271 cm-1 disappeared. It is very likely that -SH and the metal element Cu combined to form a Cu-S bond. -1 The absorption peak at 1636 cm is the stretching vibration of the NH bond. -1 and 1406cm -1 The bands are attributed to C=O and CN. 3+ The addition of made the stretching vibration peak of -OH disappear, confirming the interaction between -COOH group and Ce 3+This indicates the successful preparation of AIE Cu NCs. The FT-IR spectrum of PME shows that 1094 cm -1 Mainly caused by the stretching vibration of the CN bond, 1656cm -1 The stretching vibration peak of PME@AIE CuNCs is derived from the stretching vibration peak of C=O. The FT-IR spectrum of PME@AIE CuNCs shows that there is a stretching vibration peak of PME on the surface of AIE Cu NCs. On the other hand, the peak at 1094 cm -1 The stretching vibration peak of CN at is more obvious, which can prove the successful preparation of PME@AIE Cu NCs.

[0102] To further understand the attachment mechanism of antibiotics to metal nanoclusters, zeta potential analysis was performed. Figure 7 The negative charge of -19.27 mV on the surface of AIE Cu NCs was shown, the Zeta potential of PME was +2.68 mV, and the surface charge of AIE CuNCs loaded with PME was -14.65 mV. The results showed that PME was loaded onto AIE Cu NCs through electrostatic interaction.

[0103] Example 2

[0104] Bacterial culture;

[0105] Before preparing the culture medium, all glassware needs to be sterilized in a high-temperature autoclave. Dissolve 1g of tryptone, 0.5g of yeast extract, 1g of NaCl, and 1.5g of agarose in every 100mL of ultrapure water to prepare LB solid culture medium. After high-temperature sterilization at 121℃, wait until the temperature drops to about 60℃ before pouring the plate. Use the same method to prepare LB liquid culture medium without agarose.

[0106] Place the colony of Escherichia coli (E.coli, ATCC 25922) in 10mL LB liquid culture medium. Then place the bacteria in a constant temperature oscillator at 37°C and culture overnight at 180rpm. When the bacterial solution grows to a certain degree of turbidity, transfer 1mL of the bacterial solution to 1.5mL centrifuge tubes and prepare 3 groups in parallel. Place the bacterial solution in a high-speed centrifuge (8000rpm) and centrifuge for 3 minutes, discard the culture medium, and then wash it 3 times with 1×PBS buffer solution. Finally, re-dissolve the bacteria with 1mL of 1×PBS, and monitor the absorbance at 600nm with a UV-visible spectrophotometer. Subsequent experiments were performed according to 1OD≈10 8 CFU / mL was used for quantification.

[0107] Example 3

[0108] Construction of “sandwich structure” and sensitivity testing;

[0109] The bacterial solution was diluted in a 10-fold concentration gradient from 10 8 Dilute to 10 2 CFU / mL to obtain 7 bacterial solutions of different concentrations. Take a sterilized 1.5mL centrifuge tube, take 70μL of streptavidin-labeled magnetic beads (50mg / mL) and place them in the centrifuge tube, use a magnet for magnetic separation, and wash the magnetic beads twice with 1×PBS. After discarding the supernatant, re-dissolve with 1.4mL of 1×PBS. Distribute 1400μL of streptavidin-labeled magnetic beads evenly into 7 centrifuge tubes, take the modified PC-linker and biotinylated aptamer AptE (200μL) and add them to the above centrifuge tubes, corresponding to 7 tubes in total. Place the centrifuge tube in a constant temperature oscillator (180rpm) at 27°C and incubate for 30min. Remove the unbound aptamer by magnetic separation, and then add 1% bovine serum albumin (BSA) as a blocking solution to block the unbound sites on the surface of the magnetic beads, and incubate at 27°C for 1h. After magnetic separation and washing with PBS twice, the beads were reconstituted with 1×PBS to 200 μL to obtain aptamer-functionalized magnetic beads IMB@Apt-E. The above solutions were mixed with 7 portions of bacterial solution of different concentrations, fixed to 1 mL with 1×PBS, and incubated at 27°C for 1 h. Since PBS would affect the capture of fluorescence in the next step, the beads were washed twice with Tri-HCl (pH=7.20) containing NaCl and MgCl2 after magnetic separation, and the supernatant was discarded to wash away the free bacterial solution. 1 mL of PME-modified AIE CuNCs was added to each of the 7 centrifuge tubes, mixed and incubated at 27°C for 2 h. Excess metal nanoclusters and antibiotics were removed by magnetic separation, washed with Tris-HCl 3 times, the supernatant was discarded, the mixed precipitate was reconstituted with 1 mL of Tris-HCl, and then placed under 365 nm ultraviolet light for 4 min to break the PC-linker. After magnetic separation, the supernatant was taken and the fluorescence intensity was measured by fluorescence spectrometer.

[0110] like Figure 8 As shown in (a) and 8(b), the fluorescence intensity of Apt-E / E.coli / PME@AIE Cu NCs after magnetic separation under 330nm excitation shows a good linear relationship with the logarithmic value of the bacterial solution concentration: y = 20.48x + 7.98, R 2 =0.9946 (where y represents the fluorescence intensity and x represents Log 细菌液浓度 ), and the limit of detection (LOD) was 1.05 CFU / mL.

[0111] In order to achieve good detection results, the concentration of the aptamer, the capture time, and the UV irradiation time were optimized. During the optimization process, the concentration of the bacterial solution was 1×10 5 CFU / mL. Fig. 9As shown in (a)-(c), the final aptamer concentration of E. coli was determined to be 200 nmol / L, the capture time was 1.5 h, and the time for UV irradiation to break the PC-linker was 4 min.

[0112] Example 4

[0113] Specificity analysis

[0114] In order to explore the specificity of the “sandwich structure” sensor in detecting E. coli, Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella entericitis (S. enteriditis) were selected as interfering bacteria for determination. The concentration of all bacterial solutions during the experiment was 1×10 5 CFU / mL. Replace the E. coli bacterial solution with the above bacteria and follow the experimental procedures. Finally, detect the fluorescence signal. Fig.10 As shown, the fluorescence signal of the E. coli bacterial liquid group is the strongest, indicating that this strategy has high selectivity under the dual recognition of aptamers and antibiotics. In summary, this detection method has good sensitivity and specificity.

[0115] Example 5

[0116] In vitro antibacterial study of “sandwich structure”

[0117] The purpose of the present invention is to capture and eventually kill bacteria after dual recognition of bacteria by metal nanoclusters and antibiotics. As is well known, both metal nanoclusters and antibiotics have certain bactericidal effects. Therefore, this study uses the plate count method to explore the antibacterial activity of metal nanoclusters and antibiotics.

[0118] First, after magnetic separation, 100 μL of the sandwich solution was applied to LB solid medium and cultured overnight at 37°C. The blank control group was cultured at 37°C, and the bacterial solution was 1×10 5 CFU / mL. According to previous reports, bacterial viability was calculated according to the following formula:

[0119]

[0120] Where: BV represents bacterial activity, N c and N0 represent the colony counts of the cells treated with different antimicrobial methods and the colony count of the Control group, respectively.

[0121] like Fig.11As shown in the figure, the control group without any treatment was full of E. coli colonies. When E. coli was cultured with PME@AIE Cu NCs, the number of bacterial colonies was greatly reduced, indicating that metal nanoclusters and antibiotics had a certain antibacterial effect on bacteria. The antibacterial rate of PME@AIE Cu NCs against E. coli reached 91.0%. Metal nanoclusters and antibiotics have excellent antibacterial effects on E. coli, which can be attributed to the spectrum of antibacterial activity of Cu. Metal nanoclusters can penetrate the cell wall into the bacteria, induce the production of reactive oxygen species (ROS) to oxidize the cell membrane and interfere with the metabolism of bacteria, thereby achieving the purpose of sterilization.

[0122] In summary, the present invention establishes an antibiotic-modified AIE Cu NCs collaborative aptamer sensing technology for the integrated strategy of bacterial identification, capture and antibacterial, and prepares a "sandwich structure" material that can capture bacteria. In addition, the in vitro antibacterial study of the "sandwich structure" showed that the antibacterial rate of metal nanoclusters and antibiotics against E. coli was 91.0%, and the antibiotic-modified AIE Cu NCs had excellent luminescence performance and stability. Under the action of antibiotics and aptamers, this strategy has good sensitivity and specificity, and can achieve trace detection and directional capture of bacteria. It also has good versatility and can capture different bacteria by changing the type of aptamer. The method based on the present invention for the integration of bacterial identification, capture and antibacterial has broad application prospects.

[0123] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. An aptamer fluorescence sensing method for bacterial identification, capture and sterilization, characterized in that: The method is used for non-disease diagnosis and treatment purposes, including: preparing an AIE Cu NCs solution, and performing polymyxin E modification on the AIE Cu NCs solution to obtain polymyxin E modified AIE CuNCs; Cultivating bacteria and preparing aptamer-functionalized magnetic beads; The streptavidin-labeled magnetic beads with E. coli aptamers were linked to PME@AIE Cu NCs to recognize and capture E. coli, and the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure was obtained; The photo-controllable group PC-linker in the aptamer is broken by ultraviolet light irradiation, and the supernatant is taken after magnetic separation. The fluorescence intensity is measured by a fluorescence spectrometer to achieve recognition and capture of bacteria. The process of preparing the AIE CuNCs solution comprises: Under magnetic stirring at room temperature, anhydrous CuSO4 solution was added to 2.4 mL of ultrapure water, mixed thoroughly, and then GSH solution was added; The mixture was transferred to a 37°C water bath and magnetically stirred for 30 min, and then the pH of the system was adjusted to 4.0-5.5 with 1 M NaOH solution; The reaction solution was vigorously stirred at 37°C for 1 h to prepare a CuNCs solution; Add Ce(NO3)3·6H2O solution to the Cu NCs solution, make up to volume, and stir at room temperature for 10 min; Then dialyze for 1 h using a dialysis bag with MW = 500Da to obtain the AIE CuNCs solution; The molar ratio of the anhydrous CuSO4 solution to the GSH solution is 8:1; The pH range is 4.6 to 4.7; The amount of Ce(NO3)3·6H2O solution used is 50 μL of 20 mM solution; The process of modifying the AIE CuNCs solution with polymyxin E to obtain polymyxin E-modified AIE Cu NCs includes: Polymyxin E and cerium nitrate solution were added to Cu NCs, and the volume was adjusted to 8 mL with water and then magnetically stirred to prepare polymyxin E-modified AIE CuNCs. The amount of CuNCs used was 1.6 mL; The dosage of polymyxin E was 2.4 mL 4 mg / mL, and the volume was made up to 8 mL with water, and then stirred vigorously for 1 h; The process of culturing bacteria includes: The bacterial colony was placed in 10 mL of LB liquid medium, and then the bacteria were placed in a constant temperature shaker at 37 °C and cultured at 180 rpm overnight; The bacteria include Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes; The process of preparing aptamer-functionalized magnetic beads includes: The E. coli aptamer Apt-E labeled with biotin and photocontrollable group PC-linker was reacted with streptavidin-labeled magnetic beads at 27°C for 1 hour, and then washed twice with PBS buffer solution and magnetically separated to obtain aptamer-functionalized magnetic beads IMB@Apt-E. The process of linking the streptavidin-labeled magnetic beads with E. coli aptamers to PME@AIE Cu NCs to recognize and capture E. coli to obtain the IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure includes: The magnetic beads IMB@Apt-E were mixed with bacterial solutions of different concentrations and diluted to 1 mL with 1×PBS. The solution was incubated with shaking and washed twice with Tri-HCl after magnetic separation. The supernatant was discarded to wash away the free bacterial solution. Polymyxin E-modified AIE CuNCs were added to the solution and incubated at 27 °C with shaking for 1.5 h to form an IMB@Apt-E / E.coli / PME@AIE CuNCs sandwich structure; The concentration of the bacterial solution is: 10 8 ~10 2 CFU / mL; The temperature and time of the shaking incubation are 27° C. and 1 h, respectively; The Tri-HCl buffer contains NaCl and MgCl2 and has a pH of 7.20; The ultraviolet lamp is a 365nm ultraviolet lamp, and the irradiation time is 4 minutes.

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