A nanocomposite probe system and application thereof, and a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level

Abstract

The application belongs to the technical field of bacterial detection, and provides a nano composite probe system and application thereof, and a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level, the nano composite probe system comprising phage-embedded magnetic bead probes and AIE@Ag@MnO2 NFs composite probes. The Ag-MnO2 nanoflower surface-modified AIE (Raman reporter molecule) composite probe can realize strong Raman signals, Ag@MnO2 catalyzes the decomposition of hydrogen peroxide to push the red dye to move and realize distance signals, and the magnetic bead-phage can realize magnetic separation and enrichment as a recognition element of target bacteria. Based on the high specificity and good environmental tolerance of the phage, the Ag-MnO2 nanoflower enhances the Raman signal of the AIE Raman reporter molecule, the double-mode signal internal correction realizes the stability of the sensor and the anti-interference of environmental and experimental factors, and improves the specificity of the detection strategy and the accuracy of on-site analysis.

Description

Technical Field

[0001] This invention relates to the field of bacterial detection technology, and in particular to a nanocomposite probe system and its application, as well as a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level. Background Technology

[0002] Escherichia coli poses a serious threat to human society. Its potential for contamination in water and food sources makes it a major threat to food safety and public health. Furthermore, its drug resistance and high pathogenicity also pose serious risks to life and health. Therefore, rapid and accurate detection of E. coli and comprehensive testing for drug resistance levels are of paramount importance. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nanocomposite probe system and its application, as well as a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a nanocomposite probe system comprising phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes;

[0006] The volume ratio of the phage-embedded magnetic bead probe to the AIE@Ag@MnO2NFs composite probe is 400–600:40–60.

[0007] Preferably, the method for preparing the phage-embedded magnetic bead probe includes the following steps:

[0008] The magnetic bead solution and polydiallyldimethylammonium chloride solution were mixed and then added sequentially to the phage and bovine serum albumin solution to react, thus obtaining the phage-embedded magnetic bead probe.

[0009] The mass-to-volume ratio of magnetic beads to water in the magnetic bead solution is 5–15 mg: 0.5–1.5 mL;

[0010] The mass concentration of the polydiallyl dimethylammonium chloride solution is 0.5-1.5%;

[0011] The volume ratio of the magnetic bead solution to the polydiallyldimethylammonium chloride solution is 1-2:8-9;

[0012] The mixing speed is 2000-2500 rpm, and the time is 1-2 hours;

[0013] The abundance of the bacteriophage was 10. 13 ~15 13PFU / mL; phage addition ≥ 5 times; reaction time after each phage addition 1–2 h; phage addition volume to magnetic bead solution volume ratio 0.1:1–2;

[0014] The mass concentration of the bovine serum albumin solution is 0.5-1.5%; the volume ratio of bovine serum albumin solution to magnetic bead solution is 1:5-10; and the reaction time after adding bovine serum albumin solution is 1-2 hours.

[0015] Preferably, the preparation method of the AIE@Ag@MnO2NFs composite probe includes the following steps:

[0016] (1) TPE-C≡Cpy, iodomethane and acetonitrile were mixed and reacted to obtain the iodinated product;

[0017] (2) The iodide product, methanol and potassium hexafluorophosphate saturated solution were mixed to obtain AIE material;

[0018] (3) Potassium permanganate solution, polyvinylpyrrolidone and hydrochloric acid solution were mixed and reacted to obtain MnO2NFs;

[0019] (4) Silver nitrate solution, trisodium citrate solution, MnO2NFs solution and sodium borohydride solution are mixed and reacted to obtain Ag@MnO2NFs;

[0020] (5) The AIE material, Ag@MnO2NFs solution and Tween 80 are mixed and reacted to obtain the AIE@Ag@MnO2NFs composite probe.

[0021] Preferably, the structure of TPE-C≡Cpy in step (1) is as follows:

[0022]

[0023] The molar ratio of TPE-C≡Cpy to iodomethane in step (1) is 0.09–0.1:4.9–5;

[0024] The molar ratio of TPE-C≡Cpy to acetonitrile is 140–160:1;

[0025] The reaction in step (1) is carried out at a temperature of 80–90°C for 10–12 hours.

[0026] In step (2), the volume molar ratio of methanol to TPE-C≡Cpy in step (1) is 10–15 mL: 0.09–0.1 mmol;

[0027] In step (2), the volume ratio of methanol to saturated potassium hexafluorophosphate solution is 10–15:10–15.

[0028] The mixing temperature in step (2) is 20–30°C, and the mixing time is 1–1.5 h;

[0029] In step (3), the mass ratio of potassium permanganate to water in the potassium permanganate solution is 15-16:90-110;

[0030] In step (3), the mass ratio of polyvinylpyrrolidone to potassium permanganate is 26–26.5:15–16;

[0031] In step (3), the concentration of the hydrochloric acid solution is 0.1–0.3 M;

[0032] In step (3), the volume-to-mass ratio of hydrochloric acid solution to potassium permanganate is 80-120 mL: 15-16 g;

[0033] The reaction in step (3) is carried out at a temperature of 80–100°C for 1–2 hours.

[0034] Preferably, in step (4), the concentration of the silver nitrate solution is 0.01–0.03 M; and the concentration of the trisodium citrate solution is 0.01–0.03 M.

[0035] The concentration of the MnO2NFs solution was 5–15 mg / mL; the concentration of the sodium borohydride solution was 0.05–0.15 M.

[0036] The volume ratio of silver nitrate solution, trisodium citrate solution, MnO2NFs solution, and sodium borohydride solution is 0.5–1.5: 2–3: 20–30: 0.5–1.5;

[0037] The reaction in step (4) is carried out at a temperature of 20–25°C for 0.5–1 h.

[0038] In step (5), the concentration of the Ag@MnO2NFs solution is 120–160 μg / mL;

[0039] In step (5), the volume ratio of AIE material, Ag@MnO2NFs solution and Tween 80 is 20-30 μL: 10-15 mL: 2-3 μL;

[0040] The reaction in step (5) is carried out at a temperature of 20-25°C for 0.5-1 hour.

[0041] This invention also provides the application of the aforementioned nanocomposite probe system in the detection of bacterial count and bacterial resistance levels.

[0042] This invention also provides a single-channel microfluidic chip for detecting bacterial count and bacterial resistance levels, the structure of which is as follows:

[0043] A single-channel microfluidic chip includes a sealing glass layer and a working PDMS layer;

[0044] The working PDMS layer has three circular inlets at one end, and the other end of the three circular inlets is connected to the inlet of the chain-link micromixer.

[0045] The outlet of the chain-link micromixer is sequentially connected to the Raman signal detection chamber, the hydrogen peroxide storage chamber, and the working chamber.

[0046] The nanocomposite probe system is injected into the working PDMS layer through a circular inlet.

[0047] Preferably, the diameter of the three circular inlets is 1 to 2 mm;

[0048] The number of channel spacings in the chain-link micromixer is ≥13; the width of a single channel spacing is 1–2 mm;

[0049] The Raman signal detection chamber and the hydrogen peroxide storage chamber have independent volumes of 80–120 μL;

[0050] The working chamber consists of a dye storage chamber and a distance indicator channel connected in sequence;

[0051] The diameter of the dye storage chamber is 3-4 mm, and the width of the distance indicator channel is 0.5-1.5 mm.

[0052] This invention also provides a method for using a single-channel microfluidic chip to detect bacterial count and bacterial resistance levels, comprising the following steps:

[0053] (I) Detection of bacterial count

[0054] (a) The test bacteria, phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets respectively. After being fully mixed by a chain segment structure micromixer, Raman signals and pressure distance signals were generated to detect the number of bacteria.

[0055] (II) Detection of bacterial resistance levels

[0056] (b) The test bacteria, phage-embedded magnetic bead probes, and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets, respectively. Antibiotics were injected into the circular inlet where the test bacteria were injected. After thorough mixing by a chain segment micromixer, Raman signals and pressure distance signals were generated to detect the bacterial resistance level.

[0057] Preferably, in step (a), the volume ratio of the injected bacteria to be tested and the magnetic bead probe embedded with bacteriophage is 400-600: 400-600.

[0058] In step (b), the volume ratio of the injected bacteria to be tested and the magnetic bead probe embedded with bacteriophage is 400-600:400-600.

[0059] This invention provides a nanocomposite probe system comprising phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes. The Ag-MnO2 nanoflower surface-modified AIE (Raman reporter molecule) composite probe achieves a strong Raman signal. Ag@MnO2 catalyzes the decomposition of hydrogen peroxide, driving the movement of red dye to generate a distance signal. The magnetic beads (MB)-phage act as recognition elements for target bacteria while simultaneously achieving magnetic separation and enrichment. Based on the high specificity and good environmental tolerance of the phage, the enhancement of the Raman signal of the AIE Raman reporter molecule by the Ag-MnO2 nanoflowers, and the internal correction of the dual-mode signal, the stability of the sensor and its resistance to interference from environmental and experimental factors are achieved, thus improving the specificity of the detection strategy and the accuracy of on-site analysis.

[0060] This invention proposes a novel SERS nanoprobe for detecting bacteria by constructing a nanocomposite probe system comprising TPEC≡Cpy+(AIE) and spherical nanoflowers Ag@MnO2. MnO2 nanoflowers, with their high specific surface area, large loading rate, good biocompatibility, and ease of surface modification, are widely used in the field of biosensors. Therefore, their surface can be modified and loaded to achieve multifunctionality. Based on the Ag-MnO2NFs surface-modified AIE Raman reporter molecule composite probe, a novel biosensor was constructed, which can rapidly and sensitively detect bacterial counts through dual-mode signals (SERS signal and pressure-distance signal).

[0061] This invention presents a novel biosensor based on an Ag-MnO2NFs surface-modified AIE Raman reporter molecule composite probe, enabling rapid and sensitive detection of bacteria via dual-mode signals (SERS signal and pressure-distance signal). First, a quaternary ammonium salt TPEC≡Cpy+(AIE) molecule with Raman characteristic signals and capable of binding to bacteria was synthesized. Manganese dioxide nanoflowers (MnO2NFs) were prepared by reduction of potassium permanganate (KMnO4) using a reduction method. Then, Ag nanoparticles (AgNPs) and TPEC≡Cpy+(AIE) molecules were in situ reduced and embedded on their surface. The clustered manganese dioxide nanoflowers ensured the stability of the AgNPs. The prepared nanocomposite material consists of manganese dioxide nanoflowers, Ag nanoparticles embedded in the nanoflowers, and Raman reporter molecules (AIE), with a significant increase in signal at the SERS active site. Therefore, functionalized AIE@Ag@MnO2NFs were used as SERS nanoprobes for detecting bacteria. Furthermore, functionalized AIE@Ag@MnO2NFs and phage-modified magnetic beads formed a sandwich complex in a mixing channel, which, when enriched and separated by magnets in the reaction chamber, not only generated a significant Raman signal; but upon the addition of hydrogen peroxide, the MnO2-catalyzed decomposition of hydrogen peroxide propelled the movement of the red dye, thus generating a distance signal. Since the Raman signal and gas are generated in two different reaction chambers, the signals generated in the two sensing regions do not interfere with each other. Therefore, this dual-mode biosensing strategy enables target detection, providing a new probe design approach for developing dual-mode target biosensing detection.

[0062] This invention exhibits superior analytical performance. Under optimal conditions, different concentrations of *E. coli* were selected to test the biosensor's dual-signal detection capability. Results showed that as the bacterial count increased, both the generated SERS and distance signals increased, and the two signals generated on the two detection units did not affect the corresponding signals generated on other detection units. (10) 2 -10 8 Within the range of CFU / mL, the logarithmic concentration of bacteria [E. coli] showed a linear relationship with both SERS and distance signals. The linear regression equation for the Raman signal could be fitted as y = 777.35782Log[E. coli] + 1532.83221(R²). 2 =0.9901). The detection limit of E. coli is 17 CFU / mL. The linear regression equation for the distance signal is y = 31.0137Log[E. coli] ± 38.03813 (R = 0.9901). 2 =0.9971), and the detection limit is 35 CFU / mL (S / N = 3). Therefore, this detection method can be used for on-site quantification of target analytes with dual signals.

[0063] This invention features an extremely simple operation process, low cost, and significantly reduced detection time, thus greatly improving detection efficiency. The entire detection process is easy to implement and does not rely on large, expensive laboratory equipment. In actual testing, it allows for intuitive visualization of bacterial counts and antibiotic resistance, and further calculations using data analysis systems and linear equations can improve detection efficiency and ensure high accuracy of results. Attached Figure Description

[0064] Figure 1 This is a schematic diagram illustrating the preparation of the phage-embedded magnetic bead probe in Example 1;

[0065] Figure 2 This is a schematic diagram illustrating the preparation of the AIE@Ag@MnO2NFs composite probe in Example 1;

[0066] Figure 3 This is a schematic diagram of the application process of the microfluidic chip in Example 1;

[0067] Figure 4 This is a graph showing the test results of different concentrations of Escherichia coli in Example 1;

[0068] Figure 5 This is a graph showing the resistance distance signal response values ​​of different concentrations of antibiotics used in Example 1 against Escherichia coli. Detailed Implementation

[0069] This invention provides a nanocomposite probe system comprising phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes.

[0070] In this invention, the volume ratio of the phage-embedded magnetic bead probe and the AIE@Ag@MnO2NFs composite probe is preferably 400-600:40-60, more preferably 450-550:45-55, and even more preferably 460-540:46-54.

[0071] This invention provides a method for preparing magnetic beads, comprising the following steps:

[0072] The magnetic beads are obtained by hydrothermal synthesis of anhydrous sodium acetate, ferric chloride hexahydrate, and ethylene glycol.

[0073] In this invention, the preferred mass-to-volume ratio of anhydrous sodium acetate, ferric chloride hexahydrate, and ethylene glycol is 3.5–4 g: 1–1.5 g: 35–45 mL, more preferably 3.6–3.9 g: 1.1–1.4 g: 36–44 mL, and even more preferably 3.7–3.8 g: 1.2–1.3 g: 38–42 mL.

[0074] In this invention, the temperature of the hydrothermal synthesis is preferably 200-300°C, more preferably 220-280°C, and even more preferably 240-260°C; the time is preferably 4-8 hours, more preferably 4.5-7.5 hours, and even more preferably 5-7 hours.

[0075] In this invention, after hydrothermal synthesis, the particles are naturally cooled to room temperature and washed three times with ethanol and three times with water. The resulting magnetic nanoparticles are then vacuum dried. The vacuum drying temperature is preferably 40–60°C, more preferably 45–55°C, and even more preferably 48–52°C. The drying time is preferably 10–15 h, more preferably 11–14 h, and even more preferably 12–13 h. After drying, magnetic beads are obtained.

[0076] In this invention, the method for preparing the phage-embedded magnetic bead probe includes the following steps:

[0077] The magnetic bead solution and polydiallyldimethylammonium chloride solution are mixed and then added sequentially to the phage and bovine serum albumin solution to react, thus obtaining the phage-embedded magnetic bead probe.

[0078] In this invention, bovine serum albumin was purchased from Sigma-Aldrich.

[0079] In this invention, the preferred mass-to-volume ratio of magnetic beads to water in the magnetic bead solution is 5-15 mg: 0.5-1.5 mL, more preferably 6-14 mg: 0.6-1.4 mL, and even more preferably 8-12 mg: 0.8-1.2 mL. To ensure sufficient dispersion, the magnetic beads are dispersed in water and then subjected to ultrasonic treatment. The ultrasonic treatment time is preferably ≥10 min, more preferably ≥15 min, and even more preferably ≥20 min.

[0080] In this invention, the mass concentration of the polydiallyldimethylammonium chloride solution is preferably 0.5-1.5%, more preferably 0.6-1.4%, and even more preferably 0.8-1.2%.

[0081] In this invention, the volume ratio of the magnetic bead solution to the polydiallyldimethylammonium chloride solution is preferably 1-2:8-9, more preferably 1.2-1.8:8.2-8.8, and even more preferably 1.4-1.6:8.4-8.6.

[0082] In this invention, the mixing speed is preferably 2000-2500 rpm, more preferably 2100-2400 rpm, and even more preferably 2200-2300 rpm; the mixing time is preferably 1-2 h, more preferably 1.2-1.8 h, and even more preferably 1.4-1.6 h.

[0083] In this invention, after mixing, the MB@PDDA obtained is washed with water, preferably ≥3 times, more preferably ≥4 times, and even more preferably ≥5 times.

[0084] In this invention, the rotation speed is kept constant while bacteriophage is added; the abundance of the bacteriophage is preferably 10. 13 ~15 13 PFU / mL, further preferably 11 13 ~14 13 PFU / mL, more preferably 12 13 ~13 13 PFU / mL; the number of times the phage is added is preferably ≥5 times, more preferably ≥6 times, and even more preferably ≥7 times; the reaction time after a single phage addition is preferably 1 to 2 h, more preferably 1.2 to 1.8 h, and even more preferably 1.4 to 1.6 h; the volume ratio of the amount of phage added per addition to the volume of the magnetic bead solution is preferably 0.1:1 to 2, more preferably 0.1:1.2 to 1.8, and even more preferably 0.1:1.4 to 1.6; due to the negative charge on the phage head, the bacteria will be oriented and fixed on the surface of MB by electrostatic adsorption.

[0085] In this invention, the mass concentration of the bovine serum albumin solution is preferably 0.5-1.5%, more preferably 0.6-1.4%, and even more preferably 0.8-1.2%; the volume ratio of the bovine serum albumin solution to the magnetic bead solution is preferably 1:5-10, more preferably 1:6-9, and even more preferably 1:7-8; the reaction time after adding the bovine serum albumin solution is preferably 1-2 h, more preferably 1.2-1.8 h, and even more preferably 1.4-1.6 h. After the reaction, the solution is washed with water, preferably ≥3 times, more preferably ≥4 times, and even more preferably ≥5 times, to obtain the phage-embedded magnetic bead probe.

[0086] In this invention, the preparation method of the AIE@Ag@MnO2NFs composite probe includes the following steps:

[0087] (1) TPE-C≡Cpy, iodomethane and acetonitrile were mixed and reacted to obtain the iodinated product;

[0088] (2) The iodide product, methanol and potassium hexafluorophosphate saturated solution were mixed to obtain AIE material;

[0089] (3) Potassium permanganate solution, polyvinylpyrrolidone and hydrochloric acid solution were mixed and reacted to obtain MnO2NFs;

[0090] (4) Silver nitrate solution, trisodium citrate solution, MnO2NFs solution and sodium borohydride solution are mixed and reacted to obtain Ag@MnO2NFs;

[0091] (5) The AIE material, Ag@MnO2NFs solution and Tween 80 are mixed and reacted to obtain the AIE@Ag@MnO2NFs composite probe.

[0092] In this invention, the structure of TPE-C≡Cpy in step (1) is as follows:

[0093]

[0094] In this invention, the TPE-C≡Cpy was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0095] In this invention, the molar ratio of TPE-C≡Cpy and iodomethane in step (1) is preferably 0.09-0.1:4.9-5, more preferably 0.092-0.098:4.92-4.98, and even more preferably 0.094-0.096:4.94-4.96.

[0096] In this invention, the molar ratio of TPE-C≡Cpy to acetonitrile is preferably 140-160:1, more preferably 145-155:1, and even more preferably 148-152:1.

[0097] In this invention, the reaction described in step (1) is carried out in a protective atmosphere, preferably nitrogen, helium or neon; the temperature is preferably 80-90°C, more preferably 82-88°C, and even more preferably 84-86°C; the time is preferably 10-12h, more preferably 10.5-11.5h, and even more preferably 10.8-11.2h.

[0098] In this invention, after the reaction is completed, the mixture is cooled to room temperature, and the resulting mixture is mixed with n-hexane, then filtered and dried to obtain the iodinated product.

[0099] In this invention, the volume molar ratio of methanol in step (2) to TPE-C≡Cpy in step (1) is preferably 10-15 mL: 0.09-0.1 mmol, more preferably 11-14 mL: 0.092-0.098 mmol, and even more preferably 12-13 mL: 0.094-0.096 mmol.

[0100] In this invention, the volume ratio of methanol to potassium hexafluorophosphate saturated solution in step (2) is preferably 10-15:10-15, more preferably 11-14:11-14, and even more preferably 12-13:12-13.

[0101] In this invention, the mixing temperature in step (2) is preferably 20-30°C, more preferably 22-28°C, and even more preferably 24-26°C; the mixing time is preferably 1-1.5h, more preferably 1.1-1.4h, and even more preferably 1.2-1.3h; stirring is maintained during the mixing process; after the mixing is completed, the material is sequentially filtered, recrystallized, washed, and dried to obtain the AIE material.

[0102] In this invention, the mass ratio of potassium permanganate to water in the potassium permanganate solution in step (3) is preferably 15-16:90-110, more preferably 15.2-15.8:95-105, and even more preferably 15.4-15.6:98-102.

[0103] In this invention, the mass ratio of polyvinylpyrrolidone to potassium permanganate in step (3) is preferably 26-26.5:15-16, more preferably 26.1-26.4:15.2-15.8, and even more preferably 26.2-26.3:15.4-15.6.

[0104] In this invention, in step (3), potassium permanganate solution and polyvinylpyrrolidone are mixed first, and then hydrochloric acid solution is added.

[0105] In this invention, the concentration of the hydrochloric acid solution in step (3) is preferably 0.1 to 0.3 M, more preferably 0.15 to 0.25 M, and even more preferably 0.18 to 0.22 M.

[0106] In this invention, the volume-to-mass ratio of hydrochloric acid solution and potassium permanganate in step (3) is preferably 80-120 mL: 15-16 g, more preferably 85-115 mL: 15.2-15.8 g, and even more preferably 90-110 mL: 15.4-15.6 g.

[0107] In this invention, the temperature of the reaction in step (3) is preferably 80-100°C, more preferably 85-95°C, and even more preferably 88-92°C; the time is preferably 1-2 h, more preferably 1.2-1.8 h, and even more preferably 1.4-1.6 h.

[0108] In this invention, after the reaction in step (3) is completed, the mixture is naturally cooled to room temperature, and then centrifuged, washed and dried in sequence. The centrifugation speed is preferably 6000-10000 rpm, more preferably 7000-9000 rpm, and more preferably 7500-8500 rpm; the centrifugation time is preferably ≥30 min, more preferably ≥40 min, and more preferably ≥50 min.

[0109] In this invention, the concentration of silver nitrate solution in step (4) is preferably 0.01-0.03M, more preferably 0.015-0.025M, and even more preferably 0.018-0.022M; the concentration of trisodium citrate solution is preferably 0.01-0.03M, more preferably 0.015-0.025M, and even more preferably 0.018-0.022M.

[0110] In this invention, the concentration of the MnO2NFs solution is preferably 5-15 mg / mL, more preferably 6-14 mg / mL, and even more preferably 8-12 mg / mL; the concentration of the sodium borohydride solution is preferably 0.05-0.15 M, more preferably 0.06-0.14 M, and even more preferably 0.08-0.12 M.

[0111] In this invention, the volume ratio of silver nitrate solution, trisodium citrate solution, MnO2NFs solution and sodium borohydride solution is preferably 0.5-1.5:2-3:20-30:0.5-1.5, more preferably 0.6-1.4:2.2-2.8:22-28:0.6-1.4, and even more preferably 0.8-1.2:2.4-2.6:24-26:0.8-1.2.

[0112] In this invention, silver nitrate solution and trisodium citrate solution are mixed and stirred, then MnO2NFs solution is added, and sodium borohydride solution is added dropwise to the system to carry out the reaction. The stirring speed is preferably 400-800 rpm, more preferably 450-750 rpm, and even more preferably 500-700 rpm.

[0113] In this invention, the reaction temperature in step (4) is preferably 20-25°C, more preferably 21-24°C, and even more preferably 22-23°C; the reaction time is preferably 0.5-1h, more preferably 0.6-0.9h, and even more preferably 0.7-0.8h; after the reaction is completed, stirring is stopped, and the mixture is centrifuged, washed, and dried in sequence to obtain Ag@MnO2NFs.

[0114] In this invention, the concentration of the Ag@MnO2NFs solution in step (5) is preferably 120-160 ug / mL, more preferably 130-150 ug / mL, and even more preferably 135-145 ug / mL.

[0115] In this invention, the volume ratio of AIE material, Ag@MnO2NFs solution and Tween 80 in step (5) is preferably 20-30 μL: 10-15 mL: 2-3 μL, more preferably 22-28 μL: 11-14 mL: 2.2-2.8 μL, and even more preferably 24-26 μL: 12-13 mL: 2.4-2.6 μL.

[0116] In this invention, the AIE material and Ag@MnO2NFs solution are first mixed. The mixing temperature is preferably 20-25°C, more preferably 21-24°C, and even more preferably 22-23°C. The mixing time is preferably 2-3 hours, more preferably 2.2-2.8 hours, and even more preferably 2.4-2.6 hours. After mixing, Tween 80 is added to initiate the reaction.

[0117] In this invention, the reaction temperature in step (5) is preferably 20–25°C, more preferably 21–24°C, and even more preferably 22–23°C; the reaction time is preferably 0.5–1 h, more preferably 0.6–0.9 h, and even more preferably 0.7–0.8 h. After the reaction is completed, the AIE@Ag@MnO2NFs composite probe is obtained by washing.

[0118] This invention also provides the application of the aforementioned nanocomposite probe system in the detection of bacterial count and bacterial resistance levels.

[0119] This invention also provides a single-channel microfluidic chip for detecting bacterial count and bacterial resistance levels, the structure of which is as follows:

[0120] A single-channel microfluidic chip consists of a sealing glass layer and a working PDMS layer;

[0121] The working PDMS layer has three circular inlets at one end, and the other end of the three circular inlets is connected to the inlet of the chain-link micromixer.

[0122] The outlet of the chain-link micromixer is sequentially connected to the Raman signal detection chamber, the hydrogen peroxide storage chamber, and the working chamber.

[0123] The nanocomposite probe system is injected into the working PDMS layer through a circular inlet.

[0124] In this invention, the diameter of the three circular inlets is preferably 1 to 2 mm, more preferably 1.2 to 1.8 mm, and even more preferably 1.4 to 1.6 mm.

[0125] In this invention, the number of channel spacings in the chain-link micromixer is preferably ≥13, more preferably ≥15, and even more preferably ≥18; the width of a single channel spacing is preferably 1 to 2 mm, more preferably 1.2 to 1.8 mm, and even more preferably 1.4 to 1.6 mm.

[0126] In this invention, the Raman signal detection chamber and the hydrogen peroxide storage chamber are preferably 80-120 μL each, more preferably 90-110 μL each, and even more preferably 95-105 μL each.

[0127] In this invention, the working chamber consists of a dye storage chamber and a distance indicator channel connected in sequence.

[0128] In this invention, the diameter of the dye storage chamber is preferably 3-4 mm, more preferably 3.2-3.8 mm, and even more preferably 3.4-3.6 mm; the width of the distance indicator channel is preferably 0.5-1.5 mm, more preferably 0.6-1.4 mm, and even more preferably 0.8-1.2 mm.

[0129] In this invention, the method for preparing the working PDMS layer is as follows:

[0130] The PDMS layer was designed using 3ds Max software, then a mold for the PDMS layer was manufactured using an Object30Pro 3D printer. The PDMS prepolymer and curing agent were then mixed thoroughly and injected into the mold. The mixture was subsequently placed in a vacuum environment for degassing. The mold was then cured to obtain a complete PDMS layer.

[0131] In this invention, the mass ratio of the PDMS prepolymer to the curing agent is preferably 10-15:1, more preferably 11-14:1, and even more preferably 12-13:1; the curing temperature is preferably 60-70°C, more preferably 62-68°C, and even more preferably 64-66°C; the curing time is preferably ≥12h, more preferably ≥14h, and even more preferably ≥16h.

[0132] This invention also provides a method for using the single-channel microfluidic chip for detecting bacterial count and bacterial resistance levels, comprising the following steps:

[0133] (I) Detection of bacterial count

[0134] (a) The test bacteria, phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets respectively. After being fully mixed by a chain segment structure micromixer, Raman signals and pressure distance signals were generated to detect the number of bacteria.

[0135] (II) Detection of bacterial resistance levels

[0136] (b) The test bacteria, phage-embedded magnetic bead probes, and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets, respectively. Antibiotics were injected into the circular inlet where the test bacteria were injected. After thorough mixing by a chain segment micromixer, Raman signals and pressure distance signals were generated to detect the bacterial resistance level.

[0137] In this invention, the volume ratio of the injected bacteria to be tested and the magnetic bead probe embedded with the phage in step (a) is preferably 400-600:400-600, more preferably 450-550:450-550, and even more preferably 460-540:460-540.

[0138] In this invention, the volume ratio of the injected bacteria to be tested and the magnetic bead probe embedded with the phage in step (b) is preferably 400-600:400-600, more preferably 450-550:450-550, and even more preferably 460-540:460-540.

[0139] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0140] This invention provides methods for screening, purifying, identifying, and preserving bacteriophages, as detailed below:

[0141] The phage screening method includes the following steps:

[0142] (1) The water sample used for screening bacteriophages was taken from the medical wastewater in the sewer of the Second People's Hospital of Guangdong Province.

[0143] (2) Take 30 mL of the water sample and place it in a 50 mL centrifuge tube. Centrifuge at 8000 r / min for 30 min. After centrifugation, filter the sample through a 0.45 μm filter membrane and a 0.22 μm filter membrane, respectively. Take the supernatant into a 15 mL centrifuge tube and store it at 4 °C.

[0144] (3) Take Escherichia coli bacterial culture in the logarithmic growth phase, water sample, and LB liquid medium, mix them evenly in a ratio of 1:2:4, and incubate them in a shaker at 37℃ for 18 h. Centrifuge the cultured mixture at 8000 r / min for 30 min. After centrifugation, filter through a 0.45 μm filter membrane and a 0.22 μm filter membrane, respectively, and transfer the supernatant to a 1.5 mL centrifuge tube and store at 4℃.

[0145] (4) Prepare LB solid medium and semi-solid medium. Use LB solid medium to pour the first layer of plates for later use. Use a pipette to pipette 200 μL of Escherichia coli in the logarithmic growth phase into a 5 mL centrifuge tube, then add 100 μL of the initially enriched phage fluid, and finally add 3 mL of semi-solid medium and mix well. Pour the mixture onto the first layer of LB solid medium while it is still hot and spread it evenly. After the upper layer of semi-solid medium has cooled, invert the plate and incubate it in a 37°C incubator for 16 h to 24 h.

[0146] (5) The presence of phage plaques on a double-layer agar plate indicates the presence of Escherichia coli phages. Add 3 mL of liquid culture medium to the plate, gently scrape off and break up the upper semi-solid culture medium with an L-shaped spreader, and transfer it to a 50 mL centrifuge tube. Centrifuge at 8000 r / min for 30 min. After centrifugation, filter through a 0.45 μm filter membrane and a 0.22 μm filter membrane, respectively. Transfer the supernatant to a 1.5 mL centrifuge tube and store at 4 °C.

[0147] (6) Using a pipette, transfer 100 μL of logarithmic growth phase Escherichia coli culture into a 50 mL centrifuge tube. Then, add 100 μL of phage fluid from the supernatant obtained in the phage extraction step above to the centrifuge tube, followed by 30 mL of liquid culture medium. A phage-free negative control group is also included. After mixing thoroughly, incubate at 37°C on a shaker for 6–8 hours. If the liquid in the centrifuge tube remains turbid, it indicates that the water sample does not contain phage of the target strain; if the liquid is clear, it indicates that the water sample contains phage of the target strain and can be purified.

[0148] The purification method for bacteriophages includes the following steps:

[0149] (1) Take five 1.5 mL centrifuge tubes and add 900 μL of liquid culture medium to each tube using a pipette. Add 100 μL of phage to the first centrifuge tube and serially dilute to five different gradients. Take two more 1.5 mL centrifuge tubes and add 900 μL of liquid culture medium and 100 μL of bacterial solution to each tube and mix well to prepare a 10-fold dilution of bacterial solution.

[0150] (2) Take six 5.0 mL centrifuge tubes and add 200 μL of bacterial-broth diluent to each tube using a pipette, followed by 100 μL of phage-broth diluent. The sixth centrifuge tube should not contain phage-broth diluent as a negative control. Finally, add 3 mL of semi-solid culture medium to each tube and mix thoroughly. Label the tubes immediately. Take seven LB agar plates and quickly spread the plates evenly while still warm. Add only 3 mL of semi-solid culture medium to the seventh plate as a blank control. After the upper layer of semi-solid culture medium cools, invert the plates and incubate at 37°C for 2-5 hours. Observe the plaque growth of phage solutions with different dilution gradients to calculate phage titer and select an appropriate phage dilution gradient. The purification process generally requires at least four steps.

[0151] (3) Take the 5th gradient phage dilution from the last purification culture and add 3 mL of liquid culture medium to a double-layer agar plate. Gently scrape off and break up the upper semi-solid culture medium with an L spreader. Place it in a 15 mL centrifuge tube and centrifuge at 8000 r / min for 30 min. After centrifugation, filter through a 0.45 μm filter membrane and a 0.22 μm filter membrane, respectively. Take the supernatant (i.e., the phage stock solution) and place it in a 5.0 mL centrifuge tube. Seal the tube with a sealing film and store it at 4℃.

[0152] The identification method for bacteriophages includes the following steps:

[0153] (1) Phage titer determination: Take a brand new 96-well plate, open the cap, add 180 μL of liquid culture medium to each well in the first row, then add 20 μL of phage stock solution to the first well, and serially dilute 12 times. The operation is the same for the second and third rows, and three parallel groups are set up. Take 10 5.0 mL centrifuge tubes, add 200 μL of bacterial solution to each centrifuge tube, add 100 μL of phage dilutions from the 9th, 11th and 12th gradients to the centrifuge tubes, and make three parallel controls for each gradient. Finally, add 4 mL of semi-solid culture medium to each plate, mix well, and spread evenly on the plate while hot. At the same time, negative and positive controls are set up. After the semi-solid culture medium cools down, incubate upside down in a 37℃ light incubator for 2 h to 5 h, and mark it in time. Count the phage plaques after culture and calculate their titer. The calculation formula is:

[0154] Phage titer = (number of plaques × dilution factor) / inoculum volume

[0155] (2) Staining identification of phage-captured Escherichia coli: Add 10 μL of CYTO-13 working solution to a 1.5 mL centrifuge tube containing 990 μL of PBS buffer solution and mix well. Aliquot 50 μL of phage stock solution into a 1.5 mL sterile centrifuge tube, add 50 μL of the prepared CYTO-13 working solution, incubate at room temperature in the dark for 60 min, and wash once with PBS buffer solution. Add 50 μL of Escherichia coli bacterial culture to the phage solution containing CYTO-13 working solution, incubate at 37 °C for 10 min, use a sterile inoculation loop to spread the mixture onto a glass slide, and observe under a fluorescence microscope after it dries.

[0156] (3) Phage morphology observation: Take 10 μL of phage stock solution and drop it on the sealing film. Take out the copper mesh and cover it with the front side of the phage stock solution. Let it stand for 1 min. Take off the copper mesh and tilt it at 45° on the filter paper to remove excess phage solution. Then cover it with a drop of deionized water and let it stand for 10 s. After the filter paper absorbs the water, stain it three times with 0.5% phosphotungstic acid. After each staining, it must be dried with filter paper. After the last staining, it is placed on clean filter paper to dry for 10 s. Observe it with a transmission electron microscope, take pictures and record the results.

[0157] The method for preserving bacteriophages includes the following steps:

[0158] Prepare 80% glycerol with sterile distilled water. After autoclaving, pipette 300 μL into a 1.5 mL centrifuge tube, add 900 μL of phage stock solution, vortex, label, and place in an ice box to freeze slowly for 4 hours. Then store in a -80°C freezer.

[0159] Example 1

[0160] Synthesis of magnetic beads: First, 3.6 g of anhydrous sodium acetate and 1.3 g of finely ground FeCl3·6H2O were dissolved in 40 mL of ethylene glycol using an ultrasonic instrument, resulting in a yellow, viscous solution. The solution was stirred until homogeneous and heated at 250 °C for 6 hours. After cooling to room temperature, the beads were washed three times with ethanol and water. Finally, the obtained magnetic nanoparticles were vacuum dried at 50 °C for 12 hours to obtain magnetic beads (MB).

[0161] Preparation of phage-embedded magnetic bead probes:

[0162] 10 mg of MB was dissolved in 1.0 mL of water and sonicated for 10 min. Then, a 1% (w / w) polydiallyldimethylammonium chloride solution was added to the above solution, controlling the volume ratio of magnetic bead solution to polydiallyldimethylammonium chloride solution to be 1.5:8.5, and reacted at 2200 rpm for 1 hour. Afterwards, the obtained MB@PDDA was washed three times with H2O, and 10 mg of MB was dissolved in 1.0 mL of water and sonicated for 10 min. 13 PFU / mL phage was added and reacted for 1 hour. The product was separated using a magnet; due to the negative charge on the phage head, the cells were electrostatically immobilized on the surface of the MB. The entire process was repeated four times with phage addition (the volume ratio of phage to magnetic bead solution was 0.1:1), each reaction lasting 1 hour to ensure as many phages as possible were on the Fe3O4. The product was then washed three times with H2O, and a 1% bovine serum albumin solution was added, reacting at 2200 rpm for 1 hour to avoid nonspecific adsorption. Finally, the resulting (MP) probe was washed three times with H2O and stored at 4°C.

[0163] Preparation of AIE@Ag@MnO2NFs composite probe:

[0164] In a 50 mL two-necked flask, TPE-C≡Cpy (66 mg, 0.091 mmol) was added along with MeI (696 mg, 4.92 mmol) and acetonitrile (molar ratio of TPE-C≡Cpy to acetonitrile was 150:1). One neck was sealed with a rubber stopper. The mixture was refluxed at 85 °C for 12 g under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and poured into n-hexane. The brownish-yellow precipitate was sonicated, filtered, and dried. The crude product was redissolved in methanol (12 mL) and mixed with a saturated potassium hexafluorophosphate solution (12 mL). The mixture was stirred at 25 °C for 1 hour, filtered again, recrystallized, washed, and dried in a vacuum drying oven to obtain a yellow powder of TPEC≡Cpy+ in 53% yield.

[0165] Dissolve 15.8g of potassium permanganate (KMnO4) in 100g of deionized water. Add 26.1g of polyvinylpyrrolidone (PVP) under stirring and heat to 90℃. Then add 100mL of HCl (0.2mol / L) and react for 1 hour until a dark brown precipitate appears. Cool to room temperature and centrifuge at 8000rpm for 30 minutes. Remove the supernatant and wash three times with distilled water to obtain MnO2NFs. Dry and set aside.

[0166] Preparation of Ag@MnO2NFs: At 25℃, 1 mL of AgNO3 (0.02 mol / L) solution and 2.5 mL of trisodium citrate aqueous solution (0.02 mol / L) were added sequentially to 25 mL of MnO2NFs (10 mg / mL) aqueous solution under magnetic stirring (600 rpm). Then, 1 mL of sodium borohydride aqueous solution (0.1 mol / L) was added dropwise. After the addition was complete, stirring was continued for 0.5 hours. Stirring was stopped, and the mixture was centrifuged and washed three times with deionized water. The mixture was then dried for later use.

[0167] Preparation of AIE@Ag@MnO2NFs: First, 24 μL of AIE material (TPEC≡Cpy+) was added to 12 mL of Ag@MnO2NFs solution (140 μg / mL), and stirred at 25 °C for 2.5 hours. Then, 2.5 μL of Tween 80 was added and stirring was continued for 0.5 hours. Finally, the product was obtained. After washing three times, the AIE@Ag@MnO2NFs composite probe was obtained.

[0168] The schematic diagram of the preparation of the phage-embedded magnetic bead probe in this embodiment is shown below. Figure 1 As shown in the diagram, the fabrication of the AIE@Ag@MnO2NFs composite probe is illustrated below. Figure 2 As shown.

[0169] Fabrication of a single-channel microfluidic chip for detecting bacterial count and antibiotic resistance levels:

[0170] The microfluidic chip consists of a glass layer (thickness: 0.1cm, length: 6.5cm, width: 6.5cm) for sealing and a top PDMS layer (thickness: 0.4cm, length: 8cm, width: 7.5cm). The upper half of the top PDMS layer has three circular inlets (diameter: 1mm), and a chain-link micromixer with 13 identical channel spacings (width: 1mm) for mixing. The lower half consists of two rhombic chambers (100μL in volume, one for Raman signal detection and the other for hydrogen peroxide storage), a dye storage chamber (diameter: 3mm), and a distance indicator channel (width: 1mm). The design was first performed using 3ds Max software, then a mold for the PDMS layer was fabricated using an Object30Pro 3D printer. The PDMS prepolymer and curing agent were then mixed uniformly at a 10:1 mass ratio and injected into the mold. The mixture was subsequently degassed in a vacuum environment. Finally, the mold was transferred to a 65°C constant temperature oven to cure fully for 12 hours, thus obtaining a complete PDMS layer.

[0171] The application flow diagram of the microfluidic chip in this embodiment is shown below. Figure 3 As shown.

[0172] The following steps were taken to quantify the number of E. coli in situ using a nanocomposite probe and a dual-mode analysis strategy:

[0173] Bacterial culture: E. coli (CMCC 44484) was cultured in LB medium at 37°C in a shaker at 120 rpm for 6 hours. After culture, 1.0 mL of the bacterial suspension was transferred to a centrifuge tube and centrifuged at 5000 rpm for 5 minutes. The supernatant was then discarded, and the bacterial pellet was resuspended in PBS buffer. This washing step was repeated three times. Finally, the bacterial pellet was resuspended in 1 mL of PBS solution for later use. To determine the bacterial concentration, 200 μL of the bacterial suspension was used, and the absorbance was measured at 600 nm using a UV spectrophotometer. The actual concentration of the bacterial suspension was calculated using a pre-established standard curve of bacterial concentration versus absorbance. All processed bacterial suspensions were stored at 4°C.

[0174] Quantitative detection of E. coli: First, 500 μL of phage-embedded magnetic bead probe, 50 μL of LAIE@Ag@MnO2NFs composite probe, and 500 μL of different concentrations (10 2 -10 8E. coli O157:H7 (CFU / mL) was injected into a microfluidic chip and thoroughly mixed in a chain-structured micromixer to form an MB-PE.coli-AIE@Ag@MnO2NFs complex. The mixture was then magnetically separated for 2 minutes to remove background, and injected through the injection port with 500 μL of PBST (phosphate-buffered saline with the nonionic surfactant Tween-20) for further mixing and washing in the chain-structured micromixer to avoid nonspecific binding. Raman spectroscopy was then performed; subsequently, the complex was magnetically transferred to the distance sensing reaction chamber, and 20 μL of 5% hydrogen peroxide was injected. The complex catalyzed the oxygen production, propelling the red dye forward to quantify the distance signal.

[0175] The test results of different concentrations of E. coli are shown in the figure below. Figure 4 As shown, Figure 4 (A) shows the SERS spectra of different concentrations of Escherichia coli; (B) shows the correlation between Raman intensity and Escherichia coli concentration; (C) shows the propagation distance of red dye in pure culture of different concentrations of Escherichia coli; and (D) shows the logarithmic linear relationship between the propagation distance of red dye and the concentration of Escherichia coli in pure culture.

[0176] The results showed that, under optimal conditions, by selecting different concentrations of E. coli to test the biosensor's dual-signal detection capability, both the SERS and distance signals increased with increasing bacterial count, and the two signals generated on the two detection units did not affect the corresponding signals generated on other detection units. At 10 2 -10 8 Within the range of CFU / mL, the logarithmic concentration of bacteria [E. coli] showed a linear relationship with both SERS and distance signals. The linear regression equation for the Raman signal could be fitted as y = 777.35782Log[E. coli] + 1532.83221(R²). 2 =0.9901). The detection limit of E. coli is 17 CFU / mL. The linear regression equation for the distance signal is y = 31.0137Log[E. coli] ± 38.03813 (R = 0.9901). 2 =0.9971), and the detection limit is 35 CFU / mL (S / N = 3). Therefore, this detection method can be used for on-site quantification of target analytes with dual signals.

[0177] Taking E. coli as an example, drug resistance experiments were conducted. Ciprofloxacin (a quinolone antibiotic) was selected for the drug resistance experiment of E. coli. Bacterial culture, nanocomposite probe and microfluidic chip preparation were the same as above.

[0178] Three groups were set up to test the drug resistance of E. coli: group a contained only culture medium and ciprofloxacin without bacterial culture; group b contained the target resistant E. coli and ciprofloxacin to directly assess the sensitivity or resistance of the target strain to ciprofloxacin; and group c contained E. coli. The results are as follows: Figure 5 As shown.

[0179] As can be seen from the figure, E. coli is sensitive to ciprofloxacin, and the bacterial abundance is reduced.

[0180] As can be seen from the above embodiments, the nanocomposite probe system and single-channel microfluidic chip provided by the present invention can intuitively realize the visual detection of bacterial quantity and bacterial drug resistance in actual detection process, and can further utilize data analysis system and linear equations for combined calculation, which can improve detection efficiency and ensure high accuracy of detection results.

[0181] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nanocomposite probe system, characterized in that, The nanocomposite probe system includes phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes. The volume ratio of the phage-embedded magnetic bead probe and the AIE@Ag@MnO2NFs composite probe is 400~600:40~60; The preparation method of the AIE@Ag@MnO2NFs composite probe includes the following steps: (1) TPE-C≡Cpy, iodomethane and acetonitrile are mixed and reacted to obtain the iodinated product; (2) The iodide product, methanol and potassium hexafluorophosphate saturated solution were mixed to obtain AIE material; (3) Potassium permanganate solution, polyvinylpyrrolidone and hydrochloric acid solution are mixed and reacted to obtain MnO2NFs; (4) Silver nitrate solution, trisodium citrate solution, MnO2NFs solution and sodium borohydride solution are mixed and reacted to obtain Ag@MnO2NFs; (5) The AIE material, Ag@MnO2NFs solution and Tween 80 are mixed and reacted to obtain the AIE@Ag@MnO2NFs composite probe; The structure of TPE-C≡Cpy described in step (1) is shown below: ； The molar ratio of TPE-C≡Cpy to iodomethane in step (1) is 0.09~0.1:4.9~5; The molar ratio of TPE-C≡Cpy to acetonitrile is 140~160:1; The reaction in step (1) is carried out at a temperature of 80-90°C for 10-12 hours. In step (2), the volume molar ratio of methanol to TPE-C≡Cpy in step (1) is 10~15mL:0.09~0.1mmol; In step (2), the volume ratio of methanol to saturated potassium hexafluorophosphate solution is 10~15:10~15; The mixing temperature in step (2) is 20~30℃, and the time is 1~1.5h; In step (3), the mass ratio of potassium permanganate to water in the potassium permanganate solution is 15~16:90~110; In step (3), the mass ratio of polyvinylpyrrolidone to potassium permanganate is 26~26.5:15~16; In step (3), the concentration of the hydrochloric acid solution is 0.1~0.3M; In step (3), the volume-to-mass ratio of hydrochloric acid solution to potassium permanganate is 80-120 mL: 15-16 g; The reaction in step (3) is carried out at a temperature of 80~100℃ for 1~2 hours.

2. The nanocomposite probe system as described in claim 1, characterized in that, The method for preparing the phage-embedded magnetic bead probe includes the following steps: The magnetic bead solution and polydiallyldimethylammonium chloride solution were mixed and then added sequentially to the phage and bovine serum albumin solution to react, thus obtaining the phage-embedded magnetic bead probe. The mass-to-volume ratio of magnetic beads to water in the magnetic bead solution is 5~15 mg: 0.5~1.5 mL; The mass concentration of the polydiallyl dimethylammonium chloride solution is 0.5-1.5%; The volume ratio of the magnetic bead solution to the polydiallyldimethylammonium chloride solution is 1~2:8~9; The mixing speed is 2000~2500 rpm, and the time is 1~2 hours; The abundance of the bacteriophage was 10. 13 ~15 13 PFU / mL; phage addition ≥ 5 times; reaction time after each phage addition 1~2h; phage addition volume to magnetic bead solution volume ratio 0.1:1~2; The mass concentration of the bovine serum albumin solution is 0.5-1.5%; the volume ratio of bovine serum albumin solution to magnetic bead solution is 1:5-10; and the reaction time after adding bovine serum albumin solution is 1-2 hours.

3. The nanocomposite probe system as described in claim 2, characterized in that, In step (4), the concentration of the silver nitrate solution is 0.01~0.03M; The concentration of trisodium citrate solution is 0.01~0.03M; The concentration of the MnO2NFs solution is 5~15 mg / mL; the concentration of the sodium borohydride solution is 0.05~0.15 M; The volume ratio of silver nitrate solution, trisodium citrate solution, MnO2NFs solution, and sodium borohydride solution is 0.5~1.5:2~3:20~30:0.5~1.5; The reaction in step (4) is carried out at a temperature of 20-25°C for 0.5-1 hour. In step (5), the concentration of the Ag@MnO2NFs solution is 120~160ug / mL; In step (5), the volume ratio of AIE material, Ag@MnO2NFs solution and Tween 80 is 20~30uL: 10~15mL: 2~3uL; The reaction in step (5) is carried out at a temperature of 20-25°C for 0.5-1 h.

4. The application of the nanocomposite probe system according to any one of claims 1 to 3 in the preparation of bacterial quantity detection products and bacterial drug resistance level detection products.

5. A single-channel microfluidic chip for detecting bacterial count and bacterial drug resistance levels, characterized in that, The structure of the single-channel microfluidic chip is as follows: A single-channel microfluidic chip includes a sealing glass layer and a working PDMS layer; The working PDMS layer has three circular inlets at one end, and the other end of the three circular inlets is connected to the inlet of the chain-link micromixer. The outlet of the chain-link micromixer is sequentially connected to the Raman signal detection chamber, the hydrogen peroxide storage chamber, and the working chamber. The working chamber consists of a dye storage chamber and a distance indicator channel connected in sequence; The single-channel microfluidic chip comprises the nanocomposite probe system according to any one of claims 1 to 3; The nanocomposite probe system according to any one of claims 1 to 3 is injected into the working PDMS layer through a circular inlet.

6. The single-channel microfluidic chip for detecting bacterial count and antibiotic resistance levels as described in claim 5, characterized in that, The diameter of the three circular inlets is 1~2mm; The number of channel spacings in the chain-link micromixer is ≥13; the width of a single channel spacing is 1~2mm; The Raman signal detection chamber and the hydrogen peroxide storage chamber have independent volumes of 80~120μL; The diameter of the dye storage chamber is 3-4 mm, and the width of the distance indicator channel is 0.5-1.5 mm.

7. A method for using the single-channel microfluidic chip for detecting bacterial count and antibiotic resistance levels as described in claim 5 or 6 in a non-disease diagnostic and therapeutic method, characterized in that, Includes the following steps: (I) Detection of bacterial count (a) The test bacteria, phage-embedded magnetic bead probes and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets respectively. After being fully mixed by a chain segment structure micromixer, Raman signals and pressure distance signals were generated to detect the number of bacteria. (II) Detection of bacterial resistance levels (b) The test bacteria, phage-embedded magnetic bead probes, and AIE@Ag@MnO2NFs composite probes were injected into three circular inlets, respectively. Antibiotics were injected into the circular inlet where the test bacteria were injected. After thorough mixing by a chain segment micromixer, Raman signals and pressure distance signals were generated to detect the bacterial resistance level.

8. The method of applying the single-channel microfluidic chip for detecting bacterial count and bacterial resistance levels as described in claim 7 to a non-disease diagnostic and therapeutic method, characterized in that, In step (a), the volume ratio of the injected bacteria to be tested and the magnetic bead probe embedded with bacteriophage is 400~600:400~600; In step (b), the volume ratio of the injected bacteria to be tested and the magnetic bead probes embedded with bacteriophages is 400~600:400~600.

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