Nanometer composite probe system and application thereof, and single-channel micro-fluidic chip for detecting bacterial quantity and bacterial drug resistance level

Abstract

The invention belongs to the technical field of bacterial detection, and provides a nano-composite probe system, application thereof and a single-channel micro-fluidic chip for detecting the number of bacteria and the drug resistance level of the bacteria, and the nano-composite probe system comprises a phage embedded magnetic bead probe and an AIE (at) Ag (at) MnO2NFs composite probe. The Ag-MnO2 nanoflower surface modified AIE (Raman reporter molecule) composite probe can realize a strong Raman signal, Ag-coated MnO2 catalyzes decomposition of hydrogen peroxide to push a red dye to move so as to realize a distance signal, and magnetic bead-bacteriophage is used as a recognition element of target bacteria and realizes magnetic separation and enrichment at the same time. Based on high specificity and good environmental tolerance of the phage, the Ag-MnO2 nanoflowers enhance Raman signals of AIE Raman reporter molecules, dual-mode signal internal correction realizes the stability of the sensor and the interference resistance to factors such as environment and experiments, and the specificity of a detection strategy and the accuracy of on-site analysis are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bacterial detection, and particularly relates to a nano-composite probe system and application thereof, and a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level. BACKGROUND

[0002] Escherichia coli brings serious harm to human society, and its potential risk of being contaminated in water and food sources makes it a major threat to food safety and public health, and its drug resistance and strong pathogenicity also cause serious harm to life and health. Therefore, it is particularly important to carry out rapid and accurate detection of Escherichia coli and comprehensive detection of drug resistance level. SUMMARY

[0003] The present application aims to overcome the defects of the prior art, 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.

[0004] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0005] The present application provides a nano-composite probe system, which comprises a phage-embedded magnetic bead probe and an AIE@Ag@MnO2 NFs composite probe.

[0006] The volume ratio of the phage-embedded magnetic bead probe and the AIE@Ag@MnO2 NFs composite probe is 400-600:40-60.

[0007] Preferably, the preparation method of the phage-embedded magnetic bead probe comprises the following steps:

[0008] The phage-embedded magnetic bead probe is obtained by sequentially adding phage and bovine serum albumin solution to the mixed magnetic bead solution and polydiallyldimethylammonium chloride solution for reaction.

[0009] The mass-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 polydiallyldimethylammonium 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 rotation speed of the mixing is 2000-2500 rpm, and the time is 1-2 h.

[0013] The abundance of the phage is 10 13 -15 13PFU / mL; the number of phage addition≥5 times; the reaction time after single phage addition is 1-2 h; the volume ratio of single phage addition amount to magnetic bead solution is 0.1:1-2;

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

[0015] As preferred, the preparation method of the AIE@Ag@MnO2 NFs composite probe comprises the following steps:

[0016] (1) mixing TPE-C≡Cpy, methyl iodide and acetonitrile to react to obtain an iodination product;

[0017] (2) mixing the iodination product, methanol and a saturated potassium hexafluorophosphate solution to obtain an AIE material;

[0018] (3) mixing a potassium permanganate solution, polyvinylpyrrolidone and a hydrochloric acid solution to react to obtain MnO2 NFs;

[0019] (4) mixing a silver nitrate solution, a trisodium citrate solution, a MnO2 NFs solution and a sodium borohydride solution to react to obtain Ag@MnO2 NFs;

[0020] (5) mixing the AIE material, the Ag@MnO2 NFs solution and Tween 80 to react to obtain the AIE@Ag@MnO2 NFs composite probe.

[0021] As preferred, the structure of the TPE-C≡Cpy in step (1) is as shown below:

[0022]

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

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

[0025] The temperature of the reaction in step (1) is 80-90℃, and the time is 10-12 h;

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

[0027] The volume ratio of methanol to the saturated potassium hexafluorophosphate solution in step (2) is 10-15:10-15;

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

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

[0030] The mass ratio of polyvinylpyrrolidone and potassium permanganate in step (3) is 26-26.5:15-16;

[0031] The concentration of the hydrochloric acid solution in step (3) is 0.1-0.3 M;

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

[0033] The temperature of the reaction in step (3) is 80-100 DEG C, and the time is 1-2 h.

[0034] Preferably, the concentration of the silver nitrate solution in step (4) 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 MnO2 NFs solution is 5-15 mg / mL; and the concentration of the sodium borohydride solution is 0.05-0.15 M;

[0036] The volume ratio of the silver nitrate solution, the trisodium citrate solution, the MnO2 NFs solution and the sodium borohydride solution is 0.5-1.5:2-3:20-30:0.5-1.5;

[0037] The temperature of the reaction in step (4) is 20-25 DEG C, and the time is 0.5-1 h;

[0038] The concentration of the Ag@MnO2 NFs solution in step (5) is 120-160 ug / mL;

[0039] The volume ratio of the AIE material, the Ag@MnO2 NFs solution and the Tween 80 in step (5) is 20-30 uL:10-15 mL:2-3 uL;

[0040] The temperature of the reaction in step (5) is 20-25 DEG C, and the time is 0.5-1 h.

[0041] The application also provides application of the nano-composite probe system in detection of bacterial quantity and detection of bacterial drug resistance level.

[0042] The application also provides a single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level, and the structure of the single-channel microfluidic chip is as follows:

[0043] The single-channel microfluidic chip comprises a sealed glass layer and a working PDMS layer;

[0044] One end of the working PDMS layer is provided with three circular inlets, and the other end of the three circular inlets is connected to the inlets of the chain structure micro-mixer;

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

[0046] The nano-composite probe system is injected into the working PDMS layer through the circular inlets.

[0047] Preferably, the diameters of the three circular inlets are 1-2 mm.

[0048] The number of channel intervals of the chain structure micro-mixer is greater than or equal to 13, and the width of a single channel interval is 1-2 mm.

[0049] The volumes of the Raman signal detection chamber and the hydrogen peroxide storage chamber are independently 80-120 muL.

[0050] The working chamber is sequentially connected to a dye storage chamber and a distance indicating channel.

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

[0052] The application also provides an application method of the single-channel microfluidic chip for detecting the number and drug resistance level of bacteria, comprising the following steps:

[0053] (I) Detection of the number of bacteria

[0054] (a) Inject the bacteria to be detected, phage-embedded magnetic bead probes and AIE@Ag@MnO2 NFs composite probes into the three circular inlets respectively, and then mix them sufficiently through the chain structure micro-mixer to generate Raman signals and pressure distance signals, thereby detecting the number of bacteria.

[0055] (II) Detection of the drug resistance level of bacteria

[0056] (b) Inject the bacteria to be detected, phage-embedded magnetic bead probes and AIE@Ag@MnO2 NFs composite probes into the three circular inlets respectively, and then continue to inject antibiotics into the circular inlet of the bacteria to be detected, and then mix them sufficiently through the chain structure micro-mixer to generate Raman signals and pressure distance signals, thereby detecting the drug resistance level of bacteria.

[0057] Preferably, in step (a), the volume ratio of the injection amount of the bacteria to be detected to the phage-embedded magnetic bead probes is 400-600:400-600.

[0058] The volume ratio of the injection amount of the bacteria to be detected and the phage-embedded magnetic bead probe in step (b) is 400-600: 400-600.

[0059] The application provides a nanocomposite probe system, which comprises a phage-embedded magnetic bead probe and an AIE@Ag@MnO2 NFs composite probe. 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 realize distance signals, and the magnetic bead (MB)-phage realizes magnetic separation and enrichment as a recognition element of target bacteria. Based on the high specificity and good environmental tolerance of the phage, the Raman signal of the Ag-MnO2 nanoflower enhanced AIE Raman reporter molecule, the double-mode signal internal correction to realize the stability of the sensor and the anti-interference of environmental and experimental factors, the specificity of the detection strategy is improved and the accuracy of on-site analysis is improved.

[0060] The application constructs a nanocomposite probe system comprising TPEC≡Cpy+(AIE) and spherical nanoflower Ag@MnO2, and proposes a new SERS nanoprobe for detecting bacteria. The MnO2 nanoflower is widely used in the field of biosensors due to its high specific surface area, large loading rate, good biocompatibility and easy surface modification, so that material modification and loading can be realized on the surface thereof, multifunctionalization is realized, a new biosensor is constructed based on the Ag-MnO2 NFs surface modified AIE Raman reporter molecule composite probe, and the number of bacteria can be rapidly and sensitively detected through double-mode signals (SERS signals and pressure distance signals).

[0061] The application is based on Ag-MnO2NFs surface modification AIE Raman reporter molecule composite probe to construct a new type of biosensor, which can quickly and sensitively detect bacteria through double mode signals (SERS signal and pressure distance signal). First, a quaternary ammonium salt TPEC≡Cpy+(AIE) molecule with Raman characteristic signal and capable of binding with bacteria is synthesized, and manganese dioxide nanoflower (MnO2NFs) is prepared by reducing potassium permanganate (KMnO4), and then Ag nanoparticles (AgNPs) and TPEC≡Cpy+(AIE) molecules are embedded in situ on the surface of the nanoflower. The cluster-shaped manganese dioxide nanoflower ensures the stability of the AgNPs. The prepared nanocomposite is composed of manganese dioxide nanoflower and Ag nanoparticles and Raman reporter molecules (AIE) embedded in the nanoflower, and the signal is greatly increased at the SERS active site. Therefore, the functionalized AIE@Ag@MnO2NFs is used as a SERS nanoprobe for detecting bacteria, and in addition, the functionalized AIE@Ag@MnO2NFs and the phage-modified magnetic beads form a sandwich complex in the mixing channel, which is enriched and separated by a magnet, and in the reaction chamber, not only can a significant Raman signal be generated; after the addition of hydrogen peroxide, the decomposition of hydrogen peroxide catalyzed by MnO2 drives the movement of the red dye to realize the distance signal. Since the Raman signal and the gas are generated in two different reaction chambers, the signals generated by the two sensing regions will not affect each other. Therefore, the double-mode biosensing strategy can realize target detection, and provides a new probe design idea for the development of double-mode target biosensing detection.

[0062] The application has superior analysis performance. Under the optimal conditions, different concentrations of E. coli are selected to detect the double signal detection ability of the biosensor for the target. The results show that as the number of bacteria increases, the generated SERS signal and distance signal are rising, and the two signals generated on the two detection units will not affect the corresponding signals generated on other detection units. In the range of 10 2 -10 8 CFU / mL, the logarithmic concentration of the bacteria ([E.coli]) has a linear relationship with the SERS signal and the distance signal. The linear regression equation of the Raman signal can be fitted as y=777.35782Log[E.coli]+1532.83221(R 2 =0.9901). The detection limit of E. coli is 17 CFU / mL. The linear regression equation of the distance signal is y=31.0137Log[E.coli]+-38.03813(R 2 =0.9971), and the detection limit is 35 CFU / mL (S / N=3). Therefore, the detection method can be used for on-site quantification of the target with double signals.

[0063] The operation process of the application is extremely simple, the cost investment is low, the detection time is greatly shortened, the detection efficiency is significantly improved, and the whole detection process is easy to realize without relying on large and expensive experimental equipment. In the actual detection process, the visualization detection of the number of bacteria and the drug resistance of bacteria can be realized intuitively, and the data analysis system and the linear equation can be further combined for calculation, so that the detection efficiency is improved and the detection result is high in accuracy. BRIEF DESCRIPTION OF DRAWINGS

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

[0065] Figure 2 It is a schematic diagram for preparation of the AIE@Ag@MnO2 NFs composite probe in Example 1;

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

[0067] Figure 4 It is a test result graph of different concentrations of E. coli in Example 1;

[0068] Figure 5 It is a graph of signal response value of E. coli drug resistance distance under the use of different concentrations of antibiotics in Example 1. DETAILED DESCRIPTION

[0069] The application provides a kind of nanometer composite probe system, the nanometer composite probe system includes phage-embedded magnetic bead probe and AIE@Ag@MnO2 NFs composite probe.

[0070] In the application, the volume ratio of the phage-embedded magnetic bead probe and the AIE@Ag@MnO2 NFs composite probe is preferably 400-600:40-60, further preferably 450-550:45-55, and more preferably 460-540:46-54.

[0071] The application provides a preparation method of magnetic beads, comprising the following steps:

[0072] The anhydrous sodium acetate, ferric chloride hexahydrate and ethylene glycol are mixed and then subjected to hydrothermal synthesis to obtain the magnetic beads.

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

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

[0075] In the present application, after the hydrothermal synthesis, the magnetic nanoparticles are naturally cooled to room temperature, washed with ethanol for 3 times, and washed with water for 3 times; and then the obtained magnetic nanoparticles are vacuum dried, the temperature of the vacuum drying is preferably 40-60°C, further preferably 45-55°C, and more preferably 48-52°C; and the time is preferably 10-15h, further preferably 11-14h, and more preferably 12-13h; and the magnetic beads are obtained after the drying.

[0076] In the present application, the preparation method of the phage-embedded magnetic bead probe comprises the following steps:

[0077] The phage-embedded magnetic bead probe is obtained by sequentially adding phage and bovine serum albumin solution to the mixed magnetic bead solution and polydiallyldimethylammonium chloride solution for reaction.

[0078] In the present application, the bovine serum albumin is purchased from sigma aldrich company.

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

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

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

[0082] In the present application, the rotation speed of the mixing is preferably 2000-2500rpm, further preferably 2100-2400rpm, and more preferably 2200-2300rpm; and the time is preferably 1-2h, further preferably 1.2-1.8h, and more preferably 1.4-1.6h.

[0083] In the present application, after mixing, the MB@PDDA obtained by washing is washed for preferably ≥3 times, further preferably ≥4 times, and more preferably ≥5 times.

[0084] In the present application, the bacteriophage is added while keeping the rotation speed unchanged, and 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 bacteriophage is added for preferably ≥5 times, further preferably ≥6 times, and more preferably ≥7 times; the reaction time after single addition of the bacteriophage is preferably 1-2 h, further preferably 1.2-1.8 h, and more preferably 1.4-1.6 h; and the ratio of the amount of single addition of the bacteriophage to the volume of the magnetic bead solution is preferably 0.1:1-2, further preferably 0.1:1.2-1.8, and more preferably 0.1:1.4-1.6; due to the negative charge of the head of the bacteriophage, the bacteria will be directionally fixed on the surface of the MB by electrostatic adsorption.

[0085] In the present application, the mass concentration of the bovine serum protein solution is preferably 0.5-1.5%, further preferably 0.6-1.4%, and more preferably 0.8-1.2%; the volume ratio of the bovine serum protein solution to the magnetic bead solution is preferably 1:5-10, further preferably 1:6-9, and more preferably 1:7-8; the reaction time after addition of the bovine serum protein solution is preferably 1-2 h, further preferably 1.2-1.8 h, and more preferably 1.4-1.6 h. After the reaction, washing is performed, and the washing is performed for preferably ≥3 times, further preferably ≥4 times, and more preferably ≥5 times to obtain the bacteriophage-embedded magnetic bead probe.

[0086] In the present application, the preparation method of the AIE@Ag@MnO2 NFs composite probe comprises the following steps:

[0087] (1) mixing TPE-C≡Cpy, methyl iodide and acetonitrile to obtain an iodination product;

[0088] (2) mixing the iodination product, methanol and a saturated potassium hexafluorophosphate solution to obtain an AIE material;

[0089] (3) mixing a potassium permanganate solution, polyvinylpyrrolidone and a hydrochloric acid solution to obtain MnO2 NFs;

[0090] (4) mixing a silver nitrate solution, a trisodium citrate solution, a MnO2 NFs solution and a sodium borohydride solution to obtain Ag@MnO2 NFs;

[0091] (5) mixing the AIE material, the Ag@MnO2 NFs solution and Tween 80, and then reacting to obtain the AIE@Ag@MnO2 NFs composite probe.

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

[0093]

[0094] In the present application, the TPE-C≡Cpy is purchased from Shanghai Aladdin Biochem Technology Co., Ltd.

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

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

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

[0098] In the present application, after the reaction is completed, the mixture obtained is cooled to room temperature, mixed with n-hexane, and then subjected to suction filtration and drying to obtain the iodinated product.

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

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

[0101] In the present application, the temperature of the mixing in step (2) is preferably 20-30℃, further preferably 22-28℃, more preferably 24-26℃; the time is preferably 1-1.5h, further preferably 1.1-1.4h, more preferably 1.2-1.3h; the mixing is kept under stirring; after the mixing, filtration, recrystallization, washing and drying are sequentially performed, thereby obtaining the AIE material.

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

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

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

[0105] In the present application, the concentration of the hydrochloric acid solution in step (3) is preferably 0.1-0.3M, further preferably 0.15-0.25M, more preferably 0.18-0.22M.

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

[0107] In the present application, the temperature of the reaction in step (3) is preferably 80-100℃, further preferably 85-95℃, more preferably 88-92℃; the time is preferably 1-2h, further preferably 1.2-1.8h, more preferably 1.4-1.6h.

[0108] In the present application, after the reaction in step (3) is completed, natural cooling to room temperature is performed, and then centrifugation, washing and drying are sequentially performed; the rotation speed of the centrifugation is preferably 6000-10000rpm, further preferably 7000-9000rpm, more preferably 7500-8500rpm; the time is preferably ≥30min, further preferably ≥40min, more preferably ≥50min.

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

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

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

[0112] In the present application, the silver nitrate solution and the trisodium citrate solution are mixed while stirring, then the MnO2NFs solution is added, and the sodium borohydride solution is added dropwise into the system for reaction, and the stirring speed is preferably 400-800 rpm, further preferably 450-750 rpm, and more preferably 500-700 rpm.

[0113] In the present application, the temperature of the reaction in step (4) is preferably 20-25℃, further preferably 21-24℃, and more preferably 22-23℃; the time is preferably 0.5-1 h, further preferably 0.6-0.9 h, and more preferably 0.7-0.8 h; and after the reaction is completed, the stirring is stopped, and centrifugation, washing, and drying are sequentially performed to obtain Ag@MnO2NFs.

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

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

[0116] In the present application, the AIE material and Ag@MnO2 NFs solution are mixed first, the temperature of the mixing is preferably 20-25℃, further preferably 21-24℃, more preferably 22-23℃; the time is preferably 2-3h, further preferably 2.2-2.8h, more preferably 2.4-2.6h. After the mixing is completed, Tween 80 is added for reaction.

[0117] In the present application, the temperature of the reaction in step (5) is preferably 20-25℃, further preferably 21-24℃, more preferably 22-23℃; the time is preferably 0.5-1h, further preferably 0.6-0.9h, more preferably 0.7-0.8h. After the reaction is completed, the AIE@Ag@MnO2 NFs composite probe is obtained after washing.

[0118] The present application also provides the application of the nano-composite probe system in the detection of the number of bacteria and the detection of the drug resistance level of bacteria.

[0119] The present application also provides a single-channel microfluidic chip for detecting the number of bacteria and the drug resistance level of bacteria, and the structure of the single-channel microfluidic chip is as follows:

[0120] The single-channel microfluidic chip comprises a sealed glass layer and a working PDMS layer;

[0121] One end of the working PDMS layer is provided with three circular inlets, and the other end of the three circular inlets is connected to the inlet of the chain-link structure micro-mixer;

[0122] The outlet of the chain-link structure micro-mixer is sequentially connected to a Raman signal detection chamber, a hydrogen peroxide storage chamber and a working chamber;

[0123] The nano-composite probe system is injected into the working PDMS layer through the circular inlets.

[0124] In the present application, the diameter of the three circular inlets is preferably 1-2mm, further preferably 1.2-1.8mm, more preferably 1.4-1.6mm.

[0125] In the present application, the number of channel intervals of the chain-link structure micro-mixer is preferably ≥13, further preferably ≥15, more preferably ≥18; and the width of a single channel interval is preferably 1-2mm, further preferably 1.2-1.8mm, more preferably 1.4-1.6mm.

[0126] In the present application, the volume of the Raman signal detection chamber and the hydrogen peroxide storage chamber is independently preferably 80-120μL, further independently preferably 90-110μL, more independently preferably 95-105μL.

[0127] In the present application, the working chamber is a dye storage chamber and a distance indicating channel connected in sequence.

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

[0129] In the present application, the preparation method of the working PDMS layer is as follows:

[0130] The 3dMax software is used for design, and then the Object30Pro 3D printer is used to manufacture the mold of the PDMS layer, and then the PDMS prepolymer is mixed uniformly with the curing agent and injected into the mold, and then the mixture is placed in a vacuum environment for degassing treatment. The mold is cured, and finally the complete PDMS layer is obtained.

[0131] In the present application, the mass ratio of the PDMS prepolymer and the curing agent is preferably 10-15:1, further preferably 11-14:1, and more preferably 12-13:1; the curing temperature is preferably 60-70℃, further preferably 62-68℃, and more preferably 64-66℃; and the time is preferably ≥12h, further preferably ≥14h, and more preferably ≥16h.

[0132] The present application also provides an application method of the single-channel microfluidic chip for detecting the number of bacteria and the drug resistance level of bacteria, comprising the following steps:

[0133] (I) Detection of the number of bacteria

[0134] (a) Inject the bacteria to be tested, phage-embedded magnetic bead probes, and AIE@Ag@MnO2NFs composite probes into the three circular inlets respectively, mix them thoroughly through the chain structure micro-mixer, generate Raman signals and pressure distance signals, and detect the number of bacteria;

[0135] (II) Detection of the drug resistance level of bacteria

[0136] (b) Inject the bacteria to be tested, phage-embedded magnetic bead probes, and AIE@Ag@MnO2NFs composite probes into the three circular inlets respectively, continue to inject antibiotics into the circular inlet of the bacteria to be tested, mix them thoroughly through the chain structure micro-mixer, generate Raman signals and pressure distance signals, and detect the drug resistance level of bacteria.

[0137] In the present application, the volume ratio of the injection amount of the bacteria to be tested and the phage-embedded magnetic bead probe in step (a) is preferably 400-600:400-600, further preferably 450-550:450-550, and more preferably 460-540:460-540.

[0138] In the present application, the volume ratio of the injection amount of the bacteria to be tested and the phage-embedded magnetic bead probe in step (b) is preferably 400-600:400-600, further preferably 450-550:450-550, and more preferably 460-540:460-540.

[0139] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0140] The present application provides a method for screening, purifying, identifying and preserving phages, which is as follows:

[0141] The method for screening phages comprises the following steps:

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

[0143] (2) 30 mL of the water sample is placed in a 50 mL centrifuge tube and centrifuged at 8000 r / min for 30 min. After centrifugation, the supernatant is filtered with 0.45 μm and 0.22 μm membranes in sequence, and then transferred to a 15 mL centrifuge tube and stored at 4℃.

[0144] (3) The logarithmic growth phase E. coli liquid, the water sample and the LB liquid medium are mixed in a ratio of 1:2:4, and then placed in a 37℃ constant temperature shaking incubator for culture for 18 h. The mixed liquid after culture is centrifuged at 8000 r / min for 30 min. After centrifugation, the supernatant is filtered with 0.45 μm and 0.22 μm membranes in sequence, and then transferred to a 1.5 mL centrifuge tube and stored at 4℃.

[0145] (4) The LB solid medium and semi-solid medium are prepared. The first layer of LB solid medium is poured and reserved. 200 μL of the logarithmic growth phase E. coli liquid is taken with a pipette and transferred to a 5 mL centrifuge tube, 100 μL of the primary enriched phage liquid is added, and finally 3 mL of semi-solid medium is added and mixed uniformly, and then poured and spread on the first layer of LB solid medium while hot, and then placed in a 37℃ constant temperature incubator after the upper semi-solid medium is cooled and inverted for culture for 16-24 h.

[0146] (5) Double layer agar plate appears plaque, indicating the presence of E. coli phage. Add 3 mL liquid medium to the plate, gently scrape and stir the upper semi-solid medium with an L-shaped coating rod, and place it in a 50 mL centrifuge tube and centrifuge at 8000 r / min for 30 min. After centrifugation, filter with 0.45 μm filter and 0.22 μm filter, respectively, and take the supernatant to a 1.5 mL centrifuge tube and store at 4°C.

[0147] (6) Take 100 μL of logarithmic growth phase E. coli liquid with a pipette and place it in a 50 mL centrifuge tube, then take 100 μL of the supernatant from the above phage extraction step and add it to the centrifuge tube, and add 30 mL of liquid medium. Set up a negative control group without phage, mix well, and incubate in a 37°C constant temperature shaking incubator for 6-8 h. If the liquid in the centrifuge tube is still turbid, it indicates that there is no phage of the target strain in the water sample; if the liquid in the centrifuge tube is clear, it indicates that the water sample contains phage of the target strain, and purification can be performed.

[0148] The purification method of the phage comprises the following steps:

[0149] (1) Take 5 1.5 mL centrifuge tubes, add 900 μL of liquid medium to each with a pipette, and add 100 μL of phage to the first centrifuge tube to dilute it in 5 gradients. Take another 2 1.5 mL centrifuge tubes, add 900 μL of liquid medium and 100 μL of bacterial liquid to each and mix well to make a 10-fold dilution of the bacterial liquid.

[0150] (2) Take 6 5.0 mL centrifuge tubes, add 200 μL of bacterial-broth dilution to each with a pipette, then add 100 μL of phage-broth dilution, and the 6th centrifuge tube does not add phage-broth dilution as a negative control. Finally, add 3 mL of semi-solid medium to each centrifuge tube and mix well, and label in time. Take 7 LB plates and quickly pour them onto the plates while they are hot, and the 7th plate only adds 3 mL of semi-solid medium as a blank control. After the upper semi-solid medium cools down, invert it and incubate it in a 37°C constant temperature incubator for 2-5 h, and observe the growth of phage plaques in different gradients of phage liquid to calculate the phage titer and select the appropriate phage dilution gradient. The purification process is generally at least 4 times.

[0151] (3) Take the double layer agar plate prepared from the 5th gradient phage dilution liquid of the last purification culture, add 3 mL of liquid medium, gently scrape and stir the upper semi-solid medium with an L-shaped coating rod, and place it in a 15 mL centrifuge tube and centrifuge at 8000 r / min for 30 min. After centrifugation, filter with 0.45 μm filter and 0.22 μm filter, respectively, and take the supernatant (i.e. phage stock solution) to a 5.0 mL centrifuge tube, seal with a sealing film, and store at 4°C.

[0152] The method for identifying the bacteriophage comprises the following steps:

[0153] (1) Bacteriophage titer determination: Take a brand new 96-well plate, open the cover, and add 180 μL of liquid medium to each well in the first row. Then add 20 μL of bacteriophage stock solution to the first well and dilute it by 12 gradients. The second and third rows are operated in the same way, and three parallel groups are set. Take 10 5.0 mL centrifuge tubes, and add 200 μL of bacterial solution to each centrifuge tube. Take 100 μL of bacteriophage dilution solution of the 9th, 11th and 12th gradients and add them to the centrifuge tubes, respectively. Each gradient is made in triplicate. Finally, add 4 mL of semi-solid medium to each tube, respectively, mix well, and then pour them on the plate while the negative and positive controls are being prepared. After the semi-solid medium cools down, invert the plate and incubate it in a 37°C light incubator for 2-5 hours. Label the plate in time. Count the number of plaques on the plate and calculate the titer according to the following formula:

[0154] Bacteriophage titer = (number of plaques x dilution factor) / inoculum volume

[0155] (2) Staining to identify bacteriophage-captured E. coli: Take 10 μL of CYTO-13 working solution and add it to a 1.5 mL centrifuge tube containing 990 μL of PBS buffer solution, and mix well. Dispense 50 μL of bacteriophage stock solution into a 1.5 mL sterile centrifuge tube, add 50 μL of prepared CYTO-13 working solution, and let it stand at room temperature for 60 minutes. Wash it once with PBS buffer solution. Add 50 μL of E. coli bacterial solution to the bacteriophage solution containing CYTO-13 working solution, and incubate it at 37°C for 10 minutes. Use a sterile inoculation loop to spread the mixture on a glass slide. After drying, observe it under a fluorescence microscope.

[0156] (3) Morphological observation of bacteriophage: Take 10 μL of bacteriophage stock solution and drop it on a sealing film. Take a copper mesh and place it face up on the bacteriophage stock solution. Let it stand for 1 minute, then remove the copper mesh and tilt it at 45° on filter paper to remove excess bacteriophage solution. Add a drop of deionized water on top of the copper mesh and let it stand for 10 seconds. After the filter paper absorbs the water, stain it with 0.5% phosphotungstic acid three times, and dry it with filter paper after each staining. After the last staining for 10 seconds, place it on a clean filter paper to dry. Observe it under a transmission electron microscope, take a photo, and record it.

[0157] The method for preserving the bacteriophage comprises the following steps:

[0158] Prepare 80% glycerol with sterile distilled water, autoclave it, and then use a pipette to take 300 μL and add it to a 1.5 mL centrifuge tube. Add 900 μL of bacteriophage stock solution, vortex it, label it, and slowly freeze it in an ice box for 4 hours. Then store it in a -80°C refrigerator.

[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] Take 15.8 g of potassium permanganate KMnO4 dissolved in 100 g of deionized water, add 26.1 g of polyvinylpyrrolidone (PVP) under stirring, heat to 90℃, then add 100 mL of HCl (0.2 mol / L) and react for 1 hour to form a dark brown precipitate, cool to room temperature, centrifuge at 8000 rpm for 30 minutes, remove the supernatant, and finally wash with distilled water three times to obtain MnO2NFs, which are dried for use;

[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 into 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, and stirring was continued for 0.5 hours after the addition was completed. Stop stirring, centrifuge and wash with deionized water three times. Dry for use;

[0167] Preparation of AIE@Ag@MnO2NFs: First, 24 uL of AIE material (TPEC≡Cpy+) was added to 12 mL of Ag@MnO2NFs solution (140 ug / mL), and stirred at 25℃ for 2.5 hours, then 2.5 uL of Tween 80 was added and stirred for 0.5 hours, finally the product was obtained, and after washing three times, AIE@Ag@MnO2NFs composite probe was obtained.

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

[0169] Preparation of single-channel microfluidic chip for detecting bacterial quantity and bacterial drug resistance level:

[0170] The microfluidic chip consists of a glass layer (thickness: 0.1 cm, length: 6.5 cm, width: 6.5 cm) for sealing, a top PDMS layer (thickness: 0.4 cm, length: 8 cm, width: 7.5 cm). The top half of the PDMS layer is provided with three circular inlets (diameter: 1 mm), and the chain structure micro-mixer has the same 13 channel intervals (width: 1 mm) for mixing; the lower half is composed of two rhombic chambers (volume of 100 μL, one is a Raman signal detection chamber, and the other is a hydrogen peroxide storage chamber), a dye storage chamber (diameter: 3 mm) for dye storage, and a distance indicating channel (width: 1 mm). First, use 3dMax software to design, then use Object30Pro 3D printer to manufacture the mold of the PDMS layer, then mix the PDMS prepolymer with the curing agent in a mass ratio of 10:1, pour the mixture into the mold, and then place the mixture in a vacuum environment for degassing treatment. Finally, transfer the mold to a 65°C constant temperature box, and allow it to fully cure for 12 hours to obtain the complete PDMS layer.

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

[0172] The specific steps for using the nano-composite probe and the dual-mode analysis strategy to quantitatively determine the number of E. coli in situ are as follows:

[0173] Culturing bacteria: E. coli (CMCC 44484) was cultured in LB medium and cultured in a 37°C constant temperature shaker at a speed of 120 r / min for 6 hours. After the culture was completed, 1.0 mL of bacterial solution was transferred to a centrifuge tube and centrifuged at a speed of 5000 r / min for 5 minutes. Then, after the supernatant was discarded, the bacterial pellet was resuspended with PBS buffer, and this washing step was repeated 3 times. Finally, the bacterial pellet was resuspended in 1 mL of PBS solution for use. To determine the concentration of the bacterial solution, 200 μL of the bacterial solution was measured for absorbance at 600 nm using a UV spectrophotometer. The actual concentration of the bacterial solution was calculated by a previously established standard curve of bacterial solution concentration and absorbance. The treated bacterial solution was stored in a refrigerator at 4°C.

[0174] E. coli quantitative detection: First, 500 μL of phage-embedded magnetic bead probes, 50 μL of AIE@Ag@MnO2 NFs composite probes, and 500 μL of different concentrations (10 2 -10 8 ​E. coli O157:H7 (CFU / mL) were injected into the microfluidic chip and mixed in the chain structure micro-mixer to form the MB-P-E. coli-AIE@Ag@MnO2NFs complex. Then, the mixture was magnetically separated for 2 min to remove the background, and 500 μL of PBST (phosphate buffer solution (PBS) with the addition of non-ionic surfactant Tween-20) was injected from the sample inlet, mixed and washed in the chain structure micro-mixer to further avoid non-specific binding. Then, Raman spectrum detection was performed; subsequently, the complex was moved to the distance sensing reaction chamber by magnetic attraction, 20 μL of hydrogen peroxide (5%) was injected, and oxygen was generated after catalysis by the complex to push the red dye to move forward to realize distance signal quantification.

[0175] The test results of different concentrations of E. coli are shown in FIG. 4. Figure 4 Figure 4 (A) is the SERS spectrum of different concentrations of E. coli, (B) is the correlation graph of Raman intensity and E. coli concentration, (C) is the propagation distance graph of the red dye in pure culture of different concentrations of E. coli, and (D) is the logarithmic linear relationship graph of the propagation distance of the red dye and the concentration of E. coli in pure culture.

[0176] The results show that under the optimal conditions, different concentrations of E. coli are selected to detect the dual signal detection ability of the biosensor for the target. With the increase of the number of bacteria, the generated SERS signal and distance signal are rising, and the two signals generated on the two detection units do not affect the corresponding signals generated on other detection units. In the range of 10 2 -10 8 CFU / mL, the logarithmic concentration of the bacteria ([E. coli]) has a linear relationship with the SERS signal and the distance signal. The linear regression equation of the Raman signal can be fitted as y = 777.35782Log[E. coli] + 1532.83221 (R 2 = 0.9901). The detection limit of E. coli is 17 CFU / mL. The linear regression equation of the distance signal is y = 31.0137Log[E. coli] + -38.03813 (R 2 = 0.9971), and the detection limit is 35 CFU / mL (S / N = 3). Therefore, the detection method can be used for on-site quantification of the target with dual signals.

[0177] E. coli was used as an example to perform drug resistance experiment, and ciprofloxacin (quinolone antibiotic) was selected to perform drug resistance experiment of E. coli. The 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: a is not added bacteria liquid, only containing medium and ciprofloxacin, b is added to be tested drug resistance E. coli and ciprofloxacin, directly assess the sensitivity or drug resistance of target strain to ciprofloxacin, c is added to E. coli, and the results are shown in Figure 5

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

[0180] From the above examples, it can be seen that the nano-composite probe system and single-channel microfluidic chip provided by the present application can directly realize the visual detection of the number of bacteria and the drug resistance of bacteria in the actual detection process, and can further utilize the data analysis system and linear equation for combined calculation, so as to improve the detection efficiency and ensure the high accuracy of the detection result.

[0181] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled persons in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A nanocomposite probe system, characterized in that, The nanocomposite probe system comprises a phage-embedded magnetic bead probe and an AIE@Ag@MnO2 NFs composite probe. The volume ratio of the phage-embedded magnetic bead probe and the AIE@Ag@MnO2 NFs composite probe is 400-600:40-60.

2. The nanocomposite probe system of claim 1, wherein, The preparation method of the phage-embedded magnetic bead probe comprises the following steps: After mixing the magnetic bead solution and the polydiallyldimethylammonium chloride solution, the phage and the bovine serum albumin solution are sequentially added for reaction, so that the phage-embedded magnetic bead probe is obtained; The mass-volume ratio of the magnetic beads and water in the magnetic bead solution is 5-15 mg:0.5-1.5 mL; The mass concentration of the polydiallyldimethylammonium chloride solution is 0.5-1.5%; The volume ratio of the magnetic bead solution and the polydiallyldimethylammonium chloride solution is 1-2:8-9; The rotation speed of the mixing is 2000-2500 rpm, and the time is 1-2 h; The abundance of the phage is 10 13 ~ 15 13 PFU / mL; the number of phage addition is ≥5 times; the reaction time after single phage addition is 1~2h; the ratio of the amount of single phage addition to the volume of magnetic bead solution is 0.1:1~2; The mass concentration of the bovine serum albumin solution is 0.5-1.5%; the volume ratio of the bovine serum albumin solution and the magnetic bead solution is 1:5-10; and the reaction time after adding the bovine serum albumin solution is 1-2 h.

3. The nanocomposite probe system of claim 2, wherein, The preparation method of the AIE@Ag@MnO2 NFs composite probe comprises the following steps: (1) mixing TPE-C≡Cpy, iodomethane and acetonitrile for reaction to obtain an iodination product; (2) mixing the iodination product, methanol and a saturated potassium hexafluorophosphate solution to obtain an AIE material; (3) mixing a potassium permanganate solution, polyvinylpyrrolidone and a hydrochloric acid solution for reaction to obtain MnO2 NFs; (4) mixing a silver nitrate solution, a trisodium citrate solution, a MnO2 NFs solution and a sodium borohydride solution for reaction to obtain Ag@MnO2 NFs; (5) mixing the AIE material, the Ag@MnO2 NFs solution and Tween 80 for reaction, so that the AIE@Ag@MnO2 NFs composite probe is obtained.

4. The nanocomposite probe system of claim 3, wherein, The structure of the TPE-C≡Cpy in step (1) is as follows: The molar ratio of the TPE-C≡Cpy and iodomethane in step (1) is 0.09-0.1:4.9-5; The molar ratio of the TPE-C≡Cpy and acetonitrile is 140-160:1; The reaction temperature in step (1) is 80-90°C, and the time is 10-12 h; The volume-molar ratio of methanol and TPE-C≡Cpy in step (1) in step (2) is 10-15 mL:0.09-0.1 mmol; The volume ratio of methanol and the saturated potassium hexafluorophosphate solution in step (2) is 10-15:10-15; The mixing temperature in step (2) is 20-30°C, and the time is 1-1.5 h; The mass ratio of potassium permanganate and water in the potassium permanganate solution in step (3) is 15-16:90-110; The mass ratio of polyvinylpyrrolidone and potassium permanganate in step (3) is 26-26.5:15-16; The concentration of the hydrochloric acid solution in step (3) is 0.1-0.3 M; The volume-mass ratio of the hydrochloric acid solution and potassium permanganate in step (3) is 80-120 mL:15-16 g; The temperature of the reaction in step (3) is 80-100℃, and the time is 1-2h.

5. The nanocomposite probe system of claim 4, wherein, The concentration of the silver nitrate solution in step (4) is 0.01-0.03M; the concentration of the trisodium citrate solution is 0.01-0.03M; The concentration of the MnO2NFs solution is 5-15mg / mL; the concentration of the sodium borohydride solution is 0.05-0.15M; The volume ratio of the silver nitrate solution, the trisodium citrate solution, the MnO2NFs solution and the sodium borohydride solution is 0.5-1.5:2-3:20-30:0.5-1.5; The temperature of the reaction in step (4) is 20-25℃, and the time is 0.5-1h; The concentration of the Ag@MnO2NFs solution in step (5) is 120-160ug / mL; The volume ratio of the AIE material, the Ag@MnO2NFs solution and the Tween 80 in step (5) is 20-30uL:10-15mL:2-3uL; The temperature of the reaction in step (5) is 20-25℃, and the time is 0.5-1h.

6. The application of the nano-composite probe system in any one of claims 1-5 in the detection of the number of bacteria and the detection of the drug resistance level of bacteria.

7. A single-channel microfluidic chip for detecting the number of bacteria and the level of bacterial drug resistance, characterized in that, The structure of the single-channel microfluidic chip is as follows: The single-channel microfluidic chip comprises a sealed glass layer and a working PDMS layer; One end of the working PDMS layer is provided with three circular inlets, and the other end of the three circular inlets is connected to the inlets of the chain-link structure micro-mixer; The outlets of the chain-link structure micro-mixer are sequentially connected to a Raman signal detection chamber, a hydrogen peroxide storage chamber and a working chamber; The nano-composite probe system in any one of claims 1-5 is injected into the working PDMS layer through the circular inlets.

8. The single-channel microfluidic chip for detecting the number of bacteria and the level of bacterial drug resistance according to claim 7, wherein, The diameters of the three circular inlets are 1-2mm; The number of channel intervals of the chain-link structure micro-mixer is ≥13; the width of a single channel interval is 1-2mm; The volumes of the Raman signal detection chamber and the hydrogen peroxide storage chamber are independently 80-120μL; The working chamber is a dye storage chamber and a distance indicating channel connected in sequence; The diameter of the dye storage chamber is 3-4mm, and the width of the distance indicating channel is 0.5-1.5mm.

9. The method of claim 7 or 8, wherein the method is characterized in that, The method comprises the following steps: (I) Detection of the number of bacteria (a) The bacteria to be detected, the phage-embedded magnetic bead probe and the AIE@Ag@MnO2NFs composite probe are injected into the three circular inlets respectively, and after being fully mixed by the chain-link structure micro-mixer, Raman signals and pressure distance signals are generated to detect the number of bacteria; (II) Detection of the drug resistance level of bacteria (b) The bacteria to be detected, the phage-embedded magnetic bead probe and the AIE@Ag@MnO2NFs composite probe are injected into the three circular inlets respectively, and an antibiotic is continuously injected into the circular inlet where the bacteria to be detected are injected, and after being fully mixed by the chain-link structure micro-mixer, Raman signals and pressure distance signals are generated to detect the drug resistance level of bacteria.

10. The application method of the single-channel microfluidic chip for detecting bacterial count and bacterial resistance level as described in claim 9, characterized in that, The injection amount of the bacteria to be detected and the phage-embedded magnetic bead probe in step (a) has a volume ratio of 400-600:400-600; The volume ratio of the injection amount of the bacteria to be tested and the phage-embedded magnetic bead probe in step (b) is 400-600:400-600. The volume ratio of the injection amount of the bacteria to be tested and the phage-embedded magnetic bead probe in step (b) is 400-600:400-600.

Images