Drug-sensitive microfluidic detection platform based on double-enzyme activity reverse regulation

Abstract

The invention discloses a drug-sensitive microfluidic detection platform based on double-enzyme activity reverse regulation and control, and belongs to the technical field of microbiological pathogenic bacterium drug-sensitive detection and sensitivity judgment. The detection platform comprises a detection probe and a micro-fluidic chip; wherein the main body layer comprises an antibiotic inlet, a diluent inlet, a concentration gradient generation module and an incubation module, antibiotics and diluent enter the concentration gradient generation module through the antibiotic inlet and the diluent inlet respectively, and various antibiotic solutions with different concentrations are generated; the liquid is injected into different liquid storage tanks in the incubation module respectively; the auxiliary layer is located on the upper layer of the incubation module, a plurality of loading openings are formed in the auxiliary layer and used for adding bacteria, colorimetric auxiliary liquid and a detection probe into different liquid storage tanks respectively, and the minimum inhibitory concentration and the sensitivity degree of the bacteria to antibiotics are detected through colorimetry. And visual interpretation of a detection result can be realized through naked eyes / smart phones.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug sensitivity detection and sensitivity determination of microbiological pathogens, and specifically relates to a drug sensitivity microfluidic detection platform based on reverse regulation of dual enzyme activities. Background Art

[0002] The global spread of antibiotic resistance has become a major public health threat, resulting in over one million deaths each year. Accurately identifying and effectively inactivating pathogens in the human body in the early stages of infection is a key strategy to curb the development of bacterial infections and reduce patient mortality. In recent years, the problem of bacterial, viral, and parasite resistance to antimicrobial drugs has become increasingly serious. Failure to use appropriate antibiotics for targeted treatment in a timely manner is one of the main factors leading to the problem of drug resistance. In addition, insufficient antibiotic dosage or inappropriate selection of antibiotic types may also accelerate the formation of drug-resistant bacteria. How to effectively curb the development of antibiotic resistance has become a difficult problem that the global medical community urgently needs to solve. Antimicrobial Susceptibility Testing (AST) is an important means of assessing the sensitivity of bacteria to antibiotics. The detection of minimum inhibitory concentration (MIC) provides a scientific basis for clinical treatment and is also of great significance in curbing the development of antibiotic resistance and optimizing the use of antimicrobial drugs.

[0003] In recent years, visual detection technologies based on nanomaterials have provided new insights for the rapid detection of pathogens. By leveraging the unique physical and chemical properties of nanomaterials, they achieve rapid, sensitive, and specific detection of bacteria, laying an important foundation for the construction of colorimetric sensing platforms. Microfluidics, meanwhile, has provided powerful and precise tools for pathogenic bacteria research, enabling researchers to precisely monitor the physiological activities of pathogens at the nanoscale. Compared with traditional pathogen detection technologies, microfluidics offers unique advantages, overcoming the inherent limitations of traditional methods in sensitivity and detection speed. For example, Zhao et al. designed a visual, antibody-free, and multifunctional bacterial detection platform with a naked-eye detection limit of 10⁻ CFU / mL. Using immunomagnetic beads (IMBs) and AgPt nanoparticle-modified PCN-223-Fe metal-organic frameworks (MOFs), Shang et al. developed a biosensor for the visual detection and elimination of bacteria with high selectivity and excellent reproducibility.

[0004] However, existing visual detection systems still have significant limitations and cannot meet the dual requirements of convenience and timeliness for bedside point-of-care testing. To this end, a drug sensitivity microfluidic detection platform based on the reverse regulation of dual enzyme activities is proposed. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a drug-sensitive microfluidic detection platform based on the reverse regulation of dual enzyme activities, which solves the problems in the existing technology.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activity, comprising: a detection probe and a microfluidic chip; wherein the microfluidic chip comprises:

[0008] Main layer: includes antibiotic inlet, diluent inlet, concentration gradient generation module and incubation module. Antibiotics and diluent enter the concentration gradient generation module through the antibiotic inlet and diluent inlet respectively, generating multiple antibiotic solutions of different concentrations and injecting them into different liquid reservoirs in the incubation module respectively.

[0009] Auxiliary layer: Located on the upper layer of the incubation module, it is equipped with multiple loading ports to add bacteria, colorimetric auxiliary liquid and detection probes into different liquid reservoirs respectively, and detect the minimum inhibitory concentration and sensitivity of bacteria to antibiotics through colorimetry.

[0010] Furthermore, the colorimetric auxiliary liquid is: TMB+H2O2 or OPD.

[0011] Furthermore, the detection probe is a core-shell nanoprobe CPM-NPs, which has a three-layer core-shell structure, with a cubic Cu2O nanoparticle in the center, a polydopamine shell in the middle, and an outer layer coated with MnO2 nanosheets.

[0012] Furthermore, the preparation steps of the nanoprobe CPM-NPs include:

[0013] 1) Dissolve copper sulfate pentahydrate and sodium citrate dihydrate in deionized water and stir to dissolve; add sodium hydroxide aqueous solution and continue stirring; then add L-ascorbic acid solution, stir, age, and centrifuge to obtain Cu2O nanocubes;

[0014] 2) Dopamine and Cu2O nanocubes were dispersed in Tris-HCl buffer to obtain Cu2O@PDA; and potassium permanganate was added to ultrapure water and stirred.

[0015] Furthermore, the diluent is PBS.

[0016] Furthermore, the loading port can be used for the outflow of waste liquid.

[0017] The above-mentioned drug sensitivity microfluidic detection platform based on reverse regulation of dual enzyme activity is used to detect the minimum inhibitory concentration and sensitivity of bacteria to antibiotics.

[0018] A method for detecting the minimum inhibitory concentration and sensitivity of bacteria to antibiotics, using the above-mentioned drug sensitivity microfluidic detection platform based on reverse regulation of dual enzyme activity, is characterized by comprising the following steps:

[0019] S1, passing the antibiotic and diluent into the gradient concentration generation module respectively to generate antibiotic solutions of different concentrations;

[0020] S2, injecting the bacterial suspension into the liquid storage tank through the loading port;

[0021] S3, placing the microfluidic chip in a bacterial incubator for incubation;

[0022] S4, introducing the CPM-NPs detection probe into the reservoir through the loading port;

[0023] S5, when detecting colorimetric activity, TMB+H2O2 or OPD is injected into the reservoir through the loading port, and the color change is generated by the difference in enzyme activity, and the critical turning point of the color change is judged;

[0024] S6, based on the color change, combined with the colorimetric card, determine the minimum inhibitory concentration and sensitivity of bacteria to antibiotics.

[0025] Furthermore, the concentration of the bacterial suspension is 106 CFU / mL.

[0026] Furthermore, the bacteria is Escherichia coli or Staphylococcus aureus; and the antibiotic is amoxicillin, chloramphenicol, kanamycin, levofloxacin, sulfonamide or tetracycline for incubation.

[0027] Beneficial effects of the present invention:

[0028] 1. The platform of the present invention uses a microfluidic chip to automatically complete the incubation reaction of bacteria with multiple concentrations of antibiotics. Combined with the color development characteristics of the nanoprobe, the detection results can be visually interpreted by the naked eye or smart phone.

[0029] 2. The present invention has developed a new type of nanozyme that has both dual enzyme activity (OXD-like / POD-like) and optical properties, which dynamically regulates enzyme activity through bacterial charge to achieve multi-signal output.

[0030] 3. To address the problem that traditional drug sensitivity testing relies on a single signal and is time-consuming, the present invention has developed a dual-signal cross-validation strategy based on a microfluidic chip, which significantly improves detection reliability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0032] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention;

[0033] Figure 2 Schematic diagram of the functions of various modules of the microfluidic chip of the present invention;

[0034] Figure 3 This is a schematic diagram of the principle of the present invention using the detection platform to detect the MIC and sensitivity of pathogens;

[0035] Figure 4 is a characterization diagram of the nanoprobe of the present invention;

[0036] Figure 5 CPM-NPs probes and pathogenic bacteria (10 6 CFU / mL Escherichia coli) after co-incubation with TMB as the colorimetric substrate and OPD as the colorimetric substrate;

[0037] Figure 6 Results of the quantitative detection of peroxidase-like and oxidase-like activities of CPM-NPs nanoprobes against E. coli at different concentrations;

[0038] Figure 7 It is based on the colorimetric reaction results of 8 independent concentration gradient channels of the microfluidic chip after incubation of Escherichia coli with different concentrations of amoxicillin, chloramphenicol, kanamycin, levofloxacin, sulfonamide and tetracycline;

[0039] Figure 8 This colorimetric card is designed to show the sensitivity of Escherichia coli to different concentrations of amoxicillin, chloramphenicol, kanamycin, levofloxacin, sulfonamide and tetracycline.

[0040] Figure 9 The colorimetric reaction results of Staphylococcus aureus after incubation with different concentrations of chloramphenicol, levofloxacin, sulfonamide and tetracycline were detected based on 8 independent concentration gradient channels of the microfluidic chip;

[0041] Figure 10 A colorimetric card was designed to show the sensitivity of Staphylococcus aureus to different concentrations of chloramphenicol, levofloxacin, sulfonamide and tetracycline.

[0042] Figure 11The colorimetric reaction results of Klebsiella pneumoniae after incubation with different concentrations of amoxicillin, chloramphenicol, levofloxacin and tetracycline were detected based on 8 independent concentration gradient channels of the microfluidic chip;

[0043] Figure 12 A color chart was designed to show the sensitivity of Klebsiella pneumoniae to different concentrations of amoxicillin, chloramphenicol, levofloxacin and tetracycline.

[0044] Figure 13 The colorimetric reaction results of Pseudomonas aeruginosa after incubation with different concentrations of amoxicillin, ceftriaxone, and levofloxacin were detected based on 8 independent concentration gradient channels of the microfluidic chip;

[0045] Figure 14 A color chart was designed to show the sensitivity of Pseudomonas aeruginosa to different concentrations of amoxicillin, ceftriaxone and levofloxacin.

[0046] Figure 15 The results are colorimetric after incubation of Escherichia coli or Staphylococcus aureus (dispersed in saliva or serum) with levofloxacin and sulfonamide using the detection platform of the present invention.

[0047] Among them, 1-antibiotic inlet, 2-dilution inlet, 3-concentration gradient generation module, 4-incubation module. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0049] Example 1

[0050] like Figure 1 and Figure 2 As shown, a drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities includes: a detection probe and a microfluidic chip; wherein the microfluidic chip includes:

[0051] Main layer: includes antibiotic inlet 1, diluent inlet 2, concentration gradient generation module 3 and incubation module 4. Antibiotics and diluent enter the concentration gradient generation module 3 through antibiotic inlet 1 and diluent inlet 2 respectively, generating multiple (8) antibiotic solutions of different concentrations and injecting them into different circular liquid reservoirs in incubation module 4 respectively;

[0052] Auxiliary layer: Located on the upper layer of the incubation module 4, it is provided with multiple loading ports to add bacteria, colorimetric auxiliary liquid and detection probes into different circular liquid reservoirs respectively, and detect the minimum inhibitory concentration and sensitivity of bacteria to antibiotics by colorimetry.

[0053] In addition, the loading port can also be used for the outflow of waste liquid.

[0054] The diluent is PBS, and the colorimetric auxiliary liquid includes: 3,3',5,5'-tetramethylbenzidine (TMB) + H2O2 or o-phenylenediamine (OPD); the detection probe is a multifunctional nanoprobe Cu2O@PDA@MnO2 (CPM-NPs) with a core-shell structure, which has a three-layer core-shell structure, with cubic Cu2O nanoparticles in the center, a polydopamine (PDA) shell in the middle, and an outer layer coated with MnO2 nanosheets. The probe has dual peroxidase-like and oxidase-like activities and can be used for bacterial colorimetric detection.

[0055] The steps for using this detection platform to detect the minimum inhibitory concentration and sensitivity of bacteria to antibiotics include:

[0056] S1, passing the antibiotic and diluent into the gradient concentration generation module 3 respectively to generate antibiotic solutions of different concentrations;

[0057] S2, adjust the bacterial suspension to 10 6 CFU / mL and injected into the middle circular reservoir of incubation module 4 through the loading port;

[0058] S3, placing the microfluidic chip in a 37°C bacterial incubator for incubation;

[0059] S4, introduce CPM-NPs detection probe into the circular reservoir through the loading port and react for 20 min;

[0060] S5, when detecting colorimetric activity, TMB+H2O2 or OPD is injected into the circular reservoir through the loading port. The color change is caused by the difference in enzyme activity. The critical turning point from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0061] S6, based on the difference in color changes seen by the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, quickly determines the minimum inhibitory concentration and sensitivity of bacteria to antibiotics.

[0062] The colorimetric card design method is as follows: a colorimetric card is constructed according to the guidelines of the Clinical and Laboratory Standards Institute of the United States. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I and resistance breakpoint R; sensitive area is a green circle, intermediate area is a yellow circle, and resistant area is a red circle), drug name, and drug concentration.

[0063] The criteria for drug sensitivity determination are as follows:

[0064] Susceptible (S): When the minimum inhibitory concentration (MIC) of the test bacteria is ≤ CLSI S breakpoint, the test bacteria should be judged to be sensitive to the antibiotic, indicating that the antibiotic is clinically effective;

[0065] Resistant (R): MIC ≥ CLSI R breakpoint, directly determining that the tested bacteria are resistant to the corresponding antibiotic and the use of the drug should be avoided;

[0066] Intermediate (I): The MIC is between the S and R breakpoints, and the medication regimen needs to be evaluated in combination with pharmacokinetic / pharmacodynamic (PK / PD) parameters.

[0067] Specifically: Figure 2 and 3 As shown, the principle of using this detection platform for detection is:

[0068] First, antibiotics and diluents are introduced into gradient concentration generation module 3 to generate antibiotic solutions with different gradient concentrations; then the bacterial suspension is adjusted to 10 6 CFU / mL was injected into the microfluidic chip through the loading port and placed in a 37°C bacterial incubator for incubation. The CPM-NPs detection probe was introduced through the loading port, and the MnO2 shell with oxidase-like activity was injected through the surface Mn 2+ / Mn 3+ / Mn 4+ The redox couple-mediated electron transfer pathway can catalyze the oxidation of OPD in the absence of H2O2; the Cu2O core with peroxidase-like activity utilizes Cu + / Cu 2+Circular activation of H2O2 produces ·OH, which drives the TMB color development reaction. Pathogenic bacteria carry a large amount of negative charge on their surfaces, which can interact with CPM-NPs through electrostatic interactions, affecting the activities of these two different enzymes. This effect may be due to the polyanionic nature of the pathogenic bacteria's surface, which leads to interactions between the bacteria and the nanoparticles, causing the nanoparticles to disperse, thereby altering their enzymatic activity. When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the auxiliary liquid inlet. The difference in enzyme activity produces a color change, which can be visually interpreted as the critical turning point from dark yellow (growth) to dark blue (inhibition). Based on the difference in color change observed by the naked eye and the RGB values ​​obtained by the electronic device of the color-picking app, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of the pathogen to antibiotics can be rapidly determined.

[0069] In this embodiment, the preparation method of nanoprobe CPM-NPs includes the following steps:

[0070] 1) Dissolve 375 mg of copper sulfate pentahydrate and 147 mg of sodium citrate dihydrate in 80 mL of deionized water and stir for 15 minutes to ensure complete dissolution. Add 20 mL of 1.25 M sodium hydroxide solution and continue stirring for 15 minutes. To the mixed solution, add 50 mL of 0.03 M L-ascorbic acid solution. Stir the resulting solution for another 3 minutes, age it at room temperature for 1 hour, and centrifuge it to obtain Cu2O nanocubes.

[0071] 2) 0.119 g of dopamine and 60 mg of Cu2O nanocubes were dispersed in 60 mL of Tris-HCl buffer (pH 8.5) and stirred at room temperature for 24 hours to obtain Cu2O@PDA. 80 mg of Cu2O@PDA and 63.2 mg of potassium permanganate (KMnO4) were added to 50 mL of ultrapure water and stirred at 80°C for 10 hours to obtain a Cu2O@PDA@MnO2 (CPM-NPs) nanocomposite.

[0072] The Zeta potential of Cu2O, Cu2O@PDA and Cu2O@PDA@MnO2 prepared in this example is as follows Figure 4 As shown in Figure A, they are +10.2 mV, +1.8 mV and -12.5 mV, respectively, indicating that the coating of PDA and MnO2 makes CPM-NPs electronegative. Figure 4 Figure B shows that the mean particle size distribution of CPM-NPs is significantly larger than that of Cu2O nanocubes, confirming the successful preparation of the probe. Figure 4C in the figure shows that the peaks of Cu2O at 29.73°, 36.56°, 42.43°, 61.54° and 73.73° are respectively attributed to the (110), (111), (200), (220) and (311) planes of Cu2O nanotubes, which are consistent with the standard card; while CPM-NPs show relatively weak diffraction peaks at these positions, indicating that PDA and MnO2 are successfully coated and the modified Cu2O skeleton does not collapse. In the FT-IR spectrum (such as Figure 4 D in the figure), 631 cm -1 The absorption peak at 1600 cm is attributed to the Cu-O group, which reveals the successful synthesis of Cu2O. -1 (C=C, benzene ring skeleton vibration), 1490cm -1 (NH, amino bending vibration) and 1295 cm -1 (CO, phenol oxygen bond stretching vibration) shows a characteristic absorption peak near 526cm -1 A new absorption band (Mn-O) appeared at the α-H bond, confirming that MnO2 was anchored on the PDA surface through the Mn-OC bonding.

[0073] The feasibility of the designed CPM-NPs composite probe for detecting bacteria through dual enzyme activity was analyzed. The specific operation process was as follows: CPM-NPs dispersion (0.25 mg / mL) and HAc / NaAc buffer (0.01 M, pH = 4.0) were added to a UV cuvette. Subsequently, 50 μL TMB (5 mM) + 50 μL H2O2 (10 mM) or 50 μL OPD (10 mM) were added to the above mixture. Finally, the UV-vis absorption signal was recorded using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 5 As shown;

[0074] from Figure 5 As can be seen in Figure A, in the presence of TMB and H2O2, CPM-NPs exhibited a significant absorption peak at 652nm, confirming that the composite material has strong peroxidase-like activity. The presence of bacteria will weaken the peroxidase-like activity, causing the blue color of the solution to become lighter. Figure 5 As can be seen from Figure B, in the H2O2-free system, CPM-NPs can catalyze the oxidation of OPD to produce yellow DAP products. After the addition of bacteria, the colorimetric intensity of the solution increases, indicating that the oxidase-like catalytic activity of CPM-NPs is enhanced.

[0075] The following specific examples are used to verify the effectiveness of the detection platform of the present invention;

[0076] Example 2

[0077] The above detection platform is used to quantitatively detect E. coli at different concentrations. The specific steps are as follows:

[0078] Prepare the concentration of 10-10 8 A 0.25 mg / mL E. coli dispersion was injected into Incubation Module 4 of the microfluidic chip. Simultaneously, a 0.25 mg / mL CPM-NPs dispersion and a 0.01 M HAc / NaAc buffer (pH 4.0) were injected into Incubation Module 4. After a 20-minute incubation, 50 μL of TMB (5 mM) plus 50 μL of H2O2 (10 mM) or 50 μL of OPD (10 mM) were injected through the loading port. Finally, the RGB values ​​were extracted using a mobile colorimetric app.

[0079] Figure 6 For the quantitative detection of E. coli colorimetric and fluorescence results. Figure 6 As shown in A, as the logarithmic concentration (LogC) of bacteria increases from 10 CFU / mL to 10 8 CFU / mL, the color of the solution in the TMB+H2O2 reaction system changes from dark to light. After color extraction using the mobile phone color APP, LogC and green channel value (G) are linearly related. The calibration curve drawn is RGB G =9.20Log C+102.91(R 2 =0.990). In addition, when the oxidase-like activity of the composite material was quantitatively detected, the reaction solution showed a change from light to dark yellow, such as Figure 6 As shown in Figure B, there is a good linear relationship between the blue to green channel value (B / G) and Log C. The corresponding regression equation is further calculated as RGB B / G =-0.09Log C+0.93(R 2 =0.991). The results showed that accurate detection of Escherichia coli could be achieved by combining the peroxidase-like and oxidase-like activities of CPM-NPs.

[0080] Example 3

[0081] This example investigates the minimum inhibitory concentration and sensitivity of Escherichia coli to different antibiotics (amoxicillin, chloramphenicol, kanamycin, levofloxacin, sulfonamides, and tetracycline). The specific implementation steps are as follows:

[0082] (1) Using a syringe pump, different antibiotics and diluents are introduced into the gradient concentration generation module 3 to generate antibiotic solutions of different concentrations, which flow into eight concentration gradient branches respectively;

[0083] (2) Adjust the bacterial suspension to 10 6CFU / mL and injected into the microfluidic chip through the loading port and incubated in a 37°C bacterial incubator for 2-6 h;

[0084] (3) CPM-NPs detection probe was introduced through the loading port and reacted for 20 min;

[0085] (4) When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the auxiliary liquid injection port. The color change is caused by the difference in enzyme activity. The critical turning point of the color from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0086] (5) The colorimetric card was designed according to the CLSI guidelines. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I, and resistance breakpoint R; sensitive areas are green circles, intermediate areas are yellow circles, and resistant areas are red circles), drug name, and drug concentration;

[0087] (6) Based on the difference in color changes between the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of pathogenic bacteria to antibiotics can be quickly determined.

[0088] Colorimetric results are as follows Figure 7 As shown in the figure, the MIC of Escherichia coli to amoxicillin and chloramphenicol were both 8 μg / mL, kanamycin was 32 μg / mL, levofloxacin showed the strongest antibacterial activity (MIC = 0.5 μg / mL), the MIC of tetracycline was 4 μg / mL, and sulfonamide did not show a characteristic inflection point in all the tested gradients, indicating that the strain was completely resistant to sulfonamides. Figure 8 The sensitivity of Escherichia coli to the six antibiotics was determined by comparison (as shown in Table 1).

[0089] Table 1 Results of drug sensitivity test for Escherichia coli

[0090]

[0091] Example 4

[0092] This example explores the minimum inhibitory concentration and sensitivity of Staphylococcus aureus to chloramphenicol, levofloxacin, sulfonamides, and tetracycline. The specific implementation steps are as follows:

[0093] (1) Using a syringe pump, different antibiotics and diluents are introduced into the gradient concentration generation module 3 to generate antibiotic solutions of different concentrations, which flow into eight concentration gradient branches respectively;

[0094] (2) Adjust the bacterial suspension to 10 6CFU / mL and injected into the microfluidic chip through the loading port and incubated in a 37°C bacterial incubator for 2-6 h;

[0095] (3) CPM-NPs detection probe was introduced through the loading port and reacted for 20 min;

[0096] (4) When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the loading port, and the color change is generated by the difference in enzyme activity. The critical turning point of the color from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0097] (5) The colorimetric card was designed according to the CLSI guidelines. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I, and resistance breakpoint R; sensitive areas are green circles, intermediate areas are yellow circles, and resistant areas are red circles), drug name, and drug concentration;

[0098] (6) Based on the difference in color changes between the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of pathogenic bacteria to antibiotics can be quickly determined.

[0099] According to the attached Figure 9 The results show that the MIC of Staphylococcus aureus to chloramphenicol is 8μg / mL, the MIC to levofloxacin is 0.5μg / mL, the MIC to tetracycline is 4μg / mL, and the sulfonamide is completely resistant and no effective inhibitory concentration is detected. The experimental results are compared with the color card ( Figure 10 ) were used for comparison, and finally the antibiotic sensitivity typing of the strain was obtained. The results are shown in Table 2.

[0100] Table 2 Results of drug sensitivity test of Staphylococcus aureus

[0101]

[0102]

[0103] Example 5

[0104] This example investigates the minimum inhibitory concentration and sensitivity of the multidrug-resistant bacterium Klebsiella pneumoniae subspecies fetidronic to amoxicillin, chloramphenicol, levofloxacin, and tetracycline. The specific implementation steps are as follows:

[0105] (1) Using a syringe pump, different antibiotics and diluents are introduced into the gradient concentration generation module 3 to generate antibiotic solutions of different concentrations, which flow into eight concentration gradient branches respectively;

[0106] (2) Adjust the bacterial suspension to 10 6CFU / mL and injected into the microfluidic chip through the loading port and incubated in a 37°C bacterial incubator for 2-6 h;

[0107] (3) CPM-NPs detection probe was introduced through the loading port and reacted for 20 min;

[0108] (4) When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the loading port, and the color change is generated by the difference in enzyme activity. The critical turning point of the color from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0109] (5) The colorimetric card was designed according to the CLSI guidelines. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I, and resistance breakpoint R; sensitive areas are green circles, intermediate areas are yellow circles, and resistant areas are red circles), drug name, and drug concentration;

[0110] (6) Based on the difference in color changes between the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of pathogenic bacteria to antibiotics can be quickly determined.

[0111] according to Figure 11 The results show that the strain is highly resistant to amoxicillin, levofloxacin and tetracycline, and the MIC of chloramphenicol is 6μg / mL. Figure 12 ) for comparison, the strain was moderately sensitive to chloramphenicol, but resistant to the other three antibiotics. The results are shown in Table 3.

[0112] Table 3 Results of drug susceptibility testing of Klebsiella pneumoniae

[0113]

[0114] Example 6

[0115] This example investigates the minimum inhibitory concentration and sensitivity of Pseudomonas aeruginosa, a clinically important drug-resistant strain, to amoxicillin, ceftriaxone, and levofloxacin. The specific implementation steps are as follows:

[0116] (1) Using a syringe pump, different antibiotics and diluents are introduced into the gradient concentration generation module 3 to generate antibiotic solutions of different concentrations, which flow into eight concentration gradient branches respectively;

[0117] (2) Adjust the bacterial suspension to 10 6 CFU / mL and injected into the microfluidic chip through the loading port and incubated in a 37°C bacterial incubator for 2-6 h;

[0118] (3) CPM-NPs detection probe was introduced through the loading port and reacted for 20 min;

[0119] (4) When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the loading port, and the color change is generated by the difference in enzyme activity. The critical turning point of the color from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0120] (5) The colorimetric card was designed according to the CLSI guidelines. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I, and resistance breakpoint R; sensitive areas are green circles, intermediate areas are yellow circles, and resistant areas are red circles), drug name, and drug concentration;

[0121] (6) Based on the difference in color changes between the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of pathogenic bacteria to antibiotics can be quickly determined.

[0122] according to Figure 13 The results show that the strain is resistant to the three tested antibiotics: amoxicillin, cephalothin, and levofloxacin. Its MIC value for cephalothin is as high as 56μg / mL, and the MIC for levofloxacin is 6μg / mL. The experimental results are compared with the color card ( Figure 14 ) were used as control, and the strain showed resistance to all three antibiotics. The results are shown in Table 4.

[0123] Table 4 Results of drug sensitivity test of Pseudomonas aeruginosa

[0124]

[0125] Example 7

[0126] In this example, Escherichia coli and Staphylococcus aureus were spiked into blood or saliva samples to simulate actual samples. The samples were then tested for MIC and drug sensitivity of levofloxacin and sulfonamide. The specific implementation steps are as follows:

[0127] (1) Using a syringe pump, different antibiotics and diluents are introduced into the gradient concentration generation module 3 to generate antibiotic solutions of different concentrations, which flow into eight concentration gradient branches respectively;

[0128] (2) Adjust the bacterial suspension to 10 6 CFU / mL was injected into the microfluidic chip through the bacterial loading port and incubated in a 37°C bacterial incubator for 2-6 h;

[0129] (3) CPM-NPs detection probe was introduced through the loading port and reacted for 20 min;

[0130] (4) When detecting colorimetric activity, TMB+H2O2 or OPD chromogenic substrate is injected through the loading port, and the color change is generated by the difference in enzyme activity. The critical turning point of the color from dark yellow (growth) to dark blue (inhibition) can be judged by the naked eye;

[0131] (5) The colorimetric card was designed according to the CLSI guidelines. Its core elements include the following key information: bacterial name, CLSI standard susceptibility determination breakpoints (covering sensitivity breakpoint S, intermediate breakpoint I, and resistance breakpoint R; sensitive areas are green circles, intermediate areas are yellow circles, and resistant areas are red circles), drug name, and drug concentration;

[0132] (6) Based on the difference in color changes between the naked eye and the RGB values ​​taken by the electronic device of the color-picking APP, combined with the designed colorimetric card, the minimum inhibitory concentration and sensitivity of pathogenic bacteria to antibiotics can be quickly determined.

[0133] The results are as follows Figure 15 As shown. In saliva samples containing complex components, this method accurately identified the sensitivity of the two strains to levofloxacin (MIC = 0.5 μg / mL), and sulfonamide showed no antibacterial effect within a concentration gradient of 0-896 μg / mL. In serum samples, the MIC values ​​of both pathogens to levofloxacin remained stable at 0.5 μg / mL, while they showed complete resistance to sulfonamide.

[0134] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0135] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities, characterized in that: include: Detection probe and microfluidic chip; wherein the microfluidic chip includes: Main layer: includes antibiotic inlet, diluent inlet, concentration gradient generation module and incubation module. Antibiotics and diluent enter the concentration gradient generation module through the antibiotic inlet and diluent inlet respectively, generating multiple antibiotic solutions of different concentrations and injecting them into different liquid reservoirs in the incubation module respectively. Auxiliary layer: Located on the upper layer of the incubation module, it is equipped with multiple loading ports to add bacteria, colorimetric auxiliary liquid and detection probes into different liquid reservoirs respectively, and detect the minimum inhibitory concentration and sensitivity of bacteria to antibiotics through colorimetry.

2. A drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities according to claim 1, characterized in that: The colorimetric auxiliary liquid is: TMB+H2O2 or OPD.

3. The drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities according to claim 1, characterized in that: The detection probe is a core-shell nanoprobe CPM-NPs, which has a three-layer core-shell structure, with a cubic Cu2O nanoparticle in the center, a polydopamine shell in the middle, and an outer layer coated with MnO2 nanosheets.

4. The drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities according to claim 3, characterized in that: The preparation steps of the nanoprobe CPM-NPs include: 1) Dissolve copper sulfate pentahydrate and sodium citrate dihydrate in deionized water and stir to dissolve; add sodium hydroxide aqueous solution and continue stirring; then add L-ascorbic acid solution, stir, age, and centrifuge to obtain Cu2O nanocubes; 2) Dopamine and Cu2O nanocubes were dispersed in Tris-HCl buffer to obtain Cu2O@PDA; and potassium permanganate was added to ultrapure water and stirred.

5. The drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities according to claim 1, characterized in that: The diluent is PBS.

6. The drug-sensitive microfluidic detection platform based on reverse regulation of dual enzyme activities according to claim 1, characterized in that: The loading port can be used for the outflow of waste liquid.

7. Use of a drug sensitivity microfluidic detection platform based on reverse regulation of dual enzyme activities according to any one of claims 1 to 6 in detecting the minimum inhibitory concentration and sensitivity of bacteria to antibiotics.

8. A method for detecting the minimum inhibitory concentration and sensitivity of bacteria to antibiotics, using a drug sensitivity microfluidic detection platform based on reverse regulation of dual enzyme activities according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1, passing the antibiotic and diluent into the gradient concentration generation module respectively to generate antibiotic solutions of different concentrations; S2, injecting the bacterial suspension into the liquid storage tank through the loading port; S3, placing the microfluidic chip in a bacterial incubator for incubation; S4, introducing the CPM-NPs detection probe into the reservoir through the loading port; S5, when detecting colorimetric activity, TMB+H2O2 or OPD is injected into the reservoir through the loading port, and the color change is generated by the difference in enzyme activity, and the critical turning point of the color change is judged; S6, based on the color change, combined with the colorimetric card, determine the minimum inhibitory concentration and sensitivity of bacteria to antibiotics.

9. The method for detecting the minimum inhibitory concentration and sensitivity of bacteria to antibiotics according to claim 8, characterized in that: The concentration of the bacterial suspension is 106 CFU / mL.

10. The method for detecting the minimum inhibitory concentration and sensitivity of bacteria to antibiotics according to claim 8, characterized in that: The bacteria are Escherichia coli or Staphylococcus aureus; and the antibiotics are amoxicillin, chloramphenicol, kanamycin, levofloxacin, sulfonamide or tetracycline for incubation.

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