Droplet microfluidic detection system for detecting pathogenic bacteria based on deoxyribozyme probe

Abstract

The invention discloses a droplet microfluidic detection system for detecting pathogenic bacteria based on a deoxyribozyme probe, which belongs to the technical field of microbiological detection, and mainly comprises an automatic sample introduction device, a microfluidic droplet generation chip, a capillary tube-laser induced fluorescence detector and a signal acquisition and analysis device, the pathogen detection process comprises the following steps: respectively and automatically injecting an actual water sample, a deoxyribozyme probe, liquid drop oil, a pathogen lysis solution and a reaction buffer solution into the microfluidic liquid drop generation chip by the automatic sample injection device, stably generating liquid drops, and detecting the liquid drops through the capillary tube-laser induced fluorescence detector, and the signal acquisition and analysis device acquires and analyzes the fluorescence signal. The method has the advantages of high detection flux, high stability, high sensitivity, rapidness and the like, and can be applied to the field of environmental microbiological detection.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a droplet microfluidic detection system for detecting pathogens based on deoxyribonuclease probes. Background Technology

[0002] Waterborne pathogens can enter natural water bodies through sewage discharge, soil spread, and air deposition, seriously threatening the safety of aquatic ecosystems and human health. A survey of 204 countries and regions worldwide showed that in 2019, bacterial infections caused 7.7 million deaths globally, accounting for 13.6% of all deaths. Bacterial infections are the second leading cause of death globally, after ischemic heart disease, and are associated with one in eight deaths worldwide. The National Environmental Protection Standards of the People's Republic of China specify methods for determining total coliforms, fecal coliforms, and Escherichia coli in water bodies, indicating that environmental protection standards and planning monitoring indicators have been gradually expanding to include biological pollutants in recent years.

[0003] Existing methods for detecting pathogens mainly include culture methods, enzyme substrate methods, gene detection methods, and immunoassay methods. Culture methods are the gold standard for detecting bacterial populations in test samples, offering high accuracy and reliability in pathogen detection, but also suffer from drawbacks such as being time-consuming and cumbersome. Enzyme substrate methods, used in national standards for determining total coliforms, fecal coliforms, and Escherichia coli in water quality, offer advantages such as ease of operation and rapid detection, but their technical requirements are higher than those of culture methods. Gene detection methods are molecular biology-based detection techniques that identify and characterize pathogens by analyzing their genetic material (DNA or RNA). They offer high sensitivity and specificity, but are prone to cross-contamination and cannot accurately distinguish closely related sequences. Immunoassays are ubiquitous in medical diagnostics and food safety applications, using antibodies produced in organisms to identify pathogens for precise quantification. However, immunoassays cannot distinguish between live and dead pathogens, and due to limited sensitivity, false negative results are common. Therefore, there is a need to develop rapid, highly specific, and highly sensitive online monitoring devices for live pathogens and viruses.

[0004] Deoxyribozymes (DNAzymes) are single-stranded DNA fragments with catalytic functions synthesized using in vitro molecular evolution techniques. They possess highly efficient catalytic activity and structure recognition capabilities. DNAzymes have been widely used in fields such as biosensing, drug delivery, environmental monitoring, and disease diagnosis, and offer advantages such as low cost, ease of synthesis, chemical stability, high catalytic efficiency, and compatibility with various scale-up strategies. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a droplet microfluidic detection system for detecting pathogens based on deoxyribonuclease probes. This invention has the advantages of rapid detection of active biological pollutants, high specificity, and high sensitivity.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a deoxyribonuclease probe, wherein the deoxyribonuclease probe is composed of nucleotide sequences as shown in SEQ ID NO. 1-3, wherein R in the sequence represents RNA base A, F represents T base modified with a fluorescent group, and Q represents T base modified with a quenching group.

[0007] Based on the above technical solution, further, the fluorescent group is FAM, and the quenching group is Dabcyl.

[0008] In a first aspect, the present invention provides a droplet microfluidic detection system for detecting pathogens based on the above-mentioned deoxyribonuclease probe. The detection system mainly includes an automated sample introduction device, a microfluidic droplet generation chip 15, a capillary-laser induced fluorescence detector 16, and a signal acquisition and analysis device.

[0009] Based on the above technical solution, the detection system further includes a first aqueous phase inlet 1, a second aqueous phase inlet 2, a filter structure 3, a mixing structure 4, a fluid stabilization structure 5, an oil phase inlet 6, a droplet outlet 7, an actual water sample container 8, a reaction buffer container 9, a pathogen lysis buffer container 10, a deoxyribozyme probe container 11, a droplet oil container 12, a pressure pump 13, a flow sensor 14, a microfluidic droplet generation chip 15, a capillary-laser induced fluorescence detector 16, a waste liquid pool 17, an oscilloscope 18, and a signal analysis device 19. The microfluidic droplet generation chip 15 mainly includes a first aqueous phase inlet 1, a second aqueous phase inlet 2, an oil phase inlet 6, a filter structure 3, a fluid stabilization structure 5, a mixing structure 4, a droplet generation structure, and a droplet outlet 7. The first aqueous phase inlet 1 and the second aqueous phase inlet 2 are connected by connecting pipes and then connected to the inlet I of the droplet generation structure through pipes. The oil phase inlet 6 is connected to the inlet II of the droplet generation structure through a pipe. The outlet I of the droplet generation structure is connected to the droplet outlet 7. The filter structure 3 and the mixing structure 4 are respectively provided on the connecting pipes of the first aqueous phase inlet 1 and the second aqueous phase inlet 2. The fluid stabilization structure 5 is provided on the connecting pipe of the oil phase inlet 6. The actual water sample container 8 and the reaction buffer container 9 are respectively connected to the inlet of the mixing tank via pipes. The outlet of the mixing tank is connected to the first aqueous phase inlet 1 of the microfluidic droplet generation chip 15. The pathogen lysis buffer container 10 and the deoxyribozyme probe container 11 (containing the deoxyribozyme probe) are respectively connected to the inlet of the mixing tank via pipes. The outlet of the mixing tank is connected to the second aqueous phase inlet 1 of the microfluidic droplet generation chip 15. A pressure pump 13 is installed on the upstream pipe of the mixing tank, and a flow meter is installed on the downstream pipe of the mixing tank. Sensor 14; The droplet oil container 12 is connected to the oil phase inlet 6 of the microfluidic droplet generation chip 15 through a pipe. A pressure pump 13 and a flow sensor 14 are installed on the connecting pipe between the droplet oil container 12 and the oil phase inlet 6; The droplet outlet 7 of the microfluidic droplet generation chip 15 is connected to the inlet of the capillary-laser induced fluorescence detector 16, and the outlet of the capillary-laser induced fluorescence detector 16 is connected to the waste liquid pool 17. The oscilloscope 18 is connected to the capillary-laser induced fluorescence detector 16 and the signal analysis device 19, respectively.

[0010] Thirdly, the present invention provides the application of the above-mentioned deoxyribonuclease probe or the above-mentioned detection system in biosensing for the specific identification of pathogens.

[0011] Based on the above technical solution, the pathogens further include Escherichia coli.

[0012] Based on the above technical solution, the detection system further describes the process of specifically identifying Escherichia coli as follows: the pressure pump 13 is controlled by the flow sensor 14 to apply pressure, and the actual water sample, deoxyribozyme probe, droplet oil, pathogen lysate, and reaction buffer are automatically injected. The actual water sample and reaction buffer form the first aqueous phase, and the deoxyribozyme probe and pathogen lysate form the second aqueous phase. Together with the oil phase, the second aqueous phase is injected into the microfluidic droplet generation chip 15 through the pressure pump, and droplets are stably generated. After being detected by the capillary-laser induced fluorescence detector 16, the droplets are discharged to the waste liquid pool 17. The oscilloscope 18 collects the fluorescence signal and transmits it to the signal analysis device 19 for analysis.

[0013] Based on the above technical solution, the capillary-laser induced fluorescence detector 16 further excites the sample with excitation light of a specific wavelength, and then collects and analyzes the fluorescence signal of the sample to obtain information such as the type and concentration of the substance being tested. That is, a high-intensity laser beam is used to irradiate the component that is flowing through. If the component can be excited by the laser and produce fluorescence, it will emit light of a specific wavelength. The highly sensitive detector will capture these fluorescence signals and finally form a peak-shaped spectrum on the oscilloscope.

[0014] Based on the above technical solution, the signal analysis device 19 further analyzes the fluorescence peak value, sets 20mV, 40mV, 100mV and 200mV as thresholds respectively, collects the number of droplets above the threshold, determines droplets with fluorescence signals above 200mV as positive, and outputs the sample results.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention integrates functional nucleic acids with droplet microfluidics. Functional nucleic acids serve as recognition elements to achieve highly specific and sensitive detection of pathogens, while droplet microfluidics enables high-throughput and single-molecule-level analysis. The system features high integration, rapid detection, simple operation, and low reagent consumption. This invention can detect Escherichia coli within 60 minutes with high sensitivity. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0017] Figure 1 The graph shows the sensitivity characterization results of the Escherichia coli deoxyribonuclease fluorescent probe RFD-EC1 in Example 3; Figure 2 This is a diagram showing the specific characterization results of the Escherichia coli deoxyribonuclease fluorescent probe RFD-EC1 in Example 3; Figure 3 This is a schematic diagram of the flow channel structure of the PDMS microfluidic droplet generation chip in Example 4. In the figure, 1: first aqueous phase inlet; 2: second aqueous phase inlet; 3: filter structure; 4: mixing structure; 5: fluid stabilization structure; 6: oil phase inlet; 7: droplet outlet; Figure 4 This is a schematic diagram of the module connections of the pathogen detection system in Example 5; Figure 5 This is a schematic diagram of the pathogen detection system in Example 5. In the diagram, 8: actual water sample container; 9: reaction buffer container; 10: pathogen lysis buffer container; 11: deoxyribozyme probe container; 12: droplet oil container; 13: pressure pump; 14: flow sensor; 15: microfluidic droplet generation chip; 16: capillary-laser induced fluorescence detector; 17: waste liquid tank; 18: oscilloscope; 19: signal analysis device. Figure 6 The fluorescence signal diagram of the negative droplet in Example 6; Figure 7 The fluorescence signal diagram of the positive droplet in Example 6; Figure 8 This is a graph showing the detection and analysis results of different gradient signals for Escherichia coli in Example 7; Figure 9This is a graph showing the results of actual water sample testing in Example 8; Figure 10 This is a magnified image of the positive peak from the actual water sample tested in Example 8. Detailed Implementation

[0018] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0019] The structures, proportions, sizes, etc., shown in the accompanying drawings of this application are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed in the specification, and are not intended to limit the implementation conditions of this application. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size should still fall within the scope of the technical content disclosed in this application, provided that they do not affect the effects and purposes that this application can produce.

[0020] The names and sequences of the nucleic acids involved in the examples are shown in Table 1.

[0021] Table 1. Nucleic acid names, sequences, and uses

[0022] Example 1: Construction of a deoxyribonuclease probe (E. coli deoxyribonuclease fluorescent probe RFD-EC1) 1. Substrate purification: Take 100 µL (100 µM) of FS28-substrate chain, add 100 µL of Loading Buffer, mix well, centrifuge, heat at 90℃ for 1 min, and complete purification by polyacrylamide gel electrophoresis.

[0023] 2. Probe connection: Phosphorylation was performed on the 5' end of the enzyme / substrate chain. The phosphorylation system was 50 µL (for enzyme chain phosphorylation as an example): 100 µM enzyme chain, 10 µL 5×PNK buffer, 2.5 µL (100 mM) ATP, and 30 µL water. After mixing and centrifugation, T4 PNK enzyme was added and mixed evenly. Phosphorylation was carried out at 37 °C for 40 min before ligation.

[0024] The ligation system was 100 µL (based on phosphorylation): 7.5 µL (100 µM) of splint, 500 pmol of substrate, and 10 µL of 10×T4 ligation buffer were added at a splint:phosphorylase ratio of 1.5:1. Sterile water was added to a final volume of 95 µL. After mixing, the mixture was centrifuged, heated at 90 °C for 2 min, cooled to room temperature, and then 1 µL (100 mM) of ATP and 4 µL of T4 ligase were added and mixed thoroughly. Ligation was carried out at 37 °C for 2–3 h. After ligation, the mixture was concentrated using ice-cold anhydrous ethanol precipitation, purified by 10% dPAGE, and the probe product was determined using Nanodrop. The final E. coli deoxyribonuclease probe RFD-EC1 was obtained.

[0025] Culture of bacteria required in Example 2 The bacterial strains used in the experiment (Escherichia coli, Klebsiella pneumoniae, Burkholderia cocovenenans, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus sphaeroides) were removed at -80℃ and inoculated into 5 mL of Luria Broth (LB) liquid medium. The cultures were then incubated overnight at 37℃ with shaking to activate the bacterial strains. After activation, 200 µL of the culture was inoculated into 100 mL of Luria Broth (LB) liquid medium and incubated at 37℃ with shaking. 2 mL of the bacterial culture was then analyzed using a microplate reader. The absorbance value (OD) was recorded. 600 When the concentration reaches 0.5, stop the culture. Take 1 mL of bacterial suspension from the liquid culture medium and centrifuge at 8000 rpm at 4 ℃ for 10 min. Collect the supernatant to obtain the bacterial extracellular secretory protein mixture (CEM). Resuspend the bacterial particles in Elution buffer (5 M NaCl, 1 M Tris (pH=7.5), 0.5 M EDTA (pH=8)). Sonicate the resuspended bacterial suspension in an ultrasonicator for 30 s, then cool it on ice for 2 min. Repeat the above operation 6 times. Then centrifuge at 4 ℃ at 8000 rpm for 10 min. The supernatant after centrifugation is the bacterial intracellular secretory protein mixture (CIM).

[0026] Example 3 Characterization of deoxyribonuclease fluorescent probes 1. In the sensitivity study of the probe's response to the target, the prepared E. coli CIM stock solution (a mixture of bacterial intracellular secretory proteins) was serially diluted 5 times with sterile water at a 10-fold gradient, so that the final system represented bacterial counts of 10⁻¹⁰ and 10⁻¹⁰, respectively. 2 10 3 10 4 10 5 10 6CFU / mL. Six identical reaction systems were prepared in black 96-well microplates, including 50 µL of reaction buffer (2×RB), 25 μL of a previously prepared known concentration of bacterial intracellular secretory protein mixture (CIM-EC) at different concentrations, 10 pmol of the *E. coli* deoxyribonuclease fluorescent probe RFD-EC1 from Example 1, and sterile water to a final volume of 100 µL. Real-time fluorescence was measured using a microplate reader (test conditions: excitation light 485±20 nm, emission light 520±10 nm). The fluorescence generation principle is that the bacterial intracellular secretory protein mixture (CIM-EC) acts as a target, contacting the probe and causing it to break at the RNA, thus separating the fluorescent group (FAM) from the quenching group (Dabcyl), thereby causing the fluorescent group (FAM) to fluoresce. Reaction buffer composition (2×RB, 50 mL): 100 mM HEPES, 30 mM MgCl2, 300 mM NaCl, pH=7.5. The sensitivity of the *E. coli* deoxyribonuclease fluorescent probe RFD-EC1 is as follows: Figure 1 As shown.

[0027] 2. In the study of the selectivity of the probe against different bacteria, six identical reaction systems were prepared in a black 96-well microplate, including 50 μL of 2×RB, 10 pmol of the *E. coli* deoxyribonuclease fluorescent probe RFD-EC1 from Example 1, and 25 μL of a mixture of intracellular secreted proteins (CIM) secreted by different bacteria (*Escherichia coli*, *Klebsiella pneumoniae*, *Burkholderia cocovenenans*, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Bacillus globigii*) were added to each system. Sterile water was added to bring the volume to 100 µL, and real-time fluorescence detection was performed using a microplate reader (test conditions as above). The specificity detection results of the *E. coli* deoxyribonuclease fluorescent probe RFD-EC1 are as follows: Figure 2 As shown.

[0028] Example 4: Determining the Microfluidic Droplet Generation Chip Structure The schematic diagram of the PDMS microdroplet generation chip channel structure used in this invention is shown below. Figure 3As shown, the microfluidic droplet generation chip mainly includes a first aqueous phase inlet 1, a second aqueous phase inlet 2, an oil phase inlet 6, a filter structure 3, a fluid stabilization structure 5, a mixing structure 4, a droplet generation structure, and a droplet outlet 7. The first aqueous phase inlet 1 and the second aqueous phase inlet 2 are connected by connecting pipes and then connected to the inlet I of the droplet generation structure through pipes. The oil phase inlet 6 is connected to the inlet II of the droplet generation structure through a pipe. The outlet I of the droplet generation structure is connected to the droplet outlet 7. The filter structure 3 and the mixing structure 4 are respectively installed on the connecting pipes of the first aqueous phase inlet 1 and the second aqueous phase inlet 2. The fluid stabilization structure 5 is installed on the connecting pipe of the oil phase inlet 6. The three structural regions of the filter structure 3, the fluid stabilization structure 5, and the mixing structure 4 are interconnected by pipes to form a microfluidic chip that can stably and rapidly generate droplets. This achieves the prevention of chip clogging, stable droplet generation, and uniform mixing of fluid components. It realizes multifunctional integration in a limited space, reduces the size of the chip, and is suitable for miniaturization and portability requirements. The chip contains three inlets and one droplet outlet 7. The three inlets are an oil phase inlet 6, a first aqueous phase inlet 1, and a second aqueous phase inlet 2. The pretreated water sample to be tested, bacterial lysate, deoxyribozyme probe, and reaction buffer together form the dispersed phase, with the oil phase serving as the mobile phase. Shear force breaks the dispersed phase into uniform droplets, which are encapsulated within the droplets at the cross-shaped structure. A buffer region is also present in the chip to stabilize the liquid flow rate. The droplet generation structure of this chip adopts a cross-shaped structure, utilizing the principle of flow focusing to generate droplets, which flow out from the droplet outlet 7. The chip channel width is 50 µm, used to generate droplets with a diameter of 50 µm.

[0029] Example 5 Pathogen Detection System This embodiment demonstrates the development of a pathogen detection system through modularization and integration of equipment. A schematic diagram of the modular connection is shown below. Figure 4 The overall structural diagram of the system integration is shown below. Figure 5The actual water sample container 8 and the reaction buffer container 9 are connected to the inlet of the mixing tank via pipes. The outlet of the mixing tank is connected to the first aqueous phase inlet 1 of the microfluidic droplet generation chip 15. The pathogen lysis buffer container 10 and the deoxyribozyme probe container 11 are connected to the inlet of the mixing tank via pipes. The outlet of the mixing tank is connected to the second aqueous phase inlet 1 of the microfluidic droplet generation chip 15. A pressure pump 13 is installed on the upstream pipe of the mixing tank, and a flow sensor 14 is installed on the downstream pipe of the mixing tank. The droplet oil container 12 is connected to the microfluidic droplet generation chip 15 via a pipe. The oil phase inlet 6 of the microfluidic droplet generation chip 15 is connected to the droplet oil container 12. A pressure pump 13 and a flow sensor 14 are installed on the connecting pipe between the droplet oil container 12 and the oil phase inlet 6. The droplet outlet 7 of the microfluidic droplet generation chip 15 is connected to the inlet of the capillary-laser induced fluorescence detector 16. The outlet of the capillary-laser induced fluorescence detector 16 is connected to the waste liquid pool 17. The oscilloscope 18 is connected to the capillary-laser induced fluorescence detector 16 and the signal analysis device 19 respectively, and collects fluorescence signals and transmits them to the signal analysis device 19 for analysis. The pressure pump 13 is controlled by the flow sensor 14 via computer software to apply pressure, thereby enabling the automatic injection of actual water samples, deoxyribozyme probes, droplet oil, pathogen lysis buffer, and reaction buffer. After the relevant reagents enter the mixing tank, they form a first aqueous phase and a second aqueous phase, which, together with the oil phase, enter the microfluidic droplet generation chip 15 under the pressure of the pressure pump to stably generate droplets. After droplet generation, the pressure is reduced and the droplets are finally discharged into the waste liquid tank 17 through the capillary-laser induced fluorescence detector 16. The fluorescence signal is collected by the oscilloscope 18 and transmitted to the signal analysis device 19 for analysis.

[0030] Example 6: Determining the detection threshold To support subsequent detection of pathogens in actual water samples, signal analysis is required to correctly distinguish between positive and negative droplets. An RFD-EC1 probe at a concentration of 200 nM was used. Pure negative samples contained only the probe, with no target; pure positive samples were obtained by treating the completely broken RFD-EC1 probe with 5 M NaOH. The system described in Example 4 was used to collect 400,000 and 800,000 droplets from negative and positive samples, respectively, for fluorescence signal analysis. The fluorescence signal results for positive and negative droplets are shown below. Figure 6 and Figure 7 As shown, the fluorescence signal of negative droplets is below 40 mV, and the fluorescence signal of positive droplets is approximately 900 mV. However, the probe cannot completely break during actual sample testing. Therefore, droplets with a fluorescence signal above 200 mV when testing actual samples were ultimately determined to be positive droplets.

[0031] Example 7: Systematic detection of different concentrations of Escherichia coli CIM-EC (a mixture of intracellular secretory proteins from E. coli) was serially diluted 5 times with sterile water in a 10-fold gradient, so that the final system represented bacterial counts of 10⁻⁶, 10⁻⁶, and 10⁻⁶, respectively. 2 10 3 10 4 10 5 10 6 10 7 CFU / mL. The mixture was prepared using the automated sample introduction device of the system described in Example 4: the first aqueous phase consisted of 200 µL of the above-mentioned concentration gradients of CIM-EC (a mixture of intracellular secreted proteins from *E. coli*), and the second aqueous phase consisted of 200 µL (80 pmol) of the deoxyribonuclease probe RFD-EC1 and 200 µL of reaction buffer 2×RB (100 mM HEPES, 30 mM MgCl2, 300 mM NaCl, pH=7.5). The flow rates of the aqueous and oil phases were set to 10 µL / min and 30 µL / min respectively using computer software, thereby controlling the pressure pump to achieve liquid flow. Simultaneously, the flow rates of the three phases were monitored in real time using flow sensors, and the fluid flow stability, droplet size, and stability within the microfluidic droplet generation chip were observed in real time using a microscope. After all the sample droplets entered the capillary, the flow rates were adjusted to 1 µL / min for the aqueous phase and 5 µL / min for the oil phase, allowing the droplets to move very slowly within the capillary for incubation. This allowed for a 30-minute reaction time for the bacterial lysis and target release, and for the probe to react with the target to generate fluorescence. After the reaction, the flow rates were controlled at 10 µL / min for the aqueous phase and 30 µL / min for the oil phase, allowing the droplets to enter the capillary portion of the laser-induced fluorescence detector. Finally, the fluorescence signal from the droplets generated in the water sample was collected. After each system was tested, the first aqueous phase was replaced with sterile water to clean the channel. The detection process was repeated, ultimately generating a standard curve relating the concentration gradient of *E. coli* in the sample to the number of positive droplets, as shown in the figure. Figure 8 As shown.

[0032] Example 8: Detection of Escherichia coli in actual water samples using a system The first aqueous phase consisted of 200 µL of pretreated water sample to be tested and 200 µL of reaction buffer 4×RB (200 mM HEPES, 60 mM MgCl2, 600 mM NaCl, pH=7.5). The second aqueous phase consisted of 200 µL (80 pmol) of deoxyribozyme probe RFD-EC1 and 100 µL of SDS bacterial lysis buffer. The flow rates of the aqueous and oil phases in the system of Example 4 were set to 10 µL / min and 30 µL / min, respectively, by computer software, thereby controlling the pressure pump to apply pressure to achieve liquid flow. At the same time, the flow rate of the three channels was monitored in real time by a flow sensor, and the fluid flow stability, droplet size and stability in the microfluidic droplet generation chip were observed in real time by microscope. After all the droplets generated from the actual sample entered the capillary, the flow rates were adjusted to 1 µL / min for the aqueous phase and 5 µL / min for the oil phase, allowing the droplets to move very slowly within the capillary for incubation. This allowed for a 30-minute reaction time for the two processes: bacterial lysis and target release, and the probe reacting with the target to generate fluorescence. After the reaction, the flow rates were controlled at 10 µL / min for the aqueous phase and 30 µL / min for the oil phase, allowing the droplets to enter the capillary portion of the laser-induced fluorescence detector. To prevent droplet accumulation and signal detection errors, sheath fluid was introduced to ensure the droplets passed through the detection window in an orderly fashion. This ultimately enabled the acquisition of fluorescence signals from the droplets generated from the actual water sample, allowing analysis of the presence and quantity of *E. coli* in the actual water sample.

[0033] Figure 9 The results and peaks collected during a 1-second test of an actual water sample are shown. One positive peak was observed, indicating the presence of E. coli in the actual water sample. The concentration of E. coli in the water sample can be determined based on the total number of peaks and the number of positive peaks. Figure 10 The above-mentioned positive peaks were magnified, and four peaks were collected within 0.002 s, including three negative droplet fluorescence signal peaks and one positive droplet fluorescence signal peak.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A deoxyribozyme probe, characterized in that, The deoxyribozyme probe is connected by nucleotide sequences shown in SEQ ID NO. 1-3, wherein R represents RNA base A, F represents T base modified with a fluorescent group, and Q represents T base modified with a quenching group.

2. The deoxyribozyme probe of claim 1, wherein, The fluorescent group is FAM, and the quenching group is Dabcyl.

3. A droplet microfluidic detection system for detecting pathogenic bacteria based on the deoxyribozyme probe of claim 1 or 2, characterized in that, The detection system mainly comprises an automatic sampling device, a microfluidic droplet generation chip, a capillary-laser-induced fluorescence detector and a signal acquisition and analysis device.

4. The detection system of claim 3, wherein, The detection system mainly comprises a first water phase sampling port, a second water phase sampling port, an oil phase sampling port, a droplet outlet, an actual water sample container, a reaction buffer container, a pathogenic bacteria lysis liquid container, a deoxyribozyme probe container, a droplet oil container, a pressure pump, a flow sensor, a microfluidic droplet generation chip, a capillary-laser-induced fluorescence detector, a waste liquid pool, an oscilloscope and a signal analysis device. The microfluidic droplet generation chip mainly comprises a first water phase sampling port, a second water phase sampling port, an oil phase sampling port, a filtering structure, a fluid stabilizing structure, a mixing structure, a droplet generation structure and a droplet outlet. The actual water sample container and the reaction buffer container are connected to the inlet of the mixing pool through pipelines, and the outlet of the mixing pool is connected to the first water phase sampling port of the microfluidic droplet generation chip; the pathogenic bacteria lysis liquid container and the deoxyribozyme probe container are connected to the inlet of the mixing pool through pipelines, and the outlet of the mixing pool is connected to the second water phase sampling port of the microfluidic droplet generation chip; a pressure pump is arranged on the upstream pipeline of the mixing pool, and a flow sensor is arranged on the downstream pipeline of the mixing pool; the droplet oil container is connected to the oil phase sampling port of the microfluidic droplet generation chip through a pipeline, and a pressure pump and a flow sensor are arranged on the connecting pipeline between the droplet oil container and the oil phase sampling port; the droplet outlet of the microfluidic droplet generation chip is connected to the inlet of the capillary-laser-induced fluorescence detector, the outlet of the capillary-laser-induced fluorescence detector is connected to the waste liquid pool, and the oscilloscope is connected to the capillary-laser-induced fluorescence detector and the signal analysis device.

5. The detection system of claim 4, wherein, The connecting pipelines of the first water phase sampling port and the second water phase sampling port are respectively provided with filtering structures and mixing structures, and the connecting pipeline of the oil phase sampling port is provided with a fluid stabilizing structure.

6. Use of the deoxyribozyme probe according to claim 1 or 2 or the detection system according to any one of claims 3 to 5 in a biosensor, characterized in that, The deoxyribozyme probe is used for specifically recognizing pathogenic bacteria.

7. Use according to claim 6, characterized in that, The pathogenic bacteria include Escherichia coli.

8. Use according to claim 6, characterized in that, The detection system specifically recognizes the E. coli in the following process: a pressure pump is controlled by a flow sensor to apply pressure, and actual water sample, deoxyribozyme probe, droplet oil, pathogenic bacteria lysate and reaction buffer are automatically injected respectively; the actual water sample and the reaction buffer form a first water phase, and the deoxyribozyme probe and the pathogenic bacteria lysate form a second water phase; the two water phases and the oil phase are introduced into a microfluidic droplet generation chip through the pressure pump; droplets are stably generated; the droplets are discharged into a waste liquid pool after being detected by a capillary-laser-induced fluorescence detector; and a fluorescence signal is collected by an oscilloscope and transmitted to a signal analysis device for analysis.

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