A live and dead bacteria dual fluorescence discrimination method based on ATP-responsive ZIF-8 nanocarriers

Abstract

The present application relates to the field of microorganism detection and nanobiosensing technology, and discloses a kind of live and dead bacteria dual fluorescence discrimination method based on ATP response type ZIF-8 nano-carrier.The method first constructs magnetic bead capture probe carrying horseradish peroxidase and HEX labeled aptamer, and the signal of aptamer is blocked by complementary short chain ssDNA, for representing total bacterial content.At the same time, construct ZIF-8 nano-probe loaded with indole acetic acid and coated with polydopamine, vancomycin modified, and form complex with target microorganism, under the action of 808 nm excitation, promote live bacteria to crack and release intracellular ATP, induce nano-carrier to disintegrate and release indole acetic acid, produce ROS fluorescence signal under the catalysis of horseradish peroxidase, for representing live bacterial content.The method can realize the synchronous quantitative analysis of target microorganism total bacteria and live bacteria in the liquid sample to be measured and the discrimination of live and dead bacteria, has the advantages of simple detection process, rapid response, high specificity and high sensitivity, realizes in-situ detection of target microorganism.

Description

Technical Field

[0001] This invention relates to the field of microbial detection and nanobiosensing technology, specifically to a dual-fluorescence method for distinguishing live and dead bacteria based on nucleic acid aptamer-specific recognition, ATP response release, and enzyme catalytic signal amplification. This method can be used for the simultaneous quantitative analysis of total and live bacteria of target microorganisms in complex liquid samples. Background Technology

[0002] Staphylococcus aureus is a common foodborne pathogen and a clinically relevant microorganism. Among existing microbial detection methods, the traditional plate count method, while highly accurate, has a long detection cycle, making it difficult to meet the needs of rapid detection; detection methods based on immune recognition and nucleic acid amplification, although possessing high specificity and sensitivity, often struggle to distinguish between live and dead bacteria, easily leading to biases in evaluating sterilization effectiveness or assessing actual contamination risk; ATP-related detection methods can quickly reflect the state of live bacteria, but lack the ability to specifically identify target microorganisms.

[0003] Therefore, there is an urgent need to establish a detection system that combines specific capture capability and live bacteria response capability, so as to achieve simultaneous quantification and identification of total and live bacteria of target microorganisms in the same system. Summary of the Invention

[0004] This invention aims to address the problems of existing microbial detection methods, such as difficulty in simultaneously identifying total and live bacteria, cumbersome detection procedures, long detection cycles, significant interference from complex sample matrices, and the need to improve detection specificity and sensitivity. It provides a dual-fluorescence method for identifying live and dead bacteria based on an ATP-responsive ZIF-8 nanocarrier. This method integrates magnetic separation and enrichment, aptamer-specific recognition, short-chain ssDNA signal blocking and competitive substitution, ATP-responsive release, and enzyme-catalyzed signal amplification into a single detection system, enabling simultaneous quantitative analysis of total and live bacteria of the target microorganisms in the liquid sample.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a dual-fluorescence method for distinguishing live and dead bacteria based on ATP-responsive ZIF-8 nanocarriers, comprising the following steps: (1) Streptavidin magnetic beads were co-incubated with biotinylated horseradish peroxidase and aptamers modified with both HEX fluorescent groups and biotin groups to obtain a total bacterial capture probe MB@HRP@Apt; then, a short ssDNA partially complementary to the aptamer was hybridized with the aptamer to obtain a total bacterial capture and signal blocking probe MB@HRP@Apt / cDNA; (2) The MB@HRP@Apt / cDNA is added to the test liquid sample containing the target microorganism and incubated. The aptamer specifically binds to the target microorganism and competitively replaces the short ssDNA to form the MB@HRP@Apt-target microorganism complex, so as to achieve specific separation and enrichment of the total bacteria of the target microorganism. (3) ZIF-8@IAA was prepared using zinc acetate, 2-methylimidazole and indoleacetic acid as raw materials. Dopamine was self-polymerized and coated on the surface of ZIF-8@IAA under alkaline conditions to obtain ZIF-8@IAA@PDA. Vancomycin was then modified on the surface of ZIF-8@IAA@PDA to obtain the live bacteria quantitative probe ZIF-8@IAA@PDA@VAN. (4) Mix the MB@HRP@Apt-target microbial complex obtained in step (2) with the live bacteria quantitative probe obtained in step (3) and incubate to form MB@HRP@Apt-target microbial-ZIF-8@IAA@PDA@VAN complex; (5) The complex obtained in step (4) is subjected to signal detection. The total bacterial content of the target microorganism is characterized by the HEX fluorescence signal recovered after the short chain ssDNA is competitively replaced. The target live bacteria are lysed by 808 nm light to release intracellular ATP. The released ATP induces the disintegration of ZIF-8@IAA@PDA@VAN and releases indoleacetic acid. The indoleacetic acid generates ROS fluorescence signal under the catalysis of horseradish peroxidase. The content of live bacteria of the target microorganism is characterized by the ROS fluorescence signal, thereby realizing the identification of live and dead bacteria of the target microorganism in the liquid sample to be tested.

[0006] The biotin-modified HEX-aptor can specifically recognize the target microorganism, and its recognition site is different from the action site of the vancomycin-modified probe on the surface of the target microorganism, so as to avoid site competition and improve detection accuracy.

[0007] The aptamer has a different recognition site on the surface of the target microorganism than vancomycin.

[0008] The method described herein is not for disease diagnosis and treatment purposes.

[0009] Further, the sequence of the biotinylated HEX-aptamer in step (1) is SEQ ID No. 1, with a HEX fluorescent group attached to the 5' end of the sequence and a biotin group attached to the 3' end of the sequence, namely 5'-HEX-GCAATGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-TEG-Biotin-3'.

[0010] Furthermore, the complementary short ssDNA sequence (SEQ ID No. 2) corresponding to the aptamer is: 5'-GTACCGTACCATTGC-3'.

[0011] The short-chain ssDNA can partially hybridize with the aptamer to pre-block the HEX fluorescence signal of the aptamer; when the target microorganism is present, the short-chain ssDNA dissociates from the aptamer under competitive binding, thereby restoring the HEX fluorescence signal, which is positively correlated with the total bacterial concentration of the target microorganism.

[0012] Further, step (1) includes: washing the streptavidin magnetic beads three times with PBST to remove impurities, and resuspending them to obtain a streptavidin magnetic bead dispersion; then mixing the streptavidin magnetic bead dispersion with biotinylated horseradish peroxidase and biotinylated HEX-aptamer, and incubating at 37°C in the dark to load the biotinylated horseradish peroxidase and biotinylated HEX-aptamer onto the surface of the streptavidin magnetic beads; after incubation, washing with PBST to remove unbound free enzymes and free aptamers, and resuspending them to obtain a total bacterial capture probe MB@HRP@Apt dispersion.

[0013] Further, step (2) includes: mixing and incubating the liquid sample containing the target microorganism with the MB@HRP@Apt dispersion to allow the MB@HRP@Apt to specifically bind with the target microorganism; after incubation, performing magnetic separation using a magnetic rack, discarding the supernatant, and obtaining the MB@HRP@Apt-target microorganism complex.

[0014] Further, step (3) includes: dissolving anhydrous zinc acetate in a methanol-water mixed solvent and rapidly adding it to a methanol-water mixed solution containing 2-methylimidazole and indoleacetic acid, stirring the reaction at room temperature, and obtaining ZIF-8@IAA after centrifugation, washing and drying; dispersing the obtained ZIF-8@IAA in anhydrous ethanol, adding dopamine hydrochloride and adding Tris-HCl buffer, reacting under alkaline conditions to deposit polydopamine on the surface of ZIF-8@IAA, and obtaining ZIF-8@IAA@PDA after centrifugation and washing; dispersing the obtained ZIF-8@IAA@PDA and adding vancomycin, and continuing the reaction in a Tris-HCl buffer system, and obtaining the final product ZIF-8@IAA@PDA@VAN after centrifugation, washing and drying.

[0015] Further, step (4) includes: dispersing ZIF-8@IAA@PDA@VAN in ultrapure water, adding it to the MB@HRP@Apt-target microbial complex obtained in step (2), and mixing it by shaking to form the MB@HRP@Apt-target microorganism-ZIF-8@IAA@PDA@VAN complex.

[0016] Further, step (5) includes: irradiating the complex with an 808 nm light source to cause the target viable bacteria to lyse and release intracellular ATP; the released ATP induces the ZIF-8@IAA@PDA@VAN to disintegrate and release indoleacetic acid, which generates ROS fluorescence signal by horseradish peroxidase on the surface of the magnetic beads in the local reaction space; establishing standard curves between HEX fluorescence signal and total bacterial concentration of target microorganism, and between ROS fluorescence signal and viable bacterial concentration of target microorganism, respectively, thereby obtaining the total bacterial concentration and viable bacterial concentration of target microorganism in the liquid sample to be tested.

[0017] Furthermore, the HEX fluorescence signal is used to characterize the total bacterial content of the target microorganism in the sample, the ROS fluorescence signal is used to characterize the viable bacterial content of the target microorganism in the sample, and the dead bacterial content of the target microorganism is characterized by the difference between the total bacterial content and the viable bacterial content.

[0018] Furthermore, the target microorganism is a pathogen or a foodborne pathogen, preferably Staphylococcus aureus.

[0019] In this invention, ATP, as an important intracellular marker of lysis and release from live bacteria, can serve as a response factor triggering the disintegration of the ZIF-8 nanocarrier. Since the intracellular ATP content of dead bacteria is significantly reduced or essentially lost, it is difficult to effectively trigger the disintegration of the live bacteria quantitative probe and the subsequent ROS signal amplification response. Based on this, this invention can further achieve accurate determination of live bacteria content based on total bacterial count.

[0020] In this invention, the total bacteria capture probe and the live bacteria quantitative probe employ different recognition mechanisms. The HEX-aptamer is used for total bacteria recognition of the target microorganism, while the vancomycin-modified probe is used for live bacteria quantitative recognition. The two probes have different binding sites, which can avoid site competition during the recognition process and improve the capture efficiency and detection accuracy of the target microorganism.

[0021] In this invention, a magnetic bead enrichment unit, an ATP-responsive nanocarrier unit, and an enzyme-catalyzed signal amplification unit together constitute a composite detection system. The magnetic bead enrichment unit enables rapid separation and enrichment of target microorganisms in the sample; the ATP-responsive nanocarrier unit specifically responds to ATP released from the lysis of live bacteria; and the enzyme-catalyzed signal amplification unit converts ATP-triggered events into detectable ROS fluorescence signals, thereby significantly improving the sensitivity of live bacteria detection.

[0022] In this invention, the target microorganism is preferably a pathogenic bacterium or a foodborne pathogen, and more preferably Staphylococcus aureus. For different target microorganisms, the method can be expanded simply by changing the corresponding specific aptamer; therefore, this invention has good versatility and application prospects.

[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) By constructing a total bacteria detection channel and a live bacteria detection channel, the present invention can realize the simultaneous detection of total bacteria and live bacteria of the target microorganism in the same detection system, thereby making up for the shortcomings of the prior art that total bacteria and live bacteria need to be detected separately.

[0024] (2) The present invention adopts a dual recognition mode that combines magnetic bead aptamer specific capture with vancomycin directional recognition, which can improve the separation and enrichment efficiency of target microorganisms and effectively reduce non-specific interference caused by complex sample matrix, thereby improving the specificity and accuracy of the detection method.

[0025] (3) This invention utilizes the release of ATP from the lysis of live bacteria to trigger the disintegration of ATP-responsive ZIF-8 nanocarriers and release indoleacetic acid, which generates ROS fluorescence signal under the catalysis of horseradish peroxidase. This realizes the transformation from the change of live bacteria state to the output of fluorescence signal. The detection mechanism is clear, the response speed is fast, and the sensitivity is high.

[0026] (4) This invention integrates magnetic separation, specific recognition, ATP response release and enzyme catalytic amplification into the same system. The detection process is simple and suitable for detecting target microorganisms in liquid samples. It has good application and promotion value. Attached Figure Description

[0027] Figure 1 This is a schematic diagram illustrating the principle of the method used in a preferred embodiment of the present invention; Figure 2 This is a TEM characterization image of the ZIF-8 nanocarrier obtained in a preferred embodiment of the present invention; Figure 3 This is a SEM image of the ZIF-8 nanocarrier obtained in a preferred embodiment of the present invention. Figure 4 This is a SEM image verifying the material's ATP-responsive disintegration in a preferred embodiment of the present invention. Figure 5 This is a photothermal temperature rise curve diagram in a preferred embodiment of the present invention; Figure 6 This is a graph showing the response results of dual-channel fluorescence to live bacteria, dead bacteria, and samples with different mixing ratios in a preferred embodiment of the present invention. Figure 7 This is a diagram showing the specificity evaluation results in a preferred embodiment of the present invention; Figure 8 The method in the preferred embodiment of the present invention is applied to different concentrations (1.0 × 10⁻⁶). 2 ~1.0×10 8 The image shows the detection results of Staphylococcus aureus (CFU / mL). Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or conventional adjustments made to the conditions of each step, reagent dosage, reaction time, or detection parameters using the technical concept of the present invention shall fall within the scope of protection of the present invention.

[0029] This invention provides a dual-fluorescence method for distinguishing between live and dead bacteria based on an ATP-responsive ZIF-8 nanocarrier. This method pertains to microbial detection for purposes other than disease diagnosis and treatment. In this embodiment, Staphylococcus aureus is used as the target microorganism for illustration; for other pathogens or foodborne pathogens, simply changing the corresponding specific aptamer allows for the widespread application of this method.

[0030] In this invention, the total bacterial capture probe is MB@HRP@Apt, and the live bacterial response probe is ZIF-8@IAA@PDA@VAN. MB@HRP@Apt is composed of streptavidin magnetic beads, biotinylated horseradish peroxidase, and a biotinylated HEX-labeled aptamer, used for specific recognition of target microorganisms, magnetic separation and enrichment, and output of total bacterial fluorescence signals. ZIF-8@IAA@PDA@VAN is composed of ZIF-8 nanocarriers loaded with indoleacetic acid, a polydopamine photothermal layer, and a vancomycin recognition layer, used for surface binding, photothermal lysis, ATP-responsive disintegration, and enzyme-catalyzed fluorescence signal amplification of target live bacteria. A schematic diagram of the principle of this invention is shown below. Figure 1 Specifically, in the absence of the target microorganism, complementary short-chain ssDNA partially hybridizes with the HEX-labeled aptamer, resulting in HEX fluorescence being blocked or at a low signal level. When the target microorganism is present, it specifically binds to the aptamer, causing the complementary short-chain ssDNA to competitively dissociate from the aptamer, restoring the HEX fluorescence signal. This signal is used to characterize the total bacterial count of the target microorganism. Subsequently, ZIF-8@IAA@PDA@VAN binds to the target microorganism's cell wall-related sites via vancomycin. After irradiation with 808 nm near-infrared light, PDA generates a photothermal effect, causing viable bacteria to lyse and release intracellular ATP. ATP further induces the disintegration of the ZIF-8 backbone, releasing indoleacetic acid (IAA). The released IAA generates a ROS fluorescence signal under the localized catalytic action of HRP on the magnetic bead surface. This signal is used to characterize the viable bacterial count of the target microorganism. By jointly analyzing the total bacterial count signal and the viable bacterial count signal, dual fluorescence discrimination of viable and dead target microorganisms can be achieved.

[0031] Example 1: A dual-fluorescence method for distinguishing live and dead bacteria based on ATP-responsive ZIF-8 nanocarriers The main raw materials used in this embodiment include: streptavidin magnetic beads, biotinylated horseradish peroxidase, biotinylated HEX-aptamer, anhydrous zinc acetate, 2-methylimidazole, indoleacetic acid, dopamine hydrochloride, vancomycin, PBS buffer, PBST buffer, Tris-HCl buffer, and ROS fluorescent probe. The biotinylated HEX-aptamer can specifically recognize Staphylococcus aureus, and its recognition site differs from the action site of vancomycin on the target bacterial surface.

[0032] 1. Preparation of target microbial samples 100 μL of Staphylococcus aureus stock solution was inoculated into the corresponding liquid culture medium and cultured overnight at 35°C with shaking at 150 rpm. After incubation, the optical density of the bacterial culture at 600 nm was measured, and the OD600 was adjusted to 1.0, corresponding to a bacterial concentration of approximately 10⁻⁶. 9 CFU / mL. The bacterial culture was then centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the culture was washed three times with sterile PBS. It was then resuspended in sterile PBS and diluted to the appropriate concentration as needed. The bacterial concentration was determined using the agar plate gradient dilution method.

[0033] 2. Preparation of total bacterial capture probe MB@HRP@Apt In this embodiment, the biotinylated HEX-labeled aptamer can specifically recognize Staphylococcus aureus, and its sequence is as follows: 5'-HEX-GCAATGGGTACGGTACTTCCTCGGCACGTTCTCAGTAGCGCTCGCTGGTCATCCCACAGCTACGTCAAAAGTGCACGCTACTTTGCTAA-TEG-Biotin-3'.

[0034] The complementary short ssDNA sequence corresponding to the aptamer is as follows: 5'-GTACCGTACCATTGC-3'.

[0035] Take 100 μL of streptavidin magnetic beads (concentration 10 mg / mL), wash three times with PBST to remove impurities, and perform magnetic separation with the aid of a magnetic rack. After washing, resuspend the beads in 1 mL of PBST. Then add 0.5 μL of biotinylated horseradish peroxidase (concentration 2 mg / mL) and 200 μL of biotinylated HEX-aptamer-ssDNA (concentration 1 μM) to the system. Incubate the mixture at 37°C in the dark for 30 min to allow the biotinylated horseradish peroxidase and biotinylated HEX-aptamer to be co-loaded on the surface of the streptavidin magnetic beads. After incubation, wash three more times with PBST to remove unbound free enzyme and free aptamer. Finally, resuspend the obtained product in 1 mL of PBST to obtain a 1 mg / mL MB@HRP@Apt dispersion for later use.

[0036] 3. Preparation of live bacteria quantitative probe ZIF-8@IAA@PDA@VAN 5.5 g of anhydrous zinc acetate was dissolved in 200 mL of a methanol-water mixture with a methanol-water volume ratio of 9:1. Separately, 49.3 g of 2-methylimidazole and 1.05 g of indoleacetic acid were dissolved in 300 mL of a methanol-water mixture, also with a methanol-water volume ratio of 9:1. The anhydrous zinc acetate solution was then rapidly added to the mixture containing 2-methylimidazole and indoleacetic acid, and the mixture was magnetically stirred at room temperature for 12 h. After the reaction was complete, the mixture was centrifuged at 9500 rpm for 10 min, and the resulting white product was collected. This product was washed three times with pre-cooled pure methanol and dried at 60 °C to obtain ZIF-8@IAA.

[0037] The ZIF-8@IAA obtained above was dispersed in 100 mL of anhydrous ethanol, 100 mg of dopamine hydrochloride was added, and 200 mL of Tris-HCl buffer (pH 8.5) was added. The mixture was stirred at room temperature for 3 h to allow polydopamine to deposit on the surface of ZIF-8@IAA. After the reaction was complete, the mixture was centrifuged at 9500 rpm for 10 min, the black precipitate was collected, washed three times with anhydrous ethanol, and dried to obtain ZIF-8@IAA@PDA.

[0038] The obtained ZIF-8@IAA@PDA was then dispersed in 100 mL of anhydrous ethanol, 100 mg of vancomycin was added, and 200 mL of Tris-HCl buffer (pH 8.5) was added. The mixture was stirred at room temperature for 16 h. After the reaction was complete, the product was collected by centrifugation at 9500 rpm for 10 min, washed three times with anhydrous ethanol, and dried to obtain the final product ZIF-8@IAA@PDA@VAN.

[0039] 4. Detection of total bacterial fluorescence signal A liquid sample containing the target microorganism was placed in a 1.5 mL centrifuge tube, and 100 μL of a 1 mg / mL MB@HRP@Apt / cDNA dispersion was added. The sample was incubated under suitable conditions to allow the aptamer to specifically bind to the target microorganism. Since the specific binding between the target microorganism and the aptamer is stronger than the partial hybridization between the short-chain ssDNA and the aptamer, the short-chain ssDNA dissociates from the aptamer during competitive binding, thus releasing the blockade on the HEX fluorescence signal. After incubation, magnetic separation was performed for 5 min using a magnetic rack, and the supernatant was discarded to obtain the MB@HRP@Apt-target microorganism complex. The obtained complex was subjected to HEX fluorescence signal detection, preferably reading the HEX fluorescence emission signal at approximately 555 nm. The intensity of the HEX fluorescence signal is positively correlated with the total bacterial concentration of the target microorganism in the sample and is used to characterize the total bacterial content of the target microorganism.

[0040] 5. Detection of live bacteria fluorescence signals After obtaining the MB@HRP@Apt-target microbial complex, 100 μg of ZIF-8@IAA@PDA@VAN was dispersed in 1000 μL of ultrapure water. 100 μL of this water was then added to the MB@HRP@Apt-target microbial complex. After shaking and mixing, the live bacteria quantitative probe further bound to the surface of the target microorganism, forming the MB@HRP@Apt-target microorganism-ZIF-8@IAA@PDA@VAN complex. Since vancomycin can interact with cell wall-related sites of the target microorganism, and its binding site differs from the recognition site of the aptamer, this facilitates synergistic recognition by the dual probes.

[0041] Subsequently, the composite was irradiated with an 808 nm light source for 6 min. Because the ZIF-8@IAA@PDA@VAN surface contains a polydopamine layer, it exhibits a photothermal effect under 808 nm near-infrared light irradiation, causing the viable bacteria to lyse and release intracellular ATP. The released ATP induces the disintegration of the ZIF-8 backbone, promoting the release of indoleacetic acid contained within the ZIF-8 backbone.

[0042] Since MB@HRP@Apt has been enriched and immobilized on the surface of the target microorganism through magnetic separation, the released indoleacetic acid can undergo a catalytic coupling reaction with HRP on the surface of the magnetic beads in the local reaction space near the target microorganism, further inducing the generation of ROS signal.

[0043] A 10 mM stock solution of 2,7-dichlorodihydrofluorescein was prepared using anhydrous DMSO and then diluted with PBS to a 10 μM working solution. 1 mL of the working solution was added to 20 μL of the reaction sample irradiated at 808 nm, and the mixture was incubated at room temperature for 30 min in the dark. After incubation, fluorescence detection was performed, preferably reading the ROS-related fluorescence emission signal at approximately 500 nm. The intensity of the ROS fluorescence signal was positively correlated with the concentration of viable target microorganisms in the sample and was used to characterize the viable content of the target microorganisms.

[0044] In this embodiment, the HEX fluorescence signal is used to characterize the total bacterial count of the target microorganism in the test sample, and the ROS fluorescence signal is used to characterize the viable bacterial count of the target microorganism. By establishing standard curves for the HEX and ROS signals of standard samples with known concentrations of target microorganisms, the total bacterial count and viable bacterial count of the target microorganism in the test sample can be obtained, respectively. Furthermore, the dead bacterial count of the target microorganism can be characterized by the difference between the total bacterial count and the viable bacterial count, thereby achieving dual fluorescence discrimination of viable and dead target microorganisms.

[0045] Example 2: Performance Testing To verify the feasibility of the method of the present invention and to examine the morphology, ATP-responsive disintegration performance, near-infrared photothermal performance, live / dead bacteria discrimination ability, target microorganism specificity, and quantitative detection ability of the ZIF-8 nanocarrier, the following performance tests were conducted.

[0046] 1. Transmission electron microscopy characterization of ZIF-8@IAA@PDA@VAN nanocarriers An appropriate amount of the ZIF-8@IAA@PDA@VAN nanocarrier prepared in Example 1 was dispersed in ultrapure water, ultrasonically treated, and then dropped onto a copper grid of a transmission electron microscope. After natural drying, the nanocarrier was observed under a transmission electron microscope.

[0047] Figure 2 The TEM image shows the obtained ZIF-8@IAA@PDA@VAN nanocarriers. The results show that the nanocarriers exhibit a regular polyhedral morphology with clear particle outlines and intact structures, indicating that the method described in this invention can prepare functionalized nanocarriers with typical ZIF-8 structural characteristics.

[0048] 2. Scanning electron microscopy characterization of ZIF-8@IAA@PDA@VAN nanocarriers An appropriate amount of the ZIF-8@IAA@PDA@VAN nanocarrier prepared in Example 1 was dried and spread on the surface of the conductive adhesive. After being sputtered with gold, it was observed by scanning electron microscopy.

[0049] Figure 3The image shows the SEM characterization of the obtained ZIF-8@IAA@PDA@VAN nanocarrier. The results indicate that the nanocarrier possesses a relatively complete polyhedral structure with a certain degree of surface roughness, demonstrating that after encapsulation with indoleacetic acid, polydopamine coating, and vancomycin modification, the material maintains good particle morphology and structural integrity, providing a material basis for subsequent viable bacteria response detection.

[0050] 3. ATP response to disintegration performance test The ZIF-8@IAA@PDA@VAN prepared in Example 1 was dispersed in ultrapure water or PBS to prepare a material dispersion of 1 mg / mL. 100 μL of the material dispersion was taken, and ATP solution was added to bring the final ATP concentration in the system to 5 mM. The mixture was incubated at room temperature in the dark for 30 min. After incubation, the mixture was centrifuged at 6000 rpm for 5 min, the precipitate was collected, washed, dried, and observed under a scanning electron microscope.

[0051] Figure 4 This is a SEM image validating the material's ATP-responsive disintegration. The results show that after ATP treatment, the original regular polyhedral structure of ZIF-8@IAA@PDA@VAN was significantly disrupted, with particles collapsing, fragmenting, or irregularly accumulating. This indicates that ATP can induce the disintegration of the ZIF-8 framework, thereby promoting the release of indoleacetic acid encapsulated within it. This result demonstrates that the material possesses ATP-responsive release capability.

[0052] 4. Near-infrared photothermal heating performance test Blank group, ZIF-8@IAA group, ZIF-8@IAA@PDA group and ZIF-8@IAA@PDA@VAN group were set up respectively. The same volume and the same concentration of each group dispersion were taken and continuously irradiated with an 808 nm near-infrared light source. The temperature change of each group sample at different irradiation time was recorded using an infrared thermal imager.

[0053] Figure 5 The results show that the Blank group and the ZIF-8@IAA group showed relatively small temperature increases, while the ZIF-8@IAA@PDA group and the ZIF-8@IAA@PDA@VAN group showed significant temperature increases with irradiation time. This indicates that polydopamine modification endows the material with good near-infrared photothermal conversion capabilities. After vancomycin modification, the material still maintains good photothermal heating performance.

[0054] 5. Dual-channel response test for live and dead bacteria Live Staphylococcus aureus samples, dead samples, and mixed samples of different proportions of live and dead bacteria were prepared separately. Live samples were obtained from fresh bacterial culture that had been cultured and washed with PBS, while dead samples were obtained through heat treatment or ethanol treatment. After adjusting each group of samples to the same or similar concentrations, MB@HRP@Apt and ZIF-8@IAA@PDA@VAN were added sequentially according to the method described in Example 1 for detection, and the HEX and ROS fluorescence channels were read.

[0055] Figure 6 The image shows the response results of dual-channel fluorescence to live bacteria, dead bacteria, and samples with different mixing ratios. The results show that live bacteria samples produce a significant ROS live bacteria channel signal, while dead bacteria samples show a lower signal. As the proportion of live bacteria in the mixed sample increases, the ROS fluorescence signal correspondingly strengthens. These results demonstrate that the method of this invention can characterize the live bacteria content of target microorganisms through the ROS fluorescence channel and distinguish between live and dead bacteria.

[0056] 6. Method specificity test Select Staphylococcus aureus ( S. aureus ) was selected as the target bacterium, and Escherichia coli (E. coli) was also selected. E. coli Salmonella paratyphi B ( S. paratyphi B ), Pseudomonas aeruginosa ( P. aeruginosa Bacillus subtilis ( B. subtilis ), Enterococcus faecalis ( E. faecalis Staphylococcus epidermidis ( S. epidermidis ) and Proteus mirabilis ( P. mirabili s) as a non-target bacteria control. After adjusting each bacterial culture to the same or similar concentration, the detection was performed according to the method described in Example 1, and the HEX fluorescence channel and ROS fluorescence channel signals were recorded.

[0057] Figure 7 The figure shows the specificity evaluation results. The results indicate that the fluorescence response of the Staphylococcus aureus group was significantly higher than that of other non-target bacteria groups, while the fluorescence signals of all non-target bacteria groups were at a low level. This demonstrates that the method of this invention has good recognition specificity for Staphylococcus aureus and can reduce the interference of non-target bacteria on the detection results.

[0058] 7. Quantitative detection of Staphylococcus aureus at different concentrations Staphylococcus aureus bacterial suspensions, after plate counting calibration, were serially diluted to prepare concentrations ranging from 1.0 × 10² to 1.0 × 10⁻⁶. 8 Standard bacterial solutions of CFU / mL were collected. Standard bacterial solutions of different concentrations were tested according to the method described in Example 1. Fluorescence emission spectra and fluorescence intensity at characteristic wavelengths were collected, and a standard curve between bacterial solution concentration and fluorescence intensity was established.

[0059] Figure 8 The image shows the detection results of Staphylococcus aureus at different concentrations using the method of this invention. The results show that as the concentration of Staphylococcus aureus increases, the fluorescence signal of the detection system gradually strengthens, and within a certain concentration range, it shows a good correlation with the concentration of the target bacteria. This result indicates that the method of this invention can achieve quantitative detection of Staphylococcus aureus, with a wide detection range and good concentration response capability.

Claims

1. A dual-fluorescence method for distinguishing live and dead bacteria based on ATP-responsive ZIF-8 nanocarriers, characterized in that, Includes the following steps: (1) Streptavidin magnetic beads were co-incubated with biotinylated horseradish peroxidase and aptamers modified with both HEX fluorescent groups and biotin groups to obtain a total bacterial capture probe MB@HRP@Apt; then, a short ssDNA partially complementary to the aptamer was hybridized with the aptamer to obtain a total bacterial capture and signal blocking probe MB@HRP@Apt / cDNA; (2) The MB@HRP@Apt / cDNA is added to the test liquid sample containing the target microorganism and incubated. The aptamer specifically binds to the target microorganism and competitively replaces the short ssDNA to form the MB@HRP@Apt-target microorganism complex, so as to achieve specific separation and enrichment of the total bacteria of the target microorganism. (3) ZIF-8@IAA was prepared using zinc acetate, 2-methylimidazole and indoleacetic acid as raw materials. Dopamine was self-polymerized and coated on the surface of ZIF-8@IAA under alkaline conditions to obtain ZIF-8@IAA@PDA. Vancomycin was then modified on the surface of ZIF-8@IAA@PDA to obtain the live bacteria quantitative probe ZIF-8@IAA@PDA@VAN. (4) Mix the MB@HRP@Apt-target microbial complex obtained in step (2) with the live bacteria quantitative probe obtained in step (3) and incubate to form MB@HRP@Apt-target microbial-ZIF-8@IAA@PDA@VAN complex; (5) The complex obtained in step (4) is subjected to signal detection. The total bacterial content of the target microorganism is characterized by the HEX fluorescence signal recovered after the short chain ssDNA is competitively replaced. The target live bacteria are lysed by 808 nm light to release intracellular ATP. The released ATP induces the disintegration of ZIF-8@IAA@PDA@VAN and releases indoleacetic acid. The indoleacetic acid generates ROS fluorescence signal under the catalysis of horseradish peroxidase. The content of live bacteria of the target microorganism is characterized by the ROS fluorescence signal, thereby realizing the identification of live and dead bacteria of the target microorganism in the liquid sample to be tested. The aptamer has a different recognition site on the surface of the target microorganism than vancomycin. The method described is not for disease diagnosis or treatment purposes.

2. The method according to claim 1, characterized in that, Step (1) includes: (a) The streptavidin magnetic beads were washed with PBST to remove impurities and then resuspended in PBST to obtain a streptavidin magnetic bead dispersion for later use. (b) The streptavidin magnetic bead dispersion, the mixture of biotinylated horseradish peroxidase and biotinylated HEX-aptamer-ssDNA was incubated at 37°C in the dark for 30 min. The mixture was washed with PBST to remove unbound free probes and enzymes. Finally, it was resuspended in PBST to obtain MB@HRP@Apt dispersion. The biotinylated HEX-aptamer has the sequence SEQ ID No. 1, with a HEX fluorescent group attached to the 5' end of the sequence and a biotin group attached to the 3' end of the sequence. The sequence of the short ssDNA is SEQ ID No.

2.

3. The method according to claim 1, characterized in that, Step (2) includes: The liquid sample containing the target microorganism was placed in a 1.5 mL centrifuge tube, MB@HRP@Apt was added, and after incubation, magnetic separation was performed using a magnetic rack. The supernatant was removed to obtain the MB@HRP@Apt-target microorganism complex.

4. The method according to claim 1, characterized in that, Step (3) includes: (a) Anhydrous zinc acetate was dissolved in a methanol-water mixture and then rapidly added to a methanol-water mixture containing 2-methylimidazole and indoleacetic acid. The mixture was magnetically stirred at room temperature. The white product was then collected by centrifugation, washed with pre-cooled pure methanol, and dried to obtain ZIF-8@IAA. (b) Take the product obtained in step (a), disperse it in anhydrous ethanol, add dopamine hydrochloride, and add Tris-HCl buffer. After stirring at room temperature, centrifuge to collect the black precipitate ZIF-8@IAA@PDA, wash repeatedly with anhydrous ethanol and dry. (c) Disperse the obtained ZIF-8@IAA@PDA in anhydrous ethanol, add vancomycin and Tris-HCl buffer, stir at room temperature, collect by centrifugation, wash repeatedly with anhydrous ethanol and dry to obtain the final product ZIF-8@IAA@PDA@VAN.

5. The method according to claim 1, characterized in that, Step (4) includes: ZIF-8@IAA@PDA@VAN was added to ultrapure water. The ZIF-8@IAA@PDA@VAN dispersion was added to the MB@HRP@Apt-target microbial complex. After shaking and mixing, the MB@HRP@Apt-target microorganism-ZIF-8@IAA@PDA@VAN complex was formed.

6. The method according to claim 1, characterized in that, Step (5) includes: The MB@HRP@Apt-target microorganism-ZIF-8@IAA@PDA@VAN complex was irradiated with an 808 nm light source for 6 min, causing the target viable bacteria to lyse and release intracellular ATP. The released ATP induced the disintegration of ZIF-8@IAA@PDA@VAN and released indoleacetic acid. The indoleacetic acid was catalyzed by horseradish peroxidase on the surface of the magnetic beads to generate ROS fluorescence signal in the local reaction space. Standard curves were established between HEX fluorescence signal and total bacterial concentration of target microorganism, and between ROS fluorescence signal and viable bacterial concentration of target microorganism, respectively, to obtain the total bacterial concentration and viable bacterial concentration of target microorganism in the liquid sample to be tested.

7. The method according to claim 1, characterized in that, The short-chain ssDNA is used to block the signal of the aptamer, so that the aptamer is in a partially blocked state when it is not bound to the target microorganism; when the target microorganism is present, the short-chain ssDNA dissociates from the aptamer under competitive binding, thereby making the HEX fluorescence signal intensity positively correlated with the total bacterial concentration of the target microorganism.

8. The method according to any one of claims 1 to 7, characterized in that, The target microorganism is a pathogen or a foodborne pathogen.

9. The method according to claim 8, characterized in that, The target microorganism is Staphylococcus aureus.

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