Stable and efficient enzyme packaging method and application in bacterial broad-spectrum instant detection

Abstract

The invention discloses a preparation method of a polysaccharide-modified core-shell encapsulated enzyme MOF composite material and a real-time detection method for the broad-spectrum activity of bacteria. Through an elaborately designed heterogeneous accelerated nucleation strategy, the core-shell encapsulated enzyme MOF composite material with a polysaccharide outer layer is successfully prepared. The lectin modified by the magnetic beads is used for carrying out broad-spectrum capture on bacteria through PAMPs affinity. Furthermore, the MOF composite material is used as a competitive recognition source and a signal transduction source to participate in competitive combination. After one-step magnetic separation, the efficiently packaged enzyme and the substrate generate strong affinity, an enzyme catalysis signal with amplified signal is displayed, and the detection sensitivity is improved. And then, the mobile phone reading system analyzes the colorimetric signal on site, so that the capability of on-site quantitative analysis is improved. The enzyme encapsulation method disclosed by the invention can be used for synthesizing at normal temperature and normal pressure, and is more efficient and effectively shortened in time compared with a traditional enzyme encapsulation method. The bacteria detection method provided by the invention solves the problems that the existing bacteria detection method is long in time, is difficult to realize broad spectrum and still needs instruments and equipment.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for preparing a polysaccharide-modified core-shell enzyme encapsulated metal organic framework (MOF) composite material and a method for real-time detection of broad-spectrum bacteria. Background Art

[0002] Bacterial infection and contamination have become major pathogenic factors in public health and industrial production. At present, traditional bacterial detection methods, such as plate counts, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), are still mainly laboratory-based, requiring large-scale instruments and professional personnel to operate, and the detection time is long. The existing classic specific recognition technology relies on antibodies and aptamers, which can only detect a single type of bacterial infection in a sample at a time, while the development of the disease is related to mixed infection of multiple bacteria. In addition, in the industrial field, products must undergo rigorous screening for multiple bacterial contamination after production before they can be released. Therefore, developing a strategy that can respond immediately and has broad-spectrum detection capabilities will be an effective way to combat the serious threat of bacteria.

[0003] Metal-organic framework (MOF) materials are often used as carriers of biomolecules due to their advantages such as high specific surface area, excellent adsorption affinity, and good thermal stability. They are conducive to design and development in biological detection. With MOF as a carrier, the high specific surface area of ​​the outer layer is conducive to functional modification design, while internal encapsulation of enzymes can effectively protect the stability of the enzymes. Current encapsulation methods are limited by the slow efficiency of spontaneous nucleation, which is time-consuming and difficult to encapsulate efficiently. The activity of the enzyme is affected by the steric hindrance of the MOF carrier, and the limited electron transfer leads to the failure to enhance the enzyme activity or even inhibition, which restricts further development. In addition, the design of recognition and transduction based on MOF materials for broad-spectrum instant detection of bacteria is particularly important.

[0004] Pathogen-associated molecular patterns (PAMPs) are highly conserved molecular structures shared by bacteria, including peptidoglycans of Gram-positive bacteria and lipopolysaccharides of Gram-negative bacteria. In the immune response, pattern recognition receptors such as lectin receptors can quickly respond to the invasion of various bacteria by recognizing PAMPs. Since lipopolysaccharides and peptidoglycans do not exist in fungi, viruses, plants and mammals, PAMPs are considered to be specific biological indicators for bacterial identification and detection. At present, researchers have successfully used lectins to recognize PAMPs molecularly to achieve bacterial detection. By immobilizing lectins to capture polysaccharides in bacterial cell walls, the detection design of bacteria can be significantly improved. Therefore, the lectin-based PAMPs molecular recognition strategy can provide strong support for the transduction design of core-shell MOF-encapsulated enzymes, further realizing the instant detection of a wide spectrum of bacteria, which has broad significance. Summary of the Invention

[0005] The purpose of the present invention is first to overcome the shortcomings of the above-mentioned existing MOF encapsulation enzyme technology and provide a stable and efficient enzyme encapsulation core-shell MOF material and its preparation method. The core-shell MOF synthase composite material has excellent size uniformity, efficient cascade enzyme catalytic activity and good stability.

[0006] Secondly, polysaccharides modified with MOF synthase composites can be used for competitive recognition of bacterial capture. By combining a broad-spectrum bacterial capture magnetic probe with a 3D-printed device developed in conjunction with a smartphone readout system, this allows for instant, visual detection of a broad spectrum of bacteria on-site. This addresses the challenges of existing bacterial detection methods, which still require laboratory environments, instrumentation, and specialized personnel, resulting in lengthy testing times and the urgent need for broad-spectrum detection technology for rapid bacterial diagnosis and product release.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides a method for preparing a polysaccharide-modified core-shell encapsulated enzyme MOF material, using CTAB as a capping agent, Cyt C oxidase as an encapsulation substrate, ZIF-8 as an encapsulation carrier, and β-Dm as a modifier. Through a carefully designed heterogeneous accelerated nucleation synthesis strategy, a core-shell encapsulated enzyme MOF composite material with a polysaccharide outer layer is successfully prepared. The introduction of CTAB can achieve fine-tuning of the morphology and size of ZIF-8, so that the core MOF synthase material has a uniform nanoscale cubic structure and improved enzyme catalytic activity. Using the core MOF synthase material as a substrate, the core-shell encapsulated enzyme MOF material that further accelerates nucleation synthesis shows more excellent enzyme catalytic performance. Subsequently, the core-shell encapsulated enzyme MOF material is modified with polysaccharides to enable it to have a lectin-like capture ability similar to that of bacteria, thereby achieving the purpose of competitive binding to lectins for bacterial detection. The preparation steps are as follows:

[0009] Step 1: Dissolve dimethylimidazole in a freshly prepared CTAB aqueous solution at room temperature, mix Cyt C and zinc acetate aqueous solution, let stand, and centrifuge to obtain a core MOF synthase material with fine-tuned size and morphology;

[0010] Preferably, the concentration of the CTAB aqueous solution is 412.5 mM / mL;

[0011] Preferably, the concentration ratio of the dimethylimidazole and zinc acetate aqueous solution is 12:1;

[0012] Preferably, the mixed reaction solution is allowed to stand at room temperature for 2 hours;

[0013] Step 2: Prepare the core MOF synthase material into an aqueous solution, dissolve dimethylimidazole in a freshly prepared CTAB aqueous solution at room temperature, mix the core MOF synthase, Cyt C and zinc acetate aqueous solution, let it stand, and centrifuge and dry to collect the core-shell encapsulated enzyme MOF material;

[0014] Preferably, the core MOF synthase material concentration is 5 mg / mL;

[0015] Preferably, the mixed reaction solution is allowed to stand at room temperature for 1 hour;

[0016] Step 3: Disperse the core-shell enzyme encapsulation MOF material in the β-Dm aqueous solution, let it stand at room temperature, and centrifuge to collect the polysaccharide-modified core-shell enzyme encapsulation MOF composite material;

[0017] Preferably, the selected polysaccharide is n-dodecyl β-D-maltoside, and the concentration of the β-Dm aqueous solution is 2 mM / mL;

[0018] Preferably, the mixed reaction solution is allowed to stand at room temperature for 30 minutes;

[0019] In the second aspect, the present invention provides a method for instant detection of broad-spectrum bacteria, using carboxylated magnetic beads as a substrate and lectin concanavalin A as a modifier, and achieving broad-spectrum capture of bacteria through amide bond covalent modification. Subsequently, the polysaccharide-modified core-shell encapsulated enzyme MOF composite aqueous solution described in the first aspect is added to the reaction to compete for the excess binding sites on the magnetic beads after the lectin captures the bacteria. After one-step magnetic separation, the bound core-shell encapsulated enzyme MOF composite catalyzes the substrate to produce a visual color change, and the color change is used to detect bacteria in a broad spectrum. The steps include the following:

[0020] Step 1: Carboxylated magnetic beads are mixed with EDC / NHS carboxyl activator at room temperature, and the carboxyl groups are activated and then subjected to one-step magnetic separation and washing; the Concanavalin A aqueous solution is mixed and allowed to stand, and the magnetic beads coupled to Concanavalin A bacterial broad-spectrum capture probe are collected after one-step magnetic separation;

[0021] Preferably, the magnetic beads EDC / NHS carboxyl activation time and Concanavalin A coupling time are 1h and 30min respectively;

[0022] Step 2: Mix the magnetic bead-coupled Concanavalin A capture probe and the test substance and let it stand at room temperature; add the polysaccharide-modified core-shell enzyme-encapsulated MOF composite material to compete for binding; add the enzyme substrate after one-step magnetic separation and observe the color change;

[0023] Preferably, the concentration ratio of the magnetic bead covalent lectin probe and the polysaccharide-modified core-shell encapsulated enzyme MOF composite material is 1:2;

[0024] Preferably, the capture of bacteria and competitive binding time is 35 min;

[0025] Preferably, the enzyme substrates are ABTS and H2O2, and the color development time is 2 min;

[0026] In a third aspect, the present invention provides a 3D printing device for on-site analysis of color signals combined with a smartphone reading system. The color development result sample described in the second aspect is loaded into the sample slot of the 3D printing device, the R / G / B values ​​of the result are obtained through the smartphone camera, the working curve of the corresponding bacteria is called up, and the bacterial concentration is quantitatively output on-site.

[0027] Beneficial effects of the present invention:

[0028] This paper proposes a core-shell enzyme-encapsulating MOF material preparation technology. By modifying the substrate solution with a CTAB capping agent, the size and morphology of the MOF synthase material can be fine-tuned, influencing electron transfer between the enzyme and substrate and increasing enzyme-substrate affinity, thereby demonstrating improved catalytic performance. Further development of the core-shell enzyme-encapsulating MOF material increases enzyme loading and demonstrates more efficient enzyme catalysis, potentially enabling multi-step amplification of enzyme-catalyzed signals for bacterial detection.

[0029] The present invention proposes a method for broad-spectrum bacterial detection, which relies on the joint capture of bacteria and polysaccharide-modified core-shell enzyme MOF materials by magnetic bead-modified lectin probes, and recognizes lectin binding sites through competition. One-step magnetic separation can achieve rapid separation and detection of bacteria in complex samples (less than 40 minutes). At the same time, the capture of lectins achieves broad-spectrum bacterial capture that is free from antibodies / aptamers. The efficient enzyme catalysis significantly improves the sensitivity of bacterial detection (Gram-negative bacteria: 10 2 CFU / mL; Gram-positive bacteria: 10 3 CFU / mL).

[0030] The present invention provides a matching 3D printing device combined with a smartphone reading system. The result sample is loaded through the 3D printing device, and the bacterial concentration is quantitatively obtained on-site with the help of the mobile phone camera. It is portable and does not require additional instruments or equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of the preparation of polysaccharide-modified core-shell enzyme encapsulated MOF materials and the broad-spectrum instant detection of bacteria in Example 1 of the present invention;

[0032] Figure 2 SEM and TEM images of the core-MOF synthase, core-shell encapsulated enzyme MOF, and polysaccharide-modified core-shell encapsulated enzyme MOF composite prepared in Example 2;

[0033] Figure 3 Characterization diagram of the core MOF synthase and core-shell encapsulated enzyme MOF in Example 2;

[0034] Figure 4 This is a verification diagram for the instant detection of broad-spectrum bacteria in Example 3;

[0035] Figure 5 The concentration of each component of the instant detection of broad-spectrum bacteria in Example 4 was affected;

[0036] Figure 6 The reaction time of each component of the instant detection of broad-spectrum bacteria in Example 5 is affected;

[0037] Figure 7 The 3D printing device combined with the smartphone reading system for the instant detection of broad-spectrum bacteria in Example 6 of the present invention;

[0038] Figure 8 Detection curves of different bacterial concentrations using Escherichia coli and Staphylococcus aureus as examples in Example 7;

[0039] Figure 9 In Example 8, artificial blood and urine samples were used as examples to evaluate the test results of Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0040] The specific embodiments of the present invention are further described in detail below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. The scope of protection of the present invention is not limited to this.

[0041] Example 1

[0042] The preparation process of polysaccharide-modified core-shell enzyme encapsulated MOF materials and the detection process of broad-spectrum bacterial instant detection are as follows: Figure 1 As shown, the material preparation process includes the following steps (A):

[0043] Dissolve 1200mM dimethylimidazole in 400μL of freshly prepared 412.5mM CTAB aqueous solution at room temperature. Then, mix 200μL of 8mg / mL Cyt C and 400μL of 100mM zinc acetate aqueous solution and let stand for 2 hours. Centrifuge at 8000rpm / min, wash with pure water, and resuspend. Repeat three times before drying at 45°C to obtain the core MOF synthase.

[0044] The core-MOF synthase material was prepared into a 5 mg / mL aqueous solution. 200 μL of the solution was mixed with 1200 mM dimethylimidazole and dissolved in 400 μL of a freshly prepared 412.5 mM CTAB aqueous solution. Subsequently, 200 μL of 8 mg / mL Cyt C and 400 μL of a 100 mM zinc acetate aqueous solution were mixed and allowed to stand for 1 hour. After centrifugation at 8000 rpm / min, the solution was washed and resuspended in pure water. This was repeated three times and then dried at 45°C to obtain the core-shell encapsulated enzyme MOF.

[0045] The core-shell enzyme-encapsulated MOF material was completely dispersed in 1 mL of 2 mM β-Dm aqueous solution, allowed to stand for modification at room temperature for 6 h, centrifuged at 8000 rpm / min, washed with pure water and resuspended. This was repeated three times and then dried at 45 °C to obtain a polysaccharide-modified core-shell enzyme-encapsulated MOF composite material.

[0046] The detection process includes the following steps (B):

[0047] At room temperature, 500 μL of 10 mg / mL carboxylated magnetic beads were taken, magnetically separated, washed, mixed with 1 mL of 1 mg / mL EDC / NHS carboxyl activator and allowed to stand for 1 hour. After activating the carboxyl groups, magnetic separation and washing were performed. 1 mL of 2 mg / mL Concanavalin A aqueous solution was mixed and combined for 1 hour, magnetically separated, washed with pure water, and resuspended in 1 mL of probe protection solution (10 mM HEPES buffer (pH 7.4) mixed with 0.5% BSA) to obtain a magnetic bead-coupled Concanavalin A probe.

[0048] At room temperature, 5 μL of magnetic bead-coupled Concanavalin A capture probe was mixed with 100 μL of the test substance and allowed to stand for 20 minutes. Then, 20 μL of a polysaccharide-modified core-shell enzyme-encapsulated MOF composite was added for competitive binding for 15 minutes. After magnetic separation and washing, 5 μL of 10 mM PBS buffer (pH 7.4), 30 μL of 2 mg / mL ABTS, and 15 μL of 0.2% H₂O₂ as the enzyme substrate were added and the color change was observed for 2 minutes. The colorimetric sample was loaded into a 3D-printed sample well, which was then assembled with a smartphone and accompanying device. The camera captured the R / G / B values ​​of the result, accessed the corresponding bacterial working curve, and quantitatively output the bacterial concentration on the spot.

[0049] Example 2

[0050] Based on Example 1, the prepared core-MOF synthase material, core-shell encapsulated enzyme MOF material and polysaccharide-modified core-shell encapsulated enzyme MOF composite material were characterized.

[0051] The SEM and TEM characterization results are shown in Figure 2The figure shows a core MOF synthase material (A, D), a core-shell encapsulated enzyme MOF material with a standard cubic structure (B, E), and a polysaccharide-modified core-shell encapsulated enzyme MOF composite material with a clear polysaccharide shell wrapped around the cubic structure (C, F).

[0052] Composition and phase characterization and performance testing such as Figure 3 It is known.

[0053] The results of UV-visible absorption spectroscopy showed that the core-shell encapsulated enzyme MOF had a stronger UV absorption peak (A) than the core MOF synthase.

[0054] XRD analysis results showed that both the core MOF synthase and the core-shell encapsulated enzyme MOF had standard ZIF-8 porous structural components, corresponding to I-43m(B).

[0055] FTIR results show that the characteristic peaks at 1583 cm-1 for ZIF-8 and the core-MOF synthase and the core-shell encapsulated enzyme MOF appear coupled, attributable to the NH bending, CN, and CC stretching vibrations of the dimethylimidazole ring. The loaded Cyt C enzyme exhibits a C=O stretching band of amide I at 1670.8 cm-1, indicating the presence and encapsulation of Cyt C in the composite (C).

[0056] XPS phase characterization results showed that Cyt C contained C, N, O, and S elements, and the core-shell MOF contained C, N, Zn, and a small amount of O elements. The core-MOF synthase and the core-shell encapsulated enzyme MOF showed C, N, O, and Zn elements, indicating that they still existed in a metallic state, and the Cyt C enzyme was in an encapsulated form and was not coated on the surface of the porous structure (D).

[0057] UV-visible absorption spectroscopy and enzyme kinetic analysis revealed that the core-MOF synthase, due to its improved size and morphology, facilitated electron transfer between Cyt C and the enzyme substrate, exhibiting more efficient enzyme-substrate affinity than the ZIF-8 encapsulated enzyme material, generating a stable enzyme catalytic signal. Furthermore, the core-shell encapsulated enzyme MOF material, based on the core-MOF synthase material and further heterogeneously accelerated nucleation, exhibited even more efficient enzyme encapsulation, enabling greater enzyme loading than a single encapsulation method, thereby further enhancing enzyme catalytic activity and producing more efficient catalytic activity (E, F).

[0058] Example 3

[0059] This example studies the feasibility of broad-spectrum bacterial detection. Figure 4 It is known.

[0060] The ZETA potential analysis shows that the concanavalin A covalently modified with the amide bond of the magnetic beads exhibits a stronger negative charge than the magnetic beads, which is attributed to the formation of a complex between the protein structure and the magnetic beads (A).

[0061] The plate culture results showed that the magnetic bead concanavalin A probe could effectively enrich and capture bacteria, and the separated plates showed a small amount of bacteria (B).

[0062] UV-visible absorption spectroscopy revealed effective binding between the magnetic Concanavalin A probe and the polysaccharide-modified core-shell enzyme-encapsulated MOF composite, demonstrating a strong enzyme-catalytic signal after magnetic separation. After bacterial participation in the reaction, the bacteria in the sample bound to the magnetic Concanavalin A probe, while the polysaccharide-modified core-shell enzyme-encapsulated MOF composite was unable to compete for the binding, resulting in a diminished enzyme-catalytic signal after magnetic separation (C, D).

[0063] The results of multiple bacterial tests showed that Pseudomonas aeruginosa, Salmonella, Escherichia coli and Staphylococcus aureus in different mixing ratios all had binding competition effects, reflecting the performance of broad-spectrum bacterial detection (E).

[0064] Example 4

[0065] This example studies the concentration effect of magnetic bead concanavalin A probe and polysaccharide-modified core-shell enzyme MOF composite material. Figure 5 It is known.

[0066] The best effect was achieved when the concentration of magnetic bead concanavalin A probe was 1 mg (A). The best effect was achieved when the concentration of polysaccharide-modified core-shell enzyme-encapsulated MOF composite material was 2 mg.

[0067] Example 5

[0068] This example studies the effect of reaction time of magnetic bead concanavalin A probe, bacterial sample, polysaccharide-modified core-shell enzyme MOF composite material and enzyme substrate. Figure 6 It is known.

[0069] The magnetic bead-based concanavalin A probe was most effective at capturing bacteria for 20 minutes. The polysaccharide-modified core-shell enzyme-encapsulated MOF composite was most effective at competitive binding for 15 minutes. The enzyme substrate's catalytic color development time was optimal at 2 minutes.

[0070] Example 6

[0071] This embodiment studies the use of a 3D printing device for instant detection combined with a smartphone APP reading system. Figure 7 It is known.

[0072] The portable 3D printing device, constructed from 0.5mm white resin and using a slide rail and housing for assembly and disassembly, measures 98 × 50 × 33mm and includes a slidable sample slot (length:width:depth = 81:21:8mm). Within the slot, two 2mm-wide strips and a 1mm-diameter recess securely support the sample (A, B). This device provides reliable visual signal readout distance control, ideal for field deployment.

[0073] The customized WeChat mini-program APP named "POCT Analysis" pre-builds a built-in working curve, analyzes the green (G) value of fixed points in the positioning area, and directly calculates the bacterial concentration (C).

[0074] On-site, turn on the mobile phone to activate the flash, photograph the fixed sample through the device's photographic capture hole (35mm in diameter, 23mm in depth), read the color G value, and directly obtain the bacterial concentration (D) by calling the working curve.

[0075] Example 7

[0076] This example studies the working curves of different concentrations of Escherichia coli and Staphylococcus aureus. Figure 8 Using the above-mentioned instant detection method for broad-spectrum bacteria, the sample volume is 100 μL, and the concentration of different bacteria is diluted to 10 7 ~0CFU / mL. By quantitatively outputting bacterial concentration on site, 10 2 CFU / mL of Escherichia coli (A, C) and 10 3 CFU / mL of Staphylococcus aureus (B, D).

[0077] Example 8

[0078] This example studies the test effect of the broad-spectrum bacterial instant detection method for body fluid diagnosis. Figure 9 Using artificial blood and urine samples as diluents, add 10 3 Detection of E. coli and Staphylococcus aureus (CFU / mL) and blank dilutions was performed. Body fluid samples with color interference did not affect the color development of the system after magnetic separation (A). Both E. coli and Staphylococcus aureus demonstrated good diagnostic efficacy, showing good sensitivity and specificity (B-F). These results demonstrate that this invention can be used for rapid, broad-spectrum, and instant quantitative on-site detection of mixed bacterial infections in body fluids.

[0079] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the described embodiments. It is apparent to those skilled in the art that various changes, modifications, substitutions, and variations to these embodiments may be made without departing from the principles and spirit of the present invention, and these changes and modifications still fall within the scope of protection of the present invention.

Claims

1. A method for preparing a polysaccharide-modified core-shell enzyme encapsulated MOF material, characterized in that: The following steps are involved: (1) Dissolve 1200 mM 2-methylimidazole in 400 μL of freshly prepared 412.5 mM CTAB aqueous solution at room temperature. Then, take 200 μL of 8 mg / mL Cyt C oxidase and 400 μL of 100 mM zinc acetate aqueous solution, mix them and let them stand for 2 h. After centrifugation, wash and resuspend them in pure water. Repeat this process three times and then dry them at 45°C to obtain the core MOF synthase. (2) The core-MOF synthase material was prepared into a 5 mg / mL aqueous solution, 200 μL of which was mixed with 1200 mM dimethylimidazole and dissolved in a freshly prepared 400 μL 412.5 mM CTAB aqueous solution. Subsequently, 200 μL of 8 mg / mL Cyt C and 400 μL of 100 mM zinc acetate aqueous solution were mixed and allowed to stand for 1 h. After centrifugation, the mixture was washed with pure water and resuspended. This was repeated three times and then dried at 45 °C to obtain the core-shell encapsulated enzyme MOF. (3) The core-shell enzyme-encapsulated MOF material was completely dispersed in 1 mL of 2 mM β-Dm aqueous solution, allowed to stand for modification at room temperature for 6 h, centrifuged, washed with pure water and resuspended, and repeated three times. The material was dried at 45 °C to obtain a polysaccharide-modified core-shell enzyme-encapsulated MOF composite material.

2. The method for preparing the polysaccharide-modified core-shell enzyme encapsulated MOF material according to claim 1, characterized in that: The centrifugation procedure in steps (1), (2) and (3) is to process at 8000-10000 rpm / min for 10 min.

3. A method for real-time detection of broad-spectrum bacteria, characterized in that: The polysaccharide-modified core-shell enzyme encapsulation MOF material according to any one of claims 1 to 2 comprises the following steps: (1) Take 500 μL of 10 mg / mL carboxylated magnetic beads at room temperature, magnetically separate and wash, mix with 1 mL of 1 mg / mL EDC / NHS carboxyl activator and let stand for 1 hour, activate the carboxyl group and then magnetically separate and wash; mix with 1 mL of 2 mg / mL concanavalin A aqueous solution for 1 hour, magnetically separate and wash with pure water, and resuspend in 1 mL of probe protection solution to obtain magnetic bead-coupled concanavalin A probe; (2) Mix 5 μL of magnetic bead-coupled concanavalin A capture probe with 100 μL of the test substance and let it stand for 20 minutes at room temperature; add 20 μL of polysaccharide-modified core-shell encapsulated enzyme MOF composite material to compete for binding for 15 minutes; after magnetic separation and washing, add enzyme substrate and observe the color change after 5 minutes.

4. The method for real-time detection of broad-spectrum bacteria according to claim 3, characterized in that: The magnetic separation and washing procedures in steps (1) and (2) are to remove the supernatant by magnetic separation, resuspend in pure water, and remove the supernatant by magnetic separation again, and repeat 3 times.

5. The method for real-time detection of broad-spectrum bacteria according to claim 3, characterized in that: The probe protection solution in step (1) is 10 mM HEPES buffer (pH 7.4) mixed with 0.5% BSA.

6. The method for real-time detection of broad-spectrum bacteria according to claim 3, characterized in that: The enzyme substrate was 5 μL of 10 mM PBS buffer (pH 7.4), 30 μL of 2 mg / mL ABTS and 15 μL of 0.2% H 2 O 2 .

7. A 3D printing device for on-site analysis of color signals combined with a smartphone reading system, characterized in that: The method for instant detection of broad-spectrum bacteria according to any one of claims 1 to 5 comprises the following steps: loading the color development result sample into a 3D printed sample slot, assembling it with a smartphone and a supporting device, obtaining the R / G / B values ​​of the result through a camera, retrieving the working curve of the corresponding bacteria, and quantitatively outputting the bacterial concentration on site.

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