Magnetic hydrogel coated bacterial biosensor platform and application

Abstract

The invention discloses a magnetic hydrogel coated bacterial biosensor platform and application, and belongs to the field of synthetic biology and clinical diagnosis. The hydrogel delivery and recovery system comprises a hydrogel-coated whole-cell biosensor and magnetic particles, the hydrogel is constructed by sodium alginate and calcium chloride, and the biosensor is a bacterium for sensing heme. More specifically, engineering bacteria YES601 and magnetic Fe3O4 particles are packaged by sodium alginate hydrogel to form magnetic hydrogel microspheres, the magnetic hydrogel microspheres are delivered to intestinal tracts as a biosensor and can sense heme to emit light, and then complete microspheres are efficiently recovered from excrement in a magnetic adsorption mode for detection. The intestinal bleeding detection efficiency and safety of the whole-cell biosensor are improved. According to the overall design optimization strategy of the hydrogel delivery and recovery system, the diagnosis efficiency and the practical application value can be improved.

Description

Technical Field

[0001] The present invention relates to the fields of synthetic biology and clinical diagnosis, and in particular to a magnetic hydrogel-wrapped bacterial biosensor platform and application. Background Art

[0002] The emergence of whole-cell biosensor technology has opened up a new research direction for the early diagnosis of inflammatory bowel disease. Whole-cell biosensors (WCBs) provide an economical, efficient and environmentally friendly way to detect and report biomarkers or conditions of interest. Due to their inherent powerful functions in complex environments, more and more WCBs have been applied to various scenarios, including environmental monitoring, molecular recording, biological process monitoring, and disease diagnosis and treatment to promote human health. However, due to the harsh and complex environment in the gastrointestinal tract, the survival rate of WCBs and their sensing ability will be significantly affected. At the same time, the intestinal flora itself and other exogenous bacteria will increase the difficulty of WCB recovery and detection. Therefore, the delivery and recovery of whole-cell sensors should be systematically and rationally designed. The present invention has developed a diagnostic platform that can protect the safe delivery of engineered bacteria to the intestine and can normally exert its efficacy, while being able to quickly separate and detect from feces, increasing the clinical application value of WCB. Summary of the invention

[0003] The purpose of the present invention is to provide a magnetic hydrogel-encapsulated bacterial biosensor platform and application to solve the problems existing in the above-mentioned prior art. A biosensor for detecting intestinal inflammation is formed by using sodium alginate hydrogel to encapsulate bacterial strains and magnetic Fe3O4 particles. The bacterial strain reports the presence of the inflammatory marker heme through bioluminescent signal expression, and the presence of magnetic Fe3O4 particles can efficiently recover intact microspheres from feces by magnetic adsorption, providing a theoretical basis and new method for achieving efficient, fast and accurate detection and diagnosis of intestinal inflammation in vitro.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a magnetic hydrogel-wrapped bacterial biosensor platform, which is characterized by comprising a hydrogel-wrapped whole-cell biosensor and magnetic particles, wherein the hydrogel is constructed of sodium alginate and calcium chloride, and the biosensor is a heme-sensing bacterium.

[0006] Preferably, the bacteria include the heme-sensing engineered bacteria YES601, and the magnetic particles are magnetic Fe3O4 particles.

[0007] The present invention also provides a method for constructing a magnetic hydrogel-encapsulated bacterial biosensor platform, comprising the following steps:

[0008] Bacterial liquid and sodium alginate solution are prepared, and then the bacterial liquid, sodium alginate solution and magnetic particles are evenly mixed, and then the mixed solution is added dropwise to a calcium chloride solution, centrifuged, and the precipitate is collected to obtain magnetic hydrogel particles, which is the magnetic hydrogel-encapsulated bacterial biosensor platform.

[0009] Preferably, the volume ratio of the bacterial liquid, the sodium alginate solution and the magnetic particles is 3:5:2.

[0010] 5. The construction method according to claim 3, characterized in that the concentration of the bacterial solution is 2×10 9 CFU / mL, the mass concentration of the sodium alginate solution is 2%, the concentration of the magnetic particles is 10 mg / mL, and the mass concentration of the calcium chloride solution is 1.5%.

[0011] Preferably, the volume ratio of the mixed solution to the calcium chloride is 2:(1-1.5).

[0012] Preferably, the centrifugation condition is: 1000-1500 rpm for 15-20 minutes.

[0013] The present invention also provides application of the magnetic hydrogel-wrapped bacteria biosensor platform in preparing products for detecting enteritis.

[0014] Preferably, the feces of the recipient are recovered, the magnetic hydrogel microspheres in the feces are adsorbed by a magnet, and after washing with physiological saline, the magnetic hydrogel microspheres are collected and the luminescence values ​​are counted and measured, and the degree of intestinal inflammation is determined according to the luminescence values.

[0015] Preferably, the judgment standard is: the more severe the intestinal inflammation is, the higher the luminescence intensity of the magnetic hydrogel microspheres is.

[0016] The present invention discloses the following technical effects:

[0017] The present invention provides a magnetic hydrogel-encapsulated bacterial biosensor platform, which encapsulates bacteria and magnetic Fe3O4 particles in hydrogels. The activity and biocompatibility of bacteria in the intestine can be enhanced by hydrogel encapsulation. The magnetic adsorption method greatly improves the recovery detection efficiency, and the platform can be expanded to other whole-cell biosensor detection applications, which is universal. Experiments have found that the biosensor platform provided by the present invention can efficiently detect and diagnose enteritis bleeding as a hydrogel delivery recovery system, and the overall design optimization strategy of the hydrogel delivery recovery system can improve the diagnostic efficiency and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Schematic diagram of the delivery and recovery system and application of magnetic hydrogel-encapsulated bacteria biosensor; A: Preparation of magnetic hydrogel microspheres encapsulating engineered bacteria by centrifugation; B: Detection of intestinal bleeding in colitis mice using the magnetic hydrogel microsphere system; C: Rapid recovery of magnetic hydrogel microspheres from mouse feces by magnets for subsequent technical and luminescence value analysis;

[0020] Figure 2 Figure 3 is the morphological characterization of magnetic hydrogel microspheres; a: cross-linking mechanism of sodium alginate and calcium chloride; b: schematic diagram of hydrogel microsphere preparation; c: optical image and size distribution of hydrogel microspheres; d: confocal microscopy image of hydrogel system: (A) hydrogel; (B) hydrogel encapsulated magnetic beads; (C) hydrogel encapsulated magnetic beads and bacteria expressing sfGFP and mCherry; e: SEM image of hydrogel system: (A) hydrogel; (B) hydrogel encapsulated magnetic beads; (C) hydrogel encapsulated magnetic beads and bacteria;

[0021] Figure 3 To detect the survival rate and escape rate of magnetic hydrogel microspheres in simulated gastrointestinal fluid and the heme-induced luminescence in simulated intestinal fluid; a: survival rate in simulated gastric fluid; b: escape rate in simulated gastric fluid; c: effect of heme concentration on the luminescence of strains in magnetic hydrogel microspheres; d: schematic diagram of the photosensitization of magnetic hydrogel microspheres to different heme concentrations; e: changes in the sensing activity of magnetic hydrogel microspheres after freeze-drying;

[0022] Figure 4 Process and results of rapid detection of inflammatory bowel disease severity by hydrogel system; a: Schematic diagram of hydrogel delivery platform for detection and purification of engineered bacteria after colitis; b: Efficiency of recovering hydrogel microspheres from mouse feces; c: Statistical results of mouse weight changes over time; d: Disease activity of each group of mice on the fifth day; e: Bioluminescence of microspheres recovered from feces of mice in different groups; f: Representative H&E histological images of small intestine and colon of mice in healthy group, moderate disease group and severe disease group;

[0023] Figure 5Results of in vivo and in vitro safety assessment of magnetic hydrogel microspheres; a: RAW264.7 co-cultured with saline and YES601@ALG to produce inflammatory factors; b: Comparison of serum inflammatory factors in mice before and after oral administration of YES601@MALG; c: Comparison of serum biochemical indicators in mice before and after oral administration of YES601@MALG; d: Changes in body weight of mice five days after oral administration of saline and YES601@ALG; e: Representative H&E histological images of the small intestine and colon of mice in different groups;

[0024] Figure 6 Results of intestinal microbiota and diversity analysis of mice; ac: α diversity of intestinal microbiota of two groups of mice was compared using Chao, Shannon and Simpson indices; d: rank-abundance curve of microbial community; eg: ANOSlM between box plot showing sample distance (e), principal coordinate analysis (PCoA) plot (f) and group similarity analysis box plot (g); h bar graph showing the relative abundance of species in the two groups; Student's T-test was used to analyze the significant differences between the two groups, ns: p>0.05. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The reagents, materials, streptomycin and other chemicals used in the following examples were purchased from Sigma-Aldrich. All plasmid constructions were carried out in Escherichia coli DH5α. The strains were cultured in lysogeny broth (LB) medium containing streptomycin, and the antibiotic concentration was streptomycin (50 μg / mL). The storage solution of magnetic Fe3O4 particles (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) contained 1×PBS with 20% ethanol, which was replaced in equal proportions with lysogeny broth (LB) medium containing streptomycin (50 μg / mL).

[0031] The detection of cytokines TNF-α, IL-6, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (γ-GT) were all completed using corresponding conventional commercial kits.

[0032] The present invention takes the hemoglobin-responsive whole-cell biosensor YES601 as an example and constructs a hydrogel delivery and recovery system based on the whole-cell biosensor. By centrifugation, a sodium alginate solution mixed with a specific proportion of YES601 bacterial solution and magnetic Fe3O4 particles is added dropwise to a calcium chloride solution to form a system of hydrogel-wrapped YES601 and magnetic beads, namely magnetic hydrogel microspheres. The YES601 in the magnetic hydrogel microspheres delivered to the body reports the presence of the inflammatory marker hemoglobin through bioluminescent signal expression. The hydrogel is excreted from the body with feces and is made into a fecal homogenate by grinding and dilution. The magnetic hydrogel microspheres are recovered from the fecal homogenate by magnetic adsorption, and the luminescence value is read by an enzyme reader after washing with water. The microspheres are counted by taking a photo with a mobile phone, and the luminescence value is divided by the number of recovered microspheres to obtain a normalized luminescence value to judge the results of enteritis bleeding. The optimization strategy and application schematic diagram of the above-mentioned hydrogel delivery and recovery system based on the whole-cell biosensor are shown in Figure 1 .

[0033] Example 1 Construction method of hydrogel delivery and recovery system based on whole cell biosensor

[0034] (1) Preparation of bacterial stock solution

[0035] Strain YES601, an engineered heme-sensing Escherichia coli Nissle 1917 strain (EcN) resistant to streptomycin, was used for all other bacterial experiments. Colonies were grown in LB Miller broth with the appropriate antibiotic (100 μg / mL streptomycin) to an OD of 4. 600 =0.6 to prepare bacterial stock solution, add 50% glycerol according to the volume ratio of 1:1, and store it in frozen state at -80°C.

[0036] Take 20 μL from the frozen stock of strain YES601 and add 5 mL LB medium (100 μg / mL streptomycin) and incubate at 37°C with shaking at 220 rpm overnight. Dilute the culture 100-fold with fresh LB medium (100 μg / mL streptomycin) and incubate at 37°C with shaking at 220 rpm until OD 600 =0.6-0.8. The culture was transferred to a sterile Eppendorf tube and centrifuged at 6000 rpm for 10 minutes. The cells were washed once with sterile PBS to remove traces of LB medium, and then the cells were concentrated 3.3 times and added to LB medium (100 μg / mL streptomycin) to prepare a bacterial stock solution (2×10 9 CFU / mL).

[0037] (2) Treatment of magnetic Fe3O4 particles

[0038] The magnetic Fe3O4 microparticle stock solution with an initial concentration of 10 mg / mL was replaced in equal proportion with magnetic Fe3O4 microparticles dissolved in LB medium (100 μg / mL streptomycin) for later use.

[0039] (3) Preparation of magnetic microparticle hydrogel

[0040] Bacterial stock solution (2×10 9 CFU / mL), 2% sodium alginate solution and magnetic Fe3O4 particles (10 mg / mL) were mixed in a volume ratio of 3:5:2 and loaded into a 2mL syringe for the preparation of microspheres. The microspheres in the syringe were injected into a 15mL centrifuge tube containing 1.5mL of 1.5% calcium chloride solution. The centrifuge tube was placed in a centrifuge at a speed of 1050rpm for 15-20 minutes per round, and the precipitate was collected to obtain magnetic hydrogel microspheres, which is a hydrogel delivery and recovery system based on whole-cell biosensors.

[0041] The magnetic hydrogel microspheres are prepared by the centrifugation method. Other whole-cell biosensors can be encapsulated by the same method for application, such as biosensors for detecting thiosulfate.

[0042] Example 2 Morphological Characterization of Magnetic Hydrogel Microspheres

[0043] In order to develop a hydrogel delivery and recovery system, the present invention first determined a non-toxic and harmless method for preparing sodium alginate hydrogel microspheres: Sodium alginate, as a biodegradable and non-toxic natural polymer, will immediately cross-link to form magnetic hydrogel microspheres in the presence of calcium chloride ( Figure 2 In a and b), hydrogels based on sodium alginate and calcium chloride have been used in many aspects such as cell encapsulation and drug delivery. When gelled, the pore size is large enough to allow nutrient exchange between the encapsulated bacteria and the surrounding environment outside the gel.

[0044] Microscope imaging showed that the prepared magnetic hydrogel microspheres had a particle size between 180 and 250 μm and were roughly normally distributed ( Figure 2 In order to better observe the distribution of magnetic beads and bacteria in the hydrogel spheres, the encapsulated strains were observed by confocal microscopy using bacteria expressing both mCherry and GFP fluorescent proteins and control microspheres ( Figure 1 d), and took SEM images of blank hydrogel, hydrogel encapsulating bacteria only, and magnetic hydrogel microspheres encapsulating bacteria and magnetic beads ( Figure 1 (e) The results show that the bacteria and magnetic beads are tightly encapsulated in the hydrogel microspheres.

[0045] Example 3 Detection of survival rate and escape rate in simulated gastrointestinal fluid (SGF)

[0046] 1. Detection of bacterial survival rate in magnetic hydrogel microspheres

[0047] After oral administration, EcN probiotics encountered some challenges, including extremely low pH in the stomach and bile salts in the intestine. Therefore, maintaining a sufficient number of live bacteria until they reach the gastrointestinal tract becomes a key factor in improving the treatment of inflammatory bowel disease (IBD). In order to detect the protective effect of hydrogel microspheres on bacteria in simulated gastric fluid, 900 μL of SGF was added to the well plate using a 96-well ELISA plate, and then 100 μL of the mixed hydrogel system was added (the hydrogel system is made of magnetic hydrogel particles and LB medium containing 100 μg / mL streptomycin in a volume ratio of 1:3, and the concentration of the naked bacteria control group is 5×10 8 CFU / mL). Seal the sealing film and culture on a microplate shaker at 37°C, 100rpm for gradient time. After the digestion fluid simulation is completed, aspirate and centrifuge from the well plate, remove the digestion fluid from the naked bacteria control group, resuspend, and apply gradient dilutions. After removing the digestion fluid from the hydrogel group, add 1mL of sodium citrate to dissolve the sodium alginate spheres. Culture on a microplate shaker at 37°C, 100rpm for 30min. Aspirate and centrifuge again, remove the sodium citrate solution, resuspend, and apply gradient dilutions. Place in a 37°C incubator for overnight culture, and then take photos and count with a mobile phone. The results show ( Figure 3In a), after culturing in SGF for 30 minutes, the survival rate of the engineered bacteria wrapped in hydrogels was as high as 100%, while only 25% of the bacteria in the naked bacteria group survived. After culturing for 1 hour, the survival rate of the hydrogel group was more than 10 times that of the naked bacteria group, which fully demonstrated that the magnetic hydrogel microspheres enhanced the survival ability of bacteria in simulated gastric fluid and were more conducive to their function in the intestine.

[0048] 2. Detection of bacterial escape rate in magnetic hydrogel microspheres

[0049] In order to detect the escape rate of bacteria in magnetic hydrogel microspheres, the present invention adds 900 μL of physiological saline to a 96-well ELISA plate, and then adds 100 μL of the mixed hydrogel system, and cultured on a microplate shaker at 37°C and 100 rpm. Three parallel experiments are set up for each group. After the digestion fluid simulation is completed, 100 μL of supernatant is aspirated from the microplate and directly coated. Place in a 37°C incubator for overnight culture, and then take photos and count with a mobile phone. Figure 3 As shown in b, the results show that after 12 hours of culture, there is no significant difference in the amount of bacteria compared with the initial amount. The amount of bacteria has not decreased during the preparation and storage process, which can ensure that the effective concentration of the bacteria remains unchanged.

[0050] Example 4 Heme-induced luminescence in simulated intestinal fluid (SIF)

[0051] In order to test whether the hydrogel shell affects the performance of the biosensor, the present invention encapsulates the YES601 previously designed for sensing hemoglobin in a magnetic hydrogel, and mixes the prepared magnetic hydrogel microspheres (YES601@MALG) with LB culture medium (100μg / mL streptomycin) in a ratio of 1:3 to form a system. In order to better meet the intestinal conditions, simulated intestinal fluid (SIF) was selected as the culture environment to prepare a 40μM hemoglobin mother solution: 8μL of hemoglobin mother solution (25mM) and 4992μL of SIF (100μg / mL streptomycin) were used to prepare 5mL of 40μM hemoglobin mother solution. The 40μM hemoglobin mother solution was diluted into a gradient concentration, and 100μL was taken in turn to add to the luminescence measuring well plate. The mixed magnetic hydrogel microsphere system was mixed with a gun tip and 100μL was taken to add the well of the hemoglobin solution. Seal the sealing film, culture it on a microplate shaker at 37°C, 100rpm, and detect it every hour. Three parallel groups were set for each hemoglobin concentration. Figure 3 As shown in Figure c, the results show that the higher the hemoglobin concentration, the shorter the time to reach the highest luminescence intensity, and then the luminescence intensity decreases to varying degrees over time, proving that the hydrogel shell does not affect the ingress and egress of hemoglobin. Visualized bioluminescence images can more intuitively characterize the luminescence phenomenon of the hydrogel-wrapped biosensor sensing different hemoglobin concentrations over time ( Figure 3d), indicating that the hydrogel shell does not affect its luminescence detection. In addition, the magnetic hydrogel microspheres preserved by freeze-drying retained more than 80% of the initial sensing activity after one month ( Figure 3 In summary, the bacteria still maintain efficient sensing ability when encapsulated in hydrogel.

[0052] Example 5 Detection of recovery rate of magnetic hydrogel microspheres

[0053] Recovering engineered bacteria after in vivo testing is a difficult problem that needs to be solved urgently. After naked bacteria are tested in vivo, they will be excreted in the feces. The existing method is to homogenize the mouse feces, collect the supernatant and filter it, obtain the sample to be tested and add it to the culture medium, culture it for several hours and then detect the bioluminescence. There are problems such as engineered bacteria will remain in the intestine, less recovery, and a long time required to obtain test results. The hydrogel-encapsulated magnetic beads and engineered bacteria delivery scheme proposed in the present invention are recovered and purified by magnetic adsorption ( Figure 4 a). The magnetic rod with a protective cover adsorbs the microspheres in the fecal homogenate and then puts it into pure physiological saline. The magnetic rod is taken out and the microspheres fall off the protective cover, completing the recovery. The magnet is then used to adsorb the microspheres through the device and the microspheres are washed to complete the purification. The present invention detects the recovery rate of the microspheres. The results show that no microspheres are excreted within 0-2 hours after oral administration of mice. The recovery rate is about 20% at 2-4 hours, 4-6 hours, and 6-8 hours. The recovery rate is as high as 80% within 24 hours ( Figure 4 b). Finally, the microspheres were photographed by mobile phone and detected by ELISA. The whole process of detecting and obtaining the results took about 10 minutes, achieving the purpose of efficient recovery and purification.

[0054] Example 6 Construction and detection of mouse colitis model

[0055] Construction of a mouse enteritis model: 6-8 week old male C57BL / 6 mice (Southern Model, China) were selected and placed in a standard temperature (20-22°C) and 12h light / 12h dark cycle for one week. They were then randomly divided into four cages, and dextran sulfate sodium (DSS, molecular weight 36,000-50,000; MP Biomedals) was added to the drinking water at a final concentration of 3%. The first cage of mice had their water replaced with 3% DSS from the fourth day, the second cage of mice had their water replaced with 3% DSS from the second day, the third cage of mice had their water fed with 3% DSS from the first day, and the fourth group of mice was fed with water throughout. The weight changes of the mice were recorded every day ( Figure 4c), observe the bleeding in the mice's feces. The disease activity index (DAI) was calculated based on the weight loss of the mice and the bleeding and viscosity of the feces to assess the disease severity of each mouse. The mice were divided into four groups according to their DAI scores on the last day, with DAI of 0-1 as the healthy group, DAI of 2-5 as the mild disease group, DAI of 6-8 as the moderate disease group, and DAI of 9-12 as the severe disease group ( Figure 4 d). Then, on the fifth day, mice were gavaged with a No. 10 gavage needle, with a total volume of 200 μl (containing 100 μl of magnetic hydrogel microspheres and 100 μl of saline), to deliver the prepared magnetic hydrogel microspheres to the mouse intestines. The feces were recovered for purification and recovery detection, and the luminescence was normalized. The results showed that the more severe the disease of the mice, the higher the luminescence intensity of the microspheres, and the luminescence signal of healthy mice was the weakest. There were also significant differences in the data between different disease groups ( Figure 4 Then, pathological analysis of colon tissue sections of mice was performed, which confirmed that the enteritis in the severe disease group was the most severe, while the enteritis in the moderate disease group was milder ( Figure 4 f). These results demonstrate that the hydrogel-encapsulated engineered bacteria YES601 can be used to quickly and efficiently detect fecal bleeding in patients with different degrees of colitis.

[0056] Example 7 In vivo and in vitro safety evaluation of magnetic hydrogel microspheres

[0057] The encapsulation of hydrogel can reduce the direct contact of engineered bacteria with the mouse intestine, thereby reducing the inflammatory response caused by bacterial infection. In order to comprehensively evaluate the biocompatibility and biosafety of magnetic hydrogel microspheres, in vitro cell experiments and in vivo mouse experiments were used for verification.

[0058] (1) Add an equal volume of saline solution with a bacterial concentration of 2×10 8 The concentration changes of cytokines were detected after co-incubation of YES601 naked bacteria and magnetic hydrogel microspheres (YES601@MALG) with RAW264.7. The results of cytotoxicity test showed that ( Figure 5 In (a), compared with the saline group, the concentrations of TNF-α and IL-6 in the naked bacteria group increased significantly, while there was no significant difference in the YES601@MALG group, proving that the encapsulation of the hydrogel significantly reduced the immune recognition ability of macrophages to bacteria.

[0059] (2) The biosafety of healthy mice was then evaluated. The mice were not gavaged or gavaged with an equal volume of saline and YES601@MALG (with a bacterial count of 2×10 8 CFUs) were collected on the 5th day after the treatment to evaluate serum inflammatory factors and transaminases. Comparative analysis with the control group (administered with normal saline) showed that serum inflammatory factors (TNF-α, IL-6) levels ( Figure 5 b) or transaminase (ALT, AST, γ-GT) levels ( Figure 5 c), body weight of mice ( Figure 5 After the mice were killed on the last day, histopathological examination of the small intestine and colon showed that YES601@MALG did not cause intestinal tissue damage ( Figure 5 (e)

[0060] In addition, fecal samples were collected from each group of mice before and after oral administration on the fifth day, and then ground and analyzed by 16S ribosomal RNA (rRNA) genome sequencing. The results showed that oral administration of YES601@MALG did not cause significant changes in the composition or diversity of the mouse intestinal microbiota ( Figure 6 This confirms the high biosafety of YES601@MALG.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A magnetic hydrogel-encapsulated bacterial biosensor platform, characterized in that: The invention comprises a whole-cell biosensor wrapped in a hydrogel and magnetic particles, wherein the hydrogel is constructed of sodium alginate and calcium chloride, and the biosensor is a bacterium that senses hemoglobin.

2. The magnetic hydrogel-encapsulated bacterial biosensor platform according to claim 1, characterized in that: The bacteria include the heme-sensing engineered bacteria YES601, and the magnetic particles are magnetic Fe3O4 particles.

3. The method for constructing a magnetic hydrogel-encapsulated bacterial biosensor platform according to claim 1 or 2, characterized in that: The following steps are involved: Bacterial liquid and sodium alginate solution are prepared, and then the bacterial liquid, sodium alginate solution and magnetic particles are evenly mixed, and then the mixed solution is added dropwise to a calcium chloride solution, centrifuged, and the precipitate is collected to obtain magnetic hydrogel particles, which is the magnetic hydrogel-encapsulated bacterial biosensor platform.

4. The construction method according to claim 3, characterized in that: The volume ratio of the bacterial liquid, the sodium alginate solution and the magnetic particles is 3:5:

2.

5. The construction method according to claim 3, characterized in that: The concentration of the bacterial solution is 2×10 9 CFU / mL, the mass concentration of the sodium alginate solution is 2%, the concentration of the magnetic particles is 10 mg / mL, and the mass concentration of the calcium chloride solution is 1.5%.

6. The construction method according to claim 3, characterized in that: The volume ratio of the mixed solution to the calcium chloride is 2:(1-1.5).

7. The construction method according to claim 3, characterized in that: The centrifugal conditions are: 1000-1500 rpm for 15-20 minutes.

8. Use of a magnetic hydrogel-encapsulated bacterial biosensor platform as claimed in claim 1 or 2 in the preparation of products for detecting enteritis.

9. The use according to claim 8, characterized in that The feces of the recipient are recovered, and the magnetic hydrogel microspheres in the feces are adsorbed by a magnet. After washing with physiological saline, the magnetic hydrogel microspheres are collected and the luminescence values ​​are counted and measured, and the degree of intestinal inflammation is determined according to the luminescence values.

10. The use according to claim 9, characterized in that The judgment standard is: the more severe the intestinal inflammation is, the higher the luminescence intensity of the magnetic hydrogel microspheres is.

Images