A magnetic hydrogel-encapsulated bacterial biosensor platform and its application

Abstract

This invention discloses a magnetic hydrogel-encapsulated bacterial biosensor platform and its application, belonging to the fields of synthetic biology and clinical diagnostics. The hydrogel delivery and recovery system comprises a whole-cell biosensor encapsulated in hydrogel and magnetic microparticles. The hydrogel is constructed from sodium alginate and calcium chloride, and the biosensor is a heme-sensing bacterium. More specifically, this invention uses sodium alginate hydrogel to encapsulate engineered bacteria YES601 and magnetic Fe3O4 microparticles to form magnetic hydrogel microspheres. These microspheres are delivered to the intestine as biosensors and can emit light in response to heme. The intact microspheres are then efficiently recovered from feces via magnetic adsorption for detection, improving the efficiency and safety of whole-cell biosensors in detecting intestinal bleeding. The overall design optimization strategy of the hydrogel delivery and recovery system provided by this invention can improve diagnostic efficiency and practical application value.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and clinical diagnostics, and in particular to a magnetic hydrogel-encapsulated bacterial biosensor platform and its applications. Background Technology

[0002] The emergence of whole-cell biosensor technology has opened up new research directions for the early diagnosis of inflammatory bowel disease. Whole-cell biosensors (WCBs) offer an economical, efficient, and environmentally friendly way to detect and report biomarkers or conditions of interest. Due to their inherent powerful capabilities in complex environments, WCBs are increasingly being applied in various scenarios, including environmental monitoring, molecular recording, biological process monitoring, and disease diagnosis and treatment to promote human health. However, the survival rate and sensing ability of WCBs are significantly affected by the harsh and complex environment of the gastrointestinal tract. Furthermore, the gut microbiota itself, as well as other exogenous bacteria, increase the difficulty of WCB recovery and detection. Therefore, a systematic and rational design for the delivery and recovery of whole-cell sensors is necessary. This invention develops a diagnostic platform that can safely deliver engineered bacteria to the intestine and ensure their normal function, while also enabling rapid separation and detection from feces, thereby increasing the clinical application value of WCBs. Summary of the Invention

[0003] The purpose of this invention is to provide a magnetic hydrogel-encapsulated bacterial biosensor platform and its application to solve the problems existing in the prior art. By using sodium alginate hydrogel to encapsulate bacterial strains and magnetic Fe3O4 microparticles, a biosensor for detecting intestinal inflammation is formed. The bacterial strain reports the presence of the inflammatory marker heme through bioluminescent signal expression, while the presence of magnetic Fe3O4 microparticles allows for efficient recovery of intact microspheres from feces through magnetic adsorption. This provides a theoretical basis and new method for achieving efficient, rapid, and accurate in vitro detection and diagnosis of intestinal inflammation.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] This invention provides a magnetic hydrogel-encapsulated bacterial biosensor platform, characterized in that it includes a whole-cell biosensor encapsulated in hydrogel and magnetic microparticles, wherein the hydrogel is constructed from sodium alginate and calcium chloride, and the biosensor is a heme-sensing bacterium.

[0006] Preferably, the bacteria include the engineered bacterium YES601 that senses heme, and the magnetic particles are magnetic Fe3O4 particles.

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

[0008] A bacterial culture and a sodium alginate solution were prepared. The bacterial culture, sodium alginate solution, and magnetic microparticles were then mixed evenly. The mixture was then added dropwise to a calcium chloride solution, centrifuged, and the precipitate was collected to obtain magnetic hydrogel microparticles, which constitute the magnetic hydrogel-encapsulated bacterial biosensor platform.

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

[0010] 5. The construction method according to claim 3, wherein the concentration of the bacterial culture is 2 × 10⁻⁶. 9 The concentration of the sodium alginate solution is 2% (CFU / mL), the concentration of the magnetic particles is 10 mg / mL, and the concentration of the calcium chloride solution is 1.5% (CFU / mL).

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

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

[0013] The present invention also provides the application of the magnetic hydrogel-encapsulated bacterial biosensor platform described above in the preparation of products for detecting enteritis.

[0014] Preferably, the method involves recovering the recipient's feces, using a magnet to adsorb the magnetic hydrogel microspheres in the feces, washing them with physiological saline, collecting the magnetic hydrogel microspheres, counting and measuring the luminescence value, and judging the degree of intestinal inflammation based on the luminescence value.

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

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

[0017] This invention provides a magnetic hydrogel-encapsulated bacterial biosensor platform, encapsulating bacteria and magnetic Fe3O4 particles within a hydrogel. Hydrogel encapsulation enhances bacterial activity and biocompatibility in the gut, while magnetic adsorption significantly improves recovery and detection efficiency. Furthermore, this platform can be extended to other whole-cell biosensor applications, demonstrating its versatility. Experiments have shown that the biosensor platform provided by this invention, as a hydrogel delivery and recovery system, can efficiently detect and diagnose enteritis bleeding. The overall design optimization strategy for the hydrogel delivery and recovery system can further improve diagnostic efficiency and practical application value. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic diagram of a magnetic hydrogel-encapsulated bacterial biosensor delivery and recovery system and its application; A: Magnetic hydrogel microspheres encapsulating engineered bacteria are prepared using centrifugation; B: Intestinal bleeding in colitis mice is detected using a magnetic hydrogel microsphere system; C: Magnetic hydrogel microspheres are rapidly recovered from mouse feces using a magnet for subsequent technical and luminescence analysis.

[0020] Figure 2 Morphological characterization of magnetic hydrogel microspheres; a: Crosslinking 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 the hydrogel system: (A) Hydrogel; (B) Hydrogel-encapsulated magnetic beads; (C) Hydrogel-encapsulated magnetic beads and bacteria expressing sfGFP and mCherry; e: SEM image of the 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, as well as their heme-sensing 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 magnetic hydrogel microspheres sensing different concentrations of heme; e: changes in the sensing activity of magnetic hydrogel microspheres after lyophilization.

[0022] Figure 4 The procedure and results for rapid detection of inflammatory bowel disease severity using a hydrogel system; a: Schematic diagram of a hydrogel delivery platform for the detection and purification of engineered bacteria after colitis; b: Efficiency of recovering hydrogel microspheres from mouse feces; c: Statistical results of mouse body weight changes over time; d: Disease activity of each group of mice on day 5; e: Bioluminescence of microspheres recovered from mouse feces in different groups; f: Representative H&E histological images of the small intestine and colon of mice in the 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: Inflammatory factors generated by co-culturing RAW264.7 with saline and YES601@ALG; b: Comparison of serum inflammatory factors in mice after gavage administration of YES601@ALG and before gavage; c: Comparison of serum biochemical indicators in mice after gavage administration of YES601@ALG and before gavage; d: Weight changes in mice five days after gavage administration of saline and YES601@ALG, respectively; e: Representative H&E histological images of the small intestine and colon of mice in different groups.

[0024] Figure 6 Results of mouse gut microbiota and diversity analysis; ac: α diversity of gut microbiota between the two groups of mice was compared using the Chao, Shannon, and Simpson indices; d: rank-abundance curves of the microbial community; eg: ANOS1M showing the relationship between the box plot of sample distance (e), principal coordinate analysis (PCoA) plot (f), and group similarity analysis box plot (g); h: bar chart showing the relative abundance of species in the two groups; Student's T-test was used to analyze significant differences between the two groups, ns: p>0.05. Detailed Implementation

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

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0030] All reagents, materials, streptomycin, and other chemicals used in the following examples were purchased from Sigma-Aldrich. All plasmid constructions were performed on *E. coli* DH5α. The strains were cultured in streptomycin-containing lysogenic broth (LB) at an antibiotic concentration of 50 μg / mL. Magnetic Fe3O4 microparticle stock solution (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) containing 20% ​​ethanol in 1×PBS was replaced proportionally with lysogenic broth (LB) containing streptomycin (50 μg / mL).

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

[0032] This invention uses the heme-responsive whole-cell biosensor YES601 as an example to construct a hydrogel delivery and recovery system based on the whole-cell biosensor. A sodium alginate solution containing a specific ratio of YES601 bacterial culture and magnetic Fe3O4 particles is added dropwise to a calcium chloride solution via centrifugation, forming a hydrogel encapsulating YES601 and magnetic beads, i.e., magnetic hydrogel microspheres. YES601 within the delivered magnetic hydrogel microspheres reports the presence of the inflammatory marker heme through bioluminescent signal expression. The hydrogel is excreted with feces and diluted to form a fecal homogenate. The magnetic hydrogel microspheres are recovered from the fecal homogenate using magnet adsorption. After washing with water, the luminescence value is read using an ELISA reader, and the microspheres are counted by taking a photo with a mobile phone. The luminescence value is divided by the number of recovered microspheres to obtain a uniform luminescence value, which is used to assess the outcome of enteritis bleeding. The optimization strategy and application diagram of the above-mentioned whole-cell biosensor-based hydrogel delivery and recovery system are shown below. 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 is an engineered heme-sensitive Escherichia coli Nissle 1917 strain (EcN) resistant to streptomycin, used in all other bacterial experiments. Colonies were cultured in LB Miller broth and subjected to an appropriate antibiotic (100 μg / mL streptomycin) to OD. 600 To prepare the bacterial stock solution, use a concentration of 0.6, add 50% glycerol at a volume ratio of 1:1, and freeze at -80°C.

[0036] Take 20 μL of the cryopreserved strain YES601 and add it to 5 mL of LB medium (100 μg / mL streptomycin). Incubate overnight at 37°C with shaking at 220 rpm. 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 reaches 100%. 600 =0.6-0.8. Transfer the culture to sterile Eppendorf tubes and centrifuge at 6000 rpm for 10 minutes. Wash the cells once with sterile PBS to remove trace amounts of LB medium, then concentrate the cells 3.3 times and add them to LB medium (100 μg / mL streptomycin) to prepare a bacterial stock solution (2 × 10⁻⁶). 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 proportionally with magnetic Fe3O4 microparticles dissolved in LB medium (100 μg / mL streptomycin) for later use.

[0039] (3) Preparation of magnetic microparticle hydrogels

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

[0041] Magnetic hydrogel microspheres were prepared using the centrifugation method described above. Other whole-cell biosensors, such as those for detecting thiosulfate, can be encapsulated using the same method.

[0042] Example 2: Morphological characterization of magnetic hydrogel microspheres

[0043] To develop a hydrogel delivery and recovery system, this invention first identifies a non-toxic and harmless method for preparing sodium alginate hydrogel microspheres: sodium alginate, as a biodegradable and non-toxic natural polymer, immediately crosslinks in the presence of calcium chloride to form magnetic hydrogel microspheres. Figure 2 Hydrogels based on sodium alginate and calcium chloride (as shown in a and b) have been applied in many fields such as cell encapsulation and drug delivery. When gelled, their pore size is large enough to allow nutrient exchange between the encapsulated bacteria and the surrounding environment.

[0044] Microscopic imaging revealed that the prepared magnetic hydrogel microspheres had a particle size between 180 and 250 μm and roughly followed a normal distribution. Figure 2 (c) To better observe the distribution morphology of magnetic beads and bacteria within the hydrogel spheres, the encapsulated bacterial strains, simultaneously expressing both mCherry and GFP fluorescent proteins, were confocally observed with the control microspheres. Figure 1 (d) and captured SEM images of blank hydrogel, hydrogel containing only bacteria, and magnetic hydrogel microspheres containing both bacteria and magnetic beads. Figure 1 (e) The results showed that the bacteria and magnetic beads were 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 several challenges, including the extremely low pH in the stomach and bile salts in the intestines. Therefore, maintaining a sufficient number of live bacteria until they reach the gastrointestinal tract is a key factor in improving the treatment efficacy of inflammatory bowel disease (IBD). To assess the protective effect of hydrogel microspheres on bacteria in simulated gastric juice, 900 μL of SGF was added to each well of a 96-well ELISA plate, followed by 100 μL of a well-mixed hydrogel system (the hydrogel system was prepared by mixing magnetic hydrogel microparticles with LB medium containing 100 μg / mL streptomycin at a volume ratio of 1:3; the concentration of the naked bacterial control group was 5 × 10⁻⁶). 8 CFU / mL). Seal the plate with the sealing film and incubate in a microplate shaker at 37°C and 100 rpm for a gradient time. After the digestion simulation, aspirate the contents from the wells and centrifuge. For the naked bacterial control group, remove the digestion solution, resuspend, and perform a gradient dilution for plating. For the hydrogel group, remove the digestion solution and add 1 mL of sodium citrate to dissolve the sodium alginate balls. Incubate in a microplate shaker at 37°C and 100 rpm for 30 min. Centrifuge again, remove the sodium citrate solution, resuspend, and perform a gradient dilution for plating. Incubate overnight at 37°C, then take photos and count the cells. Results show ( Figure 3In Figure a), after 30 minutes of cultivation in SGF, the survival rate of the engineered bacteria encapsulated in the hydrogel was as high as 100%, while only 25% of the bacteria in the naked bacteria group survived. After 1 hour of cultivation, the survival rate of the hydrogel group was more than 10 times that of the naked bacteria group, which fully demonstrates that the magnetic hydrogel microspheres enhance the survival ability of bacteria in simulated gastric juice and are more conducive to their function in the intestine.

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

[0049] To detect the escape rate of bacteria in magnetic hydrogel microspheres, this invention added 900 μL of physiological saline to a 96-well microplate, followed by 100 μL of a well-mixed hydrogel system. The plate was then incubated on a shaker at 37°C and 100 rpm, with three replicates per group. After the digestion simulation, 100 μL of supernatant was aspirated from the microplate and directly plated. The plates were then incubated overnight at 37°C, and the bacteria were counted by taking photos with a mobile phone. Figure 3 As shown in Figure b, the results indicate that after 12 hours of cultivation, there was no significant difference between the bacterial count and the initial bacterial count. The bacterial count did not decrease during the preparation and storage process, ensuring that the effective bacterial concentration remained unchanged.

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

[0051] To examine whether the hydrogel shell affects the performance of the biosensor, this invention encapsulates the previously designed YES601 microspheres for sensing heme within a magnetic hydrogel. The prepared magnetic hydrogel microspheres (YES601@MALG) were mixed with LB medium (100 μg / mL streptomycin) at a 1:3 ratio to form a system. To better simulate the intestinal environment, simulated intestinal fluid (SIF) was chosen as the culture environment to prepare a 40 μM heme stock solution: 5 mL of 40 μM heme stock solution was prepared using 8 μL of 25 mM heme stock solution and 4992 μL of SIF (100 μg / mL streptomycin). The 40 μM heme stock solution was diluted to a gradient concentration, and 100 μL was added sequentially to the wells of the luminescence-measuring plate. The mixed magnetic hydrogel microsphere system was thoroughly mixed with a pipette tip, and 100 μL was added to the wells of the heme solution. The wells were sealed with a sealing film and incubated on a microplate shaker at 37°C and 100 rpm, with measurements taken every hour. Three parallel groups were set up for each concentration of heme. For example... Figure 3 As shown in Figure c, the results indicate that the higher the heme concentration, the shorter the time to reach the maximum luminescence intensity. Then, the luminescence intensity decreases to varying degrees over time, proving that the hydrogel shell does not affect the entry and exit of heme. Visualizing bioluminescence images can more intuitively characterize the luminescence phenomenon of the hydrogel-encapsulated biosensor sensing different concentrations of heme over time. Figure 3(d) indicates that the hydrogel shell does not affect its luminescence detection. Furthermore, the magnetic hydrogel microspheres, preserved by freeze-drying, retained over 80% of their initial sensing activity after one month of lyophilization. Figure 3 (e). In summary, the bacteria retain highly efficient sensing capabilities even when encapsulated in hydrogel.

[0052] Example 5: Detection of Recovery Rate of Magnetic Hydrogel Microspheres

[0053] The recovery of engineered bacteria after in vivo detection is a pressing problem. After in vivo detection, naked bacteria are excreted in feces. Current methods involve homogenizing mouse feces, collecting the supernatant, filtering, adding the sample to culture medium, culturing for several hours, and then detecting bioluminescence. This approach suffers from problems such as engineered bacteria remaining in the intestines, low recovery rates, and long lead times for obtaining results. The hydrogel-encapsulated magnetic beads and engineered bacteria delivery scheme proposed in this invention utilize magnetic adsorption for recovery and purification. Figure 4 (a) A magnetic rod with a protective sleeve adsorbs microspheres in fecal homogenate, then is placed in pure physiological saline. The magnetic rod is removed, and the microspheres detach from the protective sleeve, completing the recovery. The magnet is then used to adsorb the microspheres through a device, followed by washing to complete purification. This invention tested the recovery rate of the microspheres. The results showed that no microspheres were excreted within 0-2 hours after gavage in mice; recovery rates of approximately 20% were observed at 2-4 hours, 4-6 hours, and 6-8 hours; and the recovery rate reached as high as 80% within 24 hours. Figure 4 (b) Finally, the obtained microspheres were photographed with a mobile phone and detected by an ELISA reader. The entire process of detection and result acquisition took about 10 minutes, achieving the goal of efficient recovery and purification.

[0054] Example 6: Construction and Detection of a Mouse Colitis Model

[0055] A mouse model of enteritis was established: Six- to eight-week-old male C57BL / 6 mice (Southern model, China) were selected and placed in a standard temperature (20-22℃) and 12h light / 12h dark cycle for one week. They were then randomly divided into four cages, each with 3% sodium dextran sulfate (DSS, molecular weight 36,000–50,000; MP Biomedals) added to their drinking water. Mice in the first cage were fed 3% DSS water starting on day four; mice in the second cage were fed 3% DSS water starting on day two; mice in the third cage were fed 3% DSS water starting on day one; and mice in the fourth cage were fed water water only. Daily changes in mouse weight were recorded. Figure 4(c) Observe fecal bleeding in mice. Calculate the Disease Activity Index (DAI) based on weight loss, fecal bleeding, and stool viscosity to assess the severity of disease in each mouse. Mice are divided into four groups based on their DAI score on the last day: DAI 0-1 (healthy group), DAI 2-5 (mild disease group), DAI 6-8 (moderate disease group), and DAI 9-12 (severe disease group). Figure 4 (d). Then, on the fifth day, mice were gavage via a No. 10 gavage needle with a total volume of 200 μL (containing 100 μL of magnetic hydrogel microspheres and 100 μL of physiological saline) to deliver the prepared magnetic hydrogel microspheres into the mouse intestines. Fecal samples were collected for purification and analysis. The luminescence was normalized. The results showed that the more severe the disease in the mice, the higher the luminescence intensity of the microspheres. Healthy mice showed the weakest luminescence signal, and there were also significant differences in data between different disease groups. Figure 4 (e). Then, histopathological analysis of colon tissue sections in the mice confirmed that the severe disease group had the most severe enteritis, while the moderate disease group had milder enteritis. Figure 4 (f). These results demonstrate that the hydrogel-encapsulated engineered bacteria YES601 can be used for rapid and efficient detection of fecal bleeding in patients with varying degrees of colitis.

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

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

[0058] (1) Mix equal volumes of physiological saline with a bacterial concentration of 2×10⁻⁶. 8 The concentration changes of cytokines were detected after co-incubating YES601 naked bacteria and magnetic hydrogel microspheres (YES601@MALG) with RAW264.7. Cytotoxicity assay results showed ( Figure 5 In (a), compared with the saline group, the concentrations of TNF-α and IL-6 in the naked bacteria group were significantly increased, while there was no significant difference in the YES601@MALG group, demonstrating that the encapsulation of hydrogels significantly reduced the immune recognition ability of macrophages against bacteria.

[0059] (2) Subsequently, the biosafety of healthy mice was assessed. Mice were either not gavaged or gavaged with an equal volume of physiological saline and YES601@MALG (bacterial count of 2×10⁻⁶). 8 Blood samples were collected on day 5 after CFU administration to assess serum inflammatory factors and transaminases. Comparative analysis with the control group (administered normal saline) showed that serum inflammatory factor (TNF-α, IL-6) levels ( Figure 5 (b) or transaminase (ALT, AST, γ-GT) levels ( Figure 5 c) Mouse body weight ( Figure 5 No significant changes were observed in either d) or sd) of the mice. Histopathological examination of the small and colonic intestines after sacrifice on the last day 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 on day 5 after gavage administration and before gavage administration. The samples were then ground and subjected to 16S ribosomal RNA (rRNA) genome sequencing analysis. The results showed that gavage administration of YES601@MALG did not cause significant changes in the composition or diversity of the mouse gut microbiota. Figure 6 (Ah), which confirms the high biosafety of YES601@MALG.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a magnetic hydrogel-encapsulated bacterial biosensor platform in the preparation of products for detecting enteritis, characterized in that, The magnetic hydrogel-encapsulated bacterial biosensor platform includes a whole-cell biosensor encapsulated in hydrogel and magnetic microparticles, wherein the hydrogel is constructed from sodium alginate and calcium chloride, and the biosensor is a bacterium that senses heme. The bacteria are the engineered bacteria YES601 that senses heme, and the magnetic particles are magnetic Fe3O4 particles. By collecting the recipient's feces, magnetic hydrogel microspheres in the feces are adsorbed with a magnet. After washing with physiological saline, the magnetic hydrogel microspheres are collected, counted, and their luminescence values ​​are measured. The severity of intestinal inflammation is determined based on the luminescence values. The criterion for judgment is: the more severe the intestinal inflammation, the higher the luminescence intensity of the magnetic hydrogel microspheres.

2. A method for constructing a bacterial biosensor platform encapsulated in magnetic hydrogel as described in claim 1, characterized in that, Includes the following steps: A bacterial culture and a sodium alginate solution were prepared. The bacterial culture, sodium alginate solution, and magnetic microparticles were then mixed evenly. The mixture was then added dropwise to a calcium chloride solution, centrifuged, and the precipitate was collected to obtain magnetic hydrogel microparticles, which constitute the magnetic hydrogel-encapsulated bacterial biosensor platform.

3. The construction method as described in claim 2, characterized in that, The volume ratio of the bacterial culture, sodium alginate solution, and magnetic particles is 3:5:

2.

4. The construction method as described in claim 2, characterized in that, The concentration of the bacterial culture is 2×10⁻⁶. 9 The concentration of the sodium alginate solution is 2% (CFU / mL), the concentration of the magnetic particles is 10 mg / mL, and the concentration of the calcium chloride solution is 1.5% (CFU / mL).

5. The construction method as described in claim 2, characterized in that, The volume ratio of the mixture to the calcium chloride is 2:(1-1.5).

6. The construction method as described in claim 2, characterized in that, The centrifugation conditions are: 1000-1500 rpm for 15-20 minutes.

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