CN116515136A - Hydrogel microbead and application thereof in preparation of reagent for detecting colonitis and probiotics - Google Patents
Hydrogel microbead and application thereof in preparation of reagent for detecting colonitis and probiotics Download PDFInfo
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- CN116515136A CN116515136A CN202310280317.4A CN202310280317A CN116515136A CN 116515136 A CN116515136 A CN 116515136A CN 202310280317 A CN202310280317 A CN 202310280317A CN 116515136 A CN116515136 A CN 116515136A
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- probiotics
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Classifications
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Abstract
The invention provides a hydrogel microbead and application thereof in preparation of a reagent for detecting colonitis and a probiotic bacteria agent, and belongs to the technical field of nano materials. The hydrogel microbeads are prepared in a microfluidic manner, and the obtained hydrogel microbeads contain functional nano particles, wherein the functional nano particles have calcium alginate and chitosan crosslinked substance shells, so that the hydrogel microbeads have good water solubility and biocompatibility. In the present invention, when the inner core of the functional nanoparticle is carboxyl-modified Fe 3 O 4 When the method is used, the hydrogel microbeads have good magnetic separation performance, can be used for effectively performing biological separation, and are used for constructing fluorescent immune microspheres based on image processing of smart phones; when the inner core of the functional nano particle is an upper conversion material, the functional nano particle can be used as a substitute intracavity light source for treating a photothermal nano system after being coated with probiotics.
Description
Technical Field
The invention relates to the technical field of nano materials, in particular to a hydrogel microbead and application thereof in preparation of a reagent for detecting colonitis and a probiotic bacteria agent.
Background
With the continuous innovation of science and technology, the individuation, portability and economy of diagnostic instruments are gradually becoming demands of chronic patients, and these factors promote the development of integrated systems for home diagnosis and treatment. The nano particles have the characteristics of magnetism, optical property, radioactivity and the like, can be used as a contrast agent to enhance the contrast of imaging, and can enhance the early detection, diagnosis and treatment of diseases by influencing technology. Nanoparticles are now used in the assisted diagnosis and treatment of medical imaging such as CT, MRI and optical imaging composite slices. The optical diagnosis technology plays an important role in clinical application by the advantages of simplicity, non-contact, high precision, non-invasiveness and the like.
Colitis is also called nonspecific ulcerative colitis, and has the symptoms of slow onset and varying severity, and mainly clinically manifests as diarrhea, abdominal pain, mucous stool, bloody stool, tenesmus, constipation, and inability to relieve constipation within days. If the nanoparticle is prepared, the nanoparticle can be used for diagnosis of colonitis and treatment of colonitis, and can be used for diagnosing and treating diseases of patients at home, so that great convenience is brought to colonitis patients.
Disclosure of Invention
In view of the above, the invention aims to provide a hydrogel microbead and application thereof in preparation of a reagent for detecting colonitis and a probiotic bacterial agent. The hydrogel microbeads provided by the invention can be used for diagnosis and treatment of colonitis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of hydrogel microbeads, which comprises the following steps:
providing a microfluidic chip, wherein the microfluidic chip comprises an internal phase pipeline, an external phase pipeline and a main pipeline converging the internal phase pipeline and the external phase pipeline; the internal phase pipeline is provided with an internal phase material inlet, the external phase pipeline is provided with an external phase material inlet, and the main pipeline is provided with a nano hydrogel outlet;
injecting an internal phase material into the internal phase material inlet, injecting an external phase material into the external phase pipeline inlet, mixing the external phase material and the internal phase material in a main pipeline, irradiating the main pipeline by using an ultraviolet light source, and obtaining hydrogel microbeads at the nano hydrogel outlet;
the internal phase material comprises polyethylene glycol diacrylate, polyethylene glycol, an ultraviolet initiator and functional nano particles, wherein the functional nano particles comprise a core and a shell layer, and the core is carboxyl modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan; the up-conversion material comprises Yb 3+ 、Ho 3+ 、Er 3+ And Tm 3+ One or more of the following;
the external phase material comprises dimethyl siloxane.
Preferably, the volume ratio of the polyethylene glycol diacrylate to the polyethylene glycol is 5.8-6:1-1.2;
the volume ratio of the polyethylene glycol diacrylate to the photoinitiator is 5.8-6:1-1.2;
the mass ratio of the polyethylene glycol diacrylate to the functional nano particles is 2.7-3:1-1.4.
Preferably, the injection rate of the internal phase material is 72-80 mu L min -1 The method comprises the steps of carrying out a first treatment on the surface of the The injection rate of the external phase material is 3150-3200 mu L min -1 。
The invention provides hydrogel microbeads prepared by the preparation method, which comprise photo-curing hydrogel and functional nano particles distributed on the surface and inside of the photo-curing hydrogel, wherein the functional nano particles comprise an inner core and a shell layer, and the inner core is carboxyl modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is calcium alginate and chitosanIs a crosslinked product of (a); the photo-curing hydrogel is obtained by photo-curing polyethylene glycol diacrylate, polyethylene glycol and dimethyl siloxane.
The invention provides application of the hydrogel microbead in preparation of a reagent for detecting colonitis or preparation of a probiotic bacterial agent.
The invention provides a kit for detecting colonitis based on optical sensing of a smart phone, which comprises the magnetic hydrogel microbead, a monoclonal antibody of fecal calprotectin and a secondary antibody of phycoerythrin marked fecal calprotectin.
The invention provides a probiotic bacterial agent, which is obtained by wrapping probiotics on the up-conversion hydrogel microbeads in the scheme, wherein the inner cores of functional nano particles in the hydrogel microbeads are up-conversion materials.
Preferably, the probiotic comprises recombinant probiotic; the original bacteria of the recombinant probiotics comprise escherichia coli Nissle 1917.
Preferably, the recombinant probiotics comprise recombinant plasmids; the recombinant plasmid takes pDawn as an original vector; the recombinant plasmids comprise a first recombinant plasmid and/or a second recombinant plasmid; the coding gene of IL-10 is inserted into the first recombinant plasmid, and insertion sites are SalI and HindIII; the nucleotide sequence of the coding gene of the IL-10 is shown as SEQ ID NO. 3; the second recombinant plasmid is inserted with a reporter gene, and insertion sites are SalI and HindIII; the nucleotide sequence of the reporter gene is shown as SEQ ID NO. 4.
The invention provides application of the probiotic bacteria agent in preparing foods, health products or medicines containing probiotics; the dosage form of the medicine comprises an oral preparation.
The invention provides a preparation method of hydrogel microbeads, which adopts a microfluidic mode to prepare the hydrogel microbeads, wherein the obtained hydrogel microbeads contain functional nano particles, and the functional nano particles have calcium alginate and chitosan crosslinked substance shells, so that the hydrogel microbeads have good water solubility and biocompatibility. In the present invention, when the inner core of the functional nanoparticle is carboxyl-modified Fe 3 O 4 When the method is used, the hydrogel microbeads have good magnetic separation performance, can be used for effectively performing biological separation, and are used for constructing fluorescent immune microspheres based on image processing of smart phones; when the inner core of the functional nano particle is an upper conversion material, the functional nano particle can be used as a substitute intracavity light source for treating a photothermal nano system after being coated with probiotics.
The invention provides a kit for detecting colonitis based on optical sensing of a smart phone, which comprises magnetic hydrogel microbeads, monoclonal antibodies of fecal calprotectin (hereinafter called calprotectin, CALP) and secondary antibodies of phycoerythrin marked calprotectin. The kit provided by the invention can detect calprotectin which is a marker of the colitis based on a double-antibody sandwich immunoassay method, amino in a monoclonal antibody of the calprotectin can be combined with carboxyl in a magnetic hydrogel microbead through a covalent bond in the detection process, phycoerythrin in a secondary antibody of the calprotectin is used as a fluorescent signal probe, and the fluorescent intensity can reflect the content change of the calprotectin. The invention can realize the rapid diagnosis of the colonitis by utilizing the smart phone to acquire the fluorescence signal.
Drawings
FIG. 1 is a schematic representation of the synthesis and photocuring of magnetic hydrogel microbeads (MBMs);
FIG. 2 is Fe 3 O 4 、Fe 3 O 4 Zeta potential of-COOH, blank hydrogel microbeads and MBMs;
FIG. 3 is a graph showing the linear relationship between the quantitative detection of the concentration of CALP and FL by a smart phone;
FIG. 4 is a graph showing the results of analysis of the concentration of CALP in mouse faeces and serum samples from acute mouse colitis by ELSA;
FIG. 5 is the results of analysis of the concentration of CALP in mouse faeces and serum samples in chronic mouse colitis by ELISA;
FIG. 6 is a construction of a EcN reporter gene and EcN interleukin 10;
FIG. 7 is a flow cytometry analysis and was performed in near infrared (480 nm,4 mW.multidot.mm) -2 ) And EcN (pDawn-GFP) with GFP counted under dim light conditions;
FIG. 8 shows the concentration of mIL-10 in the supernatant of ELISA assay (n=3);
FIG. 9 is a cross-well system experimental design;
FIG. 10 is a view of UCMs observed under a fluorescence microscope;
FIG. 11 is a growth curve of ECN cultured in liquid medium, SGF and SIF, respectively, at 37 ℃;
FIG. 12 shows IL-10-His-Tag and IL-10 content in the intestinal mucosa of transplanted mice after 60min application of photothermal nanosystems in EcN;
FIG. 13 is a graph showing the results of analysis of UC mouse fecal biomarker CALP by a smartphone fluorescence sensing platform;
FIG. 14 is a graph showing IL-6 levels of mice intestinal tissue homogenates measured by ELISA;
FIG. 15 is a graph showing the concentration of CALP over time.
Detailed Description
The invention provides a preparation method of hydrogel microbeads, which comprises the following steps:
providing a microfluidic chip, wherein the microfluidic chip comprises an internal phase pipeline, an external phase pipeline and a main pipeline converging the internal phase pipeline and the external phase pipeline; the internal phase pipeline is provided with an internal phase material inlet, the external phase pipeline is provided with an external phase material inlet, and the main pipeline is provided with a nano hydrogel outlet;
injecting an internal phase material into the internal phase material inlet, injecting an external phase material into the external phase pipeline inlet, mixing the external phase material and the internal phase material in a main pipeline, irradiating the main pipeline by using an ultraviolet light source, and obtaining hydrogel microbeads at the nano hydrogel outlet;
the internal phase material comprises polyethylene glycol diacrylate, polyethylene glycol, an ultraviolet initiator and functional nano particles, wherein the functional nano particles comprise a core and a shell layer, and the core is carboxyl modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan; the up-conversion material comprises Yb 3+ 、Ho 3+ 、Er 3+ And Tm 3+ One or more of the following;
the external phase material comprises dimethyl siloxane.
In the present invention, when the inner core of the functional nanoparticle is carboxyl-modified Fe 3 O 4 When the hydrogel microbeads are magnetic hydrogel microbeads; when the inner core of the functional nanoparticle is an up-conversion material, the hydrogel microbead is an up-conversion hydrogel microbead.
The invention firstly provides a micro-fluidic chip, which comprises an internal phase pipeline, an external phase pipeline and a main pipeline converging the internal phase pipeline and the external phase pipeline; the internal phase pipeline is provided with an internal phase material inlet, the external phase pipeline is provided with an external phase material inlet, and the main pipeline is provided with a nano hydrogel outlet. In the present invention, the material of the microfluidic chip is preferably PMMA. In the present invention, the microfluidic chip preferably has an inner diameter of 530nm and an outer diameter of 690nm.
In the invention, the internal phase material comprises polyethylene glycol diacrylate, polyethylene glycol, ultraviolet initiator and functional nano particles, wherein the functional nano particles comprise an inner core and a shell layer, and the inner core is carboxyl modified Fe 3 O 4 Or upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan.
In the present invention, the preparation method of the functional nanoparticle preferably includes the steps of:
dispersing the core material in toluene to obtain a core material dispersion;
Mixing the core material dispersion liquid with sodium alginate solution to obtain a core material-sodium alginate composite;
and mixing the core material-sodium alginate complex with saturated calcium chloride solution and chitosan, and performing a crosslinking reaction to obtain the functional nano particles.
In the present invention, the concentration of the core material dispersion is preferably 9.6 to 10mol/mL, more preferably 9.8 to 10mol/mL. In the present invention, the concentration of the sodium alginate solution is preferably 0.85 to 1wt%, more preferably 0.9 to 1wt%. In the invention, the mass ratio of the chitosan to the sodium alginate is preferably 8.6-10:2.4-3, and more preferably 10:3.
In the present invention, the temperature of the crosslinking reaction is preferably 20 to 24 ℃, more preferably 21 to 23 ℃; the time is preferably 30 to 45 minutes, more preferably 35 to 40 minutes.
In the present invention, the volume ratio of the polyethylene glycol diacrylate to the polyethylene glycol in the internal phase material is preferably 5.8-6:1-1.2, more preferably 6:1.
In the present invention, the ultraviolet initiator is preferably 2-hydroxy-2-methyl acetone; the volume ratio of the polyethylene glycol diacrylate to the ultraviolet initiator is preferably 5.8-6:1-1.2, and more preferably 6:1; the mass ratio of the polyethylene glycol diacrylate to the functional nano particles is preferably 2.7-3:1-1.4, and more preferably 3:1-1.2.
In the present invention, the particle diameter of the functional nanoparticle is preferably 5.6 to 16.3nm, more preferably 8 to 12nm. In the present invention, the functional nanoparticles are preferably added in the form of an aqueous suspension, and the concentration of the aqueous suspension of functional nanoparticles is preferably 10 to 11.5mol/kg, more preferably 10.5 to 11mol/kg.
The invention simultaneously injects the internal phase material into the internal phase material inlet and the external phase material into the external phase pipeline inlet, and irradiates the main pipeline by using an ultraviolet light source to obtain the hydrogel microbeads at the nano hydrogel outlet. In the present invention, the injection rate of the internal phase material is preferably 72 to 80. Mu.L.min -1 More preferably 75 to 78. Mu.L.min -1 The method comprises the steps of carrying out a first treatment on the surface of the The injection rate of the external phase material is preferably 3150-3200 mu L.min -1 More preferably 3160 to 3180. Mu.L.min -1 . In the present invention, the wavelength of the ultraviolet light source is preferably 365nm, and the light intensity is preferably 68Uw/cm 2 。
The invention provides hydrogel microbeads prepared by the preparation method, which comprise functional nanoparticles and photo-curing hydrogel wrapping the functional nanoparticles, wherein the functional nanoparticles comprise cores and shell layers, and the cores are carboxyl-modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan; the photo-curing hydrogel is obtained by photo-curing polyethylene glycol diacrylate, polyethylene glycol and dimethyl siloxane. In the present invention, the hydrogel microbeads have particle diameters Preferably 12nm.
The invention provides application of the hydrogel microbead in preparation of a reagent for detecting colonitis or preparation of a probiotic bacterial agent.
The invention provides a kit for detecting colonitis based on optical sensing of a smart phone, which comprises the magnetic hydrogel microbead, a monoclonal antibody of calprotectin and a secondary antibody of calprotectin marked by phycoerythrin.
In the invention, the nucleotide sequence of the monoclonal antibody for encoding calprotectin is shown as SEQ ID NO.1, and specifically comprises the following steps:
atgagtggctgtagaaagcggtgtaaacgtgaaatactgaaatttgcccagtaccttctcagactattaacaggttctcttcatacag。
in the invention, the nucleotide sequence of the secondary antibody for encoding the phycoerythrin marked calprotectin is shown as SEQ ID NO.2, and specifically comprises the following steps:
atggctgactcagaagcactcccctcccttgctggggacccagtggctgtggaagccttgctccgggccgtgtttggggttgttgtggatgaggccattcagaaaggaaccagtgtctcccagaaggtctgtgagtggaaggagcctgaggagctgaagcagctgctggatttggagctgcggagccagggcgagtcacagaagcagatcctggagcggtgtcgggctgtgattcgctacagtgtcaagactggtcaccctcggttcttcaaccagctcttctctgggttggatccccatgctctggccgggcgcattatcactgagagcctcaacaccagccagtacacatatgaaatcgcccccgtgtttgtgctcatggaagaggaggtgctgaggaaactgcgggccctggtgggctggagctctggggacggaatcttctgccctggtggctccatctccaacatgtatgctgtaaatctggcccgctatcagcgctacccggattgcaagcagaggggcctccgcacactgccgcccctggccctattcacatcgaaggagtgtcactactccatccagaagggagctgcgtttctgggacttggcaccgacagtgtccgagtggtcaaggctgatgagagagggaaaatggtccccgaggatctggagaggcagattggtatggccgaggctgagggtgctgtgccgttcctggtcagtgccacctctggcaccactgtgctaggggcctttgaccccctggaggcaattgctgatgtgtgccagcgtcatgggctatggctgcatgtggatgctgcctggggtgggagcgtcctgctgtcacagacacacaggcatctcctggatgggatccagagggctgactctgtggcctggaatccccacaagctcctcgcagcaggcctgcaatgctctgcacttcttctccaggatacctcgaacctgctcaagcgctgccatgggtcccaggccagctaccttttccagcaggacaagttctacgatgtggctctggacacgggagacaaggtggtgcagtgtggccgccgtgtggactgtctgaagctgtggctcatgtggaaggcacagggcgatcaagggctggagcggcgcatcgaccaggcctttgtccttgcccggtacctggtggaggaaatgaagaagcgggaagggtttgagctagtcatggagcctgagtttgtcaatgtgtgtttctggttcgtaccccccagcctgcgagggaagcaggagagtccagattaccacgaaaggctgtcaaaggtggcccccgtgctcaaggagcgcatggtgaaggagggctccatgatgattggctaccagccccacgggacccggggcaacttcttccgtgtggttgtggccaactctgcactgacctgtgctgatatggacttcctcctcaacgagctggagcggctaggccaggacctgtgaatgagtggctgtagaaagcggtgtaaacgtgaaatactgaaatttgcccagtaccttctcagactattaacaggttctcttcatacag。
in the invention, the kit for detecting the colonitis based on the optical sensing of the smart phone also preferably comprises a buffer solution and a glass slide. In the present invention, the buffer solution is preferably a PBS buffer solution and a PBST buffer solution. Calprotectin has good sensibility to intestinal mucosa healing and has good correlation with endoscopic examination results, and the calprotectin is used as a marker for detecting colonitis and is used for reflecting the pathological change degree of colonitis.
In the invention, the using method of the kit for detecting colonitis based on optical sensing of the smart phone preferably comprises the following steps:
Performing carboxyl activation on the magnetic hydrogel microbeads to obtain carboxyl activated magnetic hydrogel microbeads;
mixing carboxyl activated magnetic hydrogel microbeads, a monoclonal antibody of calprotectin and PBS buffer solution, and performing first incubation and washing to obtain a first incubation product;
mixing a sample to be detected, the first incubation product and PBS buffer solution, and performing second incubation and washing to obtain a second incubation product;
mixing the second incubation product, the phycoerythrin-labeled secondary antibody of calprotectin and PBS buffer solution, and performing third incubation and washing to obtain fluorescent immunomagnetic hydrogel microbeads;
placing the fluorescent immunomagnetic hydrogel microbeads on a glass slide, taking an optical picture of the fluorescent immunomagnetic hydrogel microbeads by using a smart phone, and performing image processing on the optical picture by using an image processing program to obtain the fluorescence intensity of the fluorescent immunomagnetic hydrogel microbeads;
obtaining the concentration of calprotectin the sample to be detected according to the fluorescence intensity and a preset standard curve; the standard curve is a linear relation curve of calprotectin concentration logarithm and fluorescence intensity.
The invention activates the carboxyl of the magnetic hydrogel microbead to obtain the carboxyl activated magnetic hydrogel microbead. In the present invention, the present invention preferably washes the magnetic hydrogel microbeads before the carboxyl groups are activated. In the present invention, the washing preferably includes ethanol washing, ddH washing, which are sequentially performed 2 O-wash and PBS buffer wash.
In the invention, the carboxyl activating agent used for carboxyl activation is preferably EDC.HCl and sulfo NHS, and the mass ratio of EDC.HCl to sulfo NHS is preferably 1:2.
In the present invention, the temperature of the carboxyl group activation is preferably room temperature, and the time is preferably 30min.
The invention mixes carboxyl activated magnetic hydrogel microbeads, calprotectin monoclonal antibodies and PBS buffer solution, and carries out first incubation and washing to obtain a first incubation product. In the present invention, the first incubation product is a monoclonal antibody coated magnetic hydrogel microbead of calprotectin, denoted CALP-mAb-MBMs. In the present invention, the temperature of the first incubation is preferably room temperature and the time is preferably 3 hours. In the present invention, the washing liquid used for the washing is preferably a PBST buffer solution. In the present invention, the first incubation product is preferably placed in a PBST buffer containing 3% bsa and stored at 4 ℃.
The sample to be detected, the first incubation product and PBS buffer solution are mixed, and the second incubation and washing are carried out, so that a second incubation product is obtained. In the present invention, the sample to be tested is preferably serum or feces. In the present invention, the temperature of the second incubation is preferably room temperature, and the time is preferably 45min. In the present invention, the washing liquid is preferably a PBST buffer solution.
The second incubation product, the phycoerythrin marked calprotectin secondary antibody and PBS buffer solution are mixed for third incubation and washing, and the fluorescent immunomagnetic hydrogel microbeads are obtained. In the present invention, the temperature of the third incubation is preferably room temperature, and the time is preferably 30min; the third incubation is preferably performed under dark conditions. In the present invention, the washing liquid is preferably PBS at 4 ℃.
According to the invention, the fluorescent immunomagnetic hydrogel microbeads are placed on a glass slide, an optical photo of the fluorescent immunomagnetic hydrogel microbeads is taken by using a smart phone, and the optical photo is subjected to image processing by using an image processing program, so that the fluorescence intensity of the fluorescent immunomagnetic hydrogel microbeads is obtained. In the invention, when the smart phone shoots an optical photo of the fluorescent immunomagnetic hydrogel microbead, the distance between the lens of the smart phone and the glass slide is preferably 1.5mm.
According to the fluorescence intensity and a preset standard curve, the concentration of calprotectin in the sample to be detected is obtained; the standard curve is a linear relation curve of calprotectin concentration logarithm and fluorescence intensity. In the present invention, the method for obtaining a standard curve preferably includes the steps of:
Providing a standard solution of calprotectin at a known concentration in a gradient;
and (3) taking the calprotectin standard solution with the gradient and known concentration as a sample to be tested, preparing fluorescent immunomagnetic hydrogel microbeads according to the method, testing the fluorescence intensity, taking the logarithm of the calprotectin concentration as an abscissa, taking the fluorescence intensity as an ordinate, and drawing a standard curve.
As a specific embodiment of the present invention, the standard curve is: y=57.05x+8.682.
The invention also provides a probiotic bacterial agent, which is obtained by wrapping probiotics with the up-conversion hydrogel microbeads according to the scheme, wherein the inner cores of the functional nano particles in the hydrogel microbeads are up-conversion materials.
In the present invention, the probiotics preferably include recombinant probiotics; the original bacteria of the recombinant probiotics include, but are not limited to, escherichia coli Nissle 1917 (EcN). EcN is non-pathogenic and non-invasive and is thus selected as a chassis organism, and a specific safe vector can be obtained by genetic engineering EcN.
In the present invention, the recombinant probiotics comprise recombinant plasmids.
In the present invention, the recombinant plasmid preferably uses pDawn as the original vector. The pDawn vector can support flexible manipulation of EcN gene expression.
In the present invention, the recombinant plasmid includes a first recombinant plasmid and/or a second recombinant plasmid; the first recombinant plasmid is inserted with a coding gene of IL-10, and insertion sites are preferably SalI and HindIII; the nucleotide sequence of the coding gene of IL-10 is shown as SEQ ID NO.3, and specifically comprises the following steps:
atgcctggctcagcactgctatgctgcctgctcttactgactggcatgaggatcagcaggggccagtacagccgggaagacaataactgcacccacttcccagtcggccagagccacatgctcctagagctgcggactgccttcagccaggtgaagactttctttcaaacaaaggaccagctggacaacatactgctaaccgactccttaatgcaggactttaagggttacttgggttgccaagccttatcggaaatgatccagttttacctggtagaagtgatgccccaggcagagaagcatggcccagaaatcaaggagcatttgaattccctgggtgagaagctgaagaccctcaggatgcggctgaggcgctgtcatcgatttctcccctgtgaaaataagagcaaggcagtggagcaggtgaagagtgattttaataagctccaagaccaaggtgtctacaaggccatgaatgaatttgacatcttcatcaactgcatagaagcatacatgatgatcaaaatgaaaagctaa；
the reporter gene is inserted into the second recombinant plasmid; the nucleotide sequence of the reporter gene is shown as SEQ ID NO.4, and specifically comprises the following steps:
aagacgccaaaaacataaagaaaggcccggcgccattctatccgctggaagatggaaccgctggagagcaactgcataaggctatgaagagatacgccctggttcctggaacaattgcttttacagatgcacatatcgaggtggacatcacttacgctgagtacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgtatgcagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacgacatttataatgaacgtgaattgctcaacagtatgggcatttcgcagcctaccgtggtgttcgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaaaaaagctcccaatcatccaaaaaattattatcatggattctaaaacggattaccagggatttcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaatacgattttgtgccagagtccttcgatagggacaagacaattgcactgatcatgaactcctctggatctactggtctgcctaaaggtgtcgctctgcctcatagaactgcctgcgtgagattctcgcatgccagagatcctatttttggcaatcaaatcattccggatactgcgattttaagtgttgttccattccatcacggttttggaatgtttactacactcggatatttgatatgtggatttcgagtcgtcttaatgtatagatttgaagaagagctgtttctgaggagccttcaggattacaagattcaaagtgcgctgctggtgccaaccctattctccttcttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaattgcttctggtggcgctcccctctctaaggaagtcggggaagcggttgccaagaggttccatctgccaggtatcaggcaaggatatgggctcactgagactacatcagctattctgattacacccgagggggatgataaaccgggcgcggtcggtaaagttgttccattttttgaagcgaaggttgtggatctggataccgggaaaacgctgggcgttaatcaaagaggcgaactgtgtgtgagaggtcctatgattatgtccggttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctacattctggagacatagcttactgggacgaagacgaacacttcttcatcgttgaccgcctgaagtctctgattaagtacaaaggctatcaggtggctcccgctgaattggaatccatcttgctccaacaccccaacatcttcgacgcaggtgtcgcaggtcttcccgacgatgacgccggtgaacttcccgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaagggcggaaagatcgccgtgtaa。
in the invention, the preparation method of the probiotic bacteria agent preferably comprises the following steps:
dispersing the upper conversion material in toluene to obtain an upper conversion material dispersion liquid;
mixing probiotics, the dispersion liquid of the upper conversion material and sodium alginate solution to obtain an upper conversion material-sodium alginate complex;
mixing the upper conversion material-sodium alginate complex with saturated calcium chloride solution and chitosan, and performing a crosslinking reaction to obtain functional nano particles loaded with probiotics;
and taking the functional nano particles with the embedded probiotics as raw materials of inward materials, simultaneously injecting the inward materials into an inward material inlet, injecting the outward materials into an outward pipeline inlet, irradiating a main pipeline by using an ultraviolet light source, and obtaining the up-conversion hydrogel microbeads with the embedded probiotics at a nano hydrogel outlet.
In the present invention, the probiotics are preferably used as raw materials in the form of bacterial liquid, and the effective viable cell number concentration of the probiotics in the bacterial liquid is preferably (1-9) ×10 9 CFU/mL; the ratio of the mass of the up-conversion material to the volume of the probiotics is preferably 100g:1mL.
The probiotics entrapment success rate of the preparation method is 60 percent on average.
The invention also provides application of the probiotic bacteria agent in preparation of foods, health products or medicines containing probiotics; the dosage form of the medicine comprises an oral preparation.
The food, health care product or medicine prepared based on the probiotics preparation can safely survive in early Gastrointestinal (GI) tract, and the bioavailability of the probiotics can be improved.
The hydrogel microbeads and the application thereof in preparing the reagent for detecting colonitis and the probiotic bacteria are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of magnetic hydrogel microbeads (MBMs) comprising the steps of:
(1) Fe is added to 3 O 4 -COOH Magnetic Nanoparticles (MNPs) were dispersed in toluene at a mass concentration of 10mol/mL.
(2) 5 ml of Fe 3 O 4 Washing and re-suspending the COOH magnetic nanoparticle dispersion in a 1% Sodium Alginate (SA) solution to obtain MNPs@SA; the mnps@sa solution was then dissolved in a saturated calcium chloride solution using the syringe thrust. Finally, magnetically functional nanoparticles (MGPs) were prepared by electrostatic interaction of calcium alginate with chitosan. The MGPs were vacuum freeze-dried and stored at room temperature.
(3) A microfluidic chip was fabricated with two syringe needles inserted into the inner and outer inlet ports, the outlet side connected to a polyethylene tube, the inlet port connected to a syringe containing prepolymer and the outlet port connected to an empty centrifuge tube.
(4) An ultraviolet light source with an ultraviolet driver is arranged on an external light source interface of the fluorescent inverted microscope. PEGDA-700 liquid, polyethylene glycol 200 liquid, ultraviolet initiator 2-hydroxy-2-methyl acetone (HMPP) and magnetic functional nanoparticle suspension (Fe 3 O 4 -COOH suspension concentration 10 mol/mL) at 6:1:1:2 in a volume ratio to form a prepolymer solutionAnd (3) liquid. The prepolymer was injected at an inlet in the syringe at a rate of 80. Mu.L.min -1 . Injecting dimethylsiloxane into the external inlet of the syringe at a rate of 3200. Mu.L.min -1 After uv curing, magnetic hydrogel microbeads (MBMs) were obtained. The collected hydrogel microbeads were treated with 1 XPhosphate buffer (PBS) (0.01 mol.L) -1 Ph 7.4), resuspended in 1 x phosphate buffer and stored in 4 ℃ buffer until use.
The synthesis and photocuring of magnetic hydrogel microbeads (MBMs) is schematically shown in fig. 1.
After modification by calcium alginate and chitosan, fe 3 O 4 TEM of-COOH shows that it has good monodispersed water solubility, a uniform particle size of about 12nm.
Optical images (MBMs) of magnetic hydrogel microbeads under fluorescence microscopy show that the average diameter of the MBMs is about 200 μm, and the internal structure is compact and dense.
Omitting the addition of the magnetic functional nanoparticle suspension, and preparing the blank hydrogel microbeads according to the method of the step (4). Fe (Fe) 3 O 4 、Fe 3 O 4 The zeta potential of-COOH, blank hydrogel microbeads and MBMs is shown in FIG. 2. As can be seen from FIG. 2, fe 3 O 4 The carboxylic group of-COOH was successfully coupled and subsequently available for attachment to the hydroxyl group of PEGDA-700 to form MBMs. Loaded with Fe 3 O 4 after-COOH, the Zeta potential of MBMs was significantly altered (-12.52-24.83 mV). Due to the presence of carboxyl groups (-COOH) on the surface of MBMs, their characteristic absorption of delta (O-H) and delta (C-O) in Fourier transform infrared spectroscopy (FTIR) is 3450 and 1643cm, respectively -1 . In conclusion, the research results show that MBMs can be prepared in batches, have multifunctional groups on the surfaces, and have wide application prospects in sensing application.
Example 2
The method for detecting the colonitis based on the optical sensing of the smart phone comprises the following steps:
(1) MBMs pretreatment: ethanol and ddH 2 O and 1 XPBS were washed sequentially with 100mg MBMs in 1.5ml tubes. MBMs were dissolved in 1mL1 XPBS, and 1.2mL EDC. HCl (0.17 g. ML) -1 ) And 1.2mL of sulfoNHS (0.34 g·ml -1 ). The solution was incubated at room temperature for 30 minutes to activate the carboxyl groups and washed three times with 1×pbs.
(2) MBMs were added to 500. Mu.L of 1 XPBS of a monoclonal antibody (CALP) -mAb to calprotectin and incubated for 2h at room temperature. The CALP-mAb coated MBMs were washed with PBST (pbs+0.02% tween 20) and stored overnight at 4 ℃ in 200 μl PBST with 3% bsa before further use.
(3) First, the CALP diluted standard was placed in a 1.5ml test tube. Then, CALP-mAb coated MBMs were added and incubated at room temperature for 45 minutes. Then, 200. Mu.L of PBST was washed twice, and phycoerythrin-labeled secondary antibody (PcAb-PE) of 200. Mu.L of CALP, which was 320-fold diluted, was added thereto, and incubated in darkness at room temperature for 30min. Finally, MBMs are washed again with PBST and dissolved in ddH 2 O, then drop-in onto a clean glass slide, take an image with a cell phone, and use an image processing application to obtain fluorescence intensity.
The linear relationship between the quantitative detection of the CALP and the concentration of the FL by the smart phone is shown in figure 3. As can be seen from fig. 3, the CALP concentration has a good linear correlation with the fluorescence intensity (FL).
The interference effect was evaluated in a practical simulated environment by selecting C-reactive protein (CRP) and matrix metalloproteinase 9 (Mmp-9). The results showed that only CALP induced reaction, corresponding to mAb, was found in the reaction solution at the same concentration (100 ng. Ml -1 ) The Mmp-9 and CRP of (C) have no obvious effect on the reaction. The kit for detecting the colonitis based on the optical sensing of the smart phone has good specificity.
The concentration of sodium dextran sulfate (DSS) in the feces and serum of mice chronic UC and acute or acute myocardial infarction patients was determined. The results of analysis of the concentration of CALP in the mouse faeces and serum samples of acute mouse colitis by ELSA are shown in fig. 4, and the results of analysis of the concentration of CALP in the mouse faeces and serum samples of chronic mouse colitis by ELISA are shown in fig. 5. As can be seen from FIGS. 4 and 5, the concentration of CALP in feces and serum reached 90 and 250 ng.ml, respectively -1 。
The recovery rates (n=3) of CALP, each of which had different concentrations, were added to the samples to be tested, are shown in table 1.
Recovery of CALP at different concentrations in the sample of table 1 (n=3)
As can be seen from Table 1, the variability (CV) was from 102% to 109% below 6%, and the detection time was shortened to 1.5h. The kit for detecting the colonitis based on the optical sensing of the smart phone has higher stability, and shows that the fluorescent signal acquisition system based on the smart phone can be used for quantitative detection of the conventional fecal disease biomarker, and provides user-friendly point-of-care testing (POCT) equipment for patients.
Example 3
1. Construction of light-responsive E.coli Nissle1917-mIL-10 and bioavailability test thereof
The light as an induction factor has the advantages of being fast and nontoxic, and being capable of rapidly transmitting an induction signal into cells, and can realize accurate expression of a target gene in time space on the premise of not changing host metabolism conditions, thereby having better application potential. In actual operation, the expression level of the target gene can be regulated according to the illumination intensity and time, or the light-dependent conformational change of the light effect protein can be caused by switching illumination and darkness conditions, so that the reversible induction effect can be achieved.
1) The light response EcN strain (pDawn-reporter gene) was genetically engineered to explore the light response efficiency of pDawn (fig. 6). The blue fluorescent gene fragment is respectively connected to pDAWN-reporter gene and pDAWN-mIL10 plasmid, and insertion sites are SalI and HindIII; the constructed plasmid was transformed into strain EcN to give luciferase expression EcN (i.e., ecN-LuxCdABE).
Verification of light response EcN strain: in the near infrared (480 nm,4 mW.multidot.mm) -2 ) After 30min of irradiation, the luminescence of the constructed EcN-LuxCdABE is increased by 106 times. Flow cytometry examined fluorescence at different times, indicating that light-responsive Green Fluorescent Protein (GFP) reached saturated expression within 1 h. Strains in logarithmic growth phase (od=0.6) were irradiated with near infrared light for 1hAfter that, confocal microscopy showed that the fluorescence intensity of GFP also reached saturation. By fusing ssrA protease tag to the C-terminus of GFP, an unstable GFP protein, ecN-GFP-ssrA, was constructed in which the amino acid sequence of GFP is shown in SEQ ID NO.5, specifically:
MKPSDDKAQLSGLAQSEESSLDVDHQSFPCSPSIQPVASGCTHTENSAAYFLWPTSNLQHCAAEGRANYFGNLQKGLLPRHPGRLPKGQQANSLLDLMTIRAFHSKILRRFSLGTAVGFRIRKGDLTDIPAILVFVARKVHKKWLNPAQCLPAILEGPGGVWCDVDVVEFSYYGAPAQTPKEQMFSELVDKLCGSDECIGSGSQVASHETFGTLGAIVKRRTGNKQVGFLTNHHVAVDLDYPNQKMFHPLPPNLGPGVYLGAVERATSFITDDVWYGIYAGTNPDMPPHYGFAETFVRADGAFIPFADDFDISTVTTVVRGVGDIGDVKVIDLQCPLNSLIGRQVCKVGRSSGHTTGTVMAYALEYNDEKGICFFTDILVVGENRQTFDLEGDSGSLIILTSQDGEKPRPIGIIWGGTANRGRLKLTSDHGPENWTSGVDLGRLLDRLELDIIITNESLQDAVQQQRFALVAAVTSAVGESSGVPVAIPEEKIEEIFEPLGIQIQQLPRHDVAASGTEGEEASNTVVNVEEHQFISNFVGMSPVRDDQDAPRSITNLNNPSEEELAMSLHLGDREPKRLRSDSGSSLDLEK。
EcN-GFP-ssrA increases fluorescence by two times after irradiation with near infrared light for 6 hours, and decreases fluorescence intensity by 2 times after 2 hours of cultivation in a dark environment; subsequently, the fluorescence increased by a factor of 1.5 again in the next round of near infrared light (fig. 7). These results indicate that the pDawn module can support flexible manipulation of EcN gene expression.
After observing the time course of population expansion using the reporter construct, it was replaced with mouse interleukin 10 (mIL-10), constituting a EcN strain secreting mIL-10 (pDAWN-mIL-10).
The construction process of pDawn-mIL-10 is as follows:
(1) 2 μl of recombinant plasmid pDAWN-mIL-10 (concentration higher than 1000 ng/. Mu.l) and 50 μl of competent cell suspension of Escherichia coli are taken, mixed gently, added into a 0.2cm electric shock cup with coldness, and electric shock conditions are set to be 1.5lV,400 Ω;
(2) Transferring the bacterial liquid after electric shock into an EP tube, adding 0.9mL of a culture medium containing resistance, culturing for 3 hours at 37 ℃, centrifuging, precipitating bacterial bodies, reserving 100 mu l of the culture medium, blowing and resuspension bacterial bodies, coating the bacterial bodies on a flat plate, and culturing overnight at 37 ℃;
(3) Positive colonies were picked.
Light-induced 1h post-culture by ELISA assayThe concentration of mIL-10 in the supernatant of the nutrient solution is 3 ng.ml -1 (FIG. 8). The same results were obtained for measuring the content of mIL-10 in the intracellular supernatant by Coomassie brilliant blue staining. To verify the biological activity of the secreted mIL-10 of EcN, we co-cultured EcN with RAW264.7 by a trans-well system and exposed it to near infrared light for 1h. Meanwhile, the thermal performance of near infrared light was studied by tissue simulation. Continuous near infrared light irradiation produced a temperature fluctuation of 4 ℃, while pulsed near infrared radiation produced a temperature fluctuation of 1.2 ℃ during the 1h amount. Based on the known effect of mIL-10 on limiting and ultimately stopping inflammatory responses, design experiments showed that EcN strain (pDawn-mIL-10) and UCMs were placed in the upper lumen of the trans-well system, the lower lumen of which was inoculated with RAW264.7, and induced with Lipopolysaccharide (LPS) to form an inflammatory cell model (FIG. 9). RAW264.7 cytokine secretion was examined by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The ability of EcN, which secretes mIL-10, to scavenge Reactive Oxygen Species (ROS) was investigated by introducing a fluorescent probe. The fluorescence of LPS+ EcN-MIL-10+NIR was reduced compared to the LPS group. EcN-mIL-10 was shown to be effective in inhibiting intracellular ROS production. LPS+ EcN-mIL-10+NIR down-regulated eNOs and MHC class II mRNA levels compared to LPS-induced RAW264.7, indicating that recombinant mIL-10 from EcN-culture supernatant showed full specific bioactivity. Successful conversion of strain EcN (pDawn-mIL-10) to subsequent testing in animal models for its ability to modulate cytokine levels provides evidence for an effective optogenetic system.
2. Preparation of up-conversion hydrogel microbeads loaded with photogenetic engineering strains, construction of crosslinked hydrogels and delivery of dual-component MGPs of up-conversion microgels
1) The steps of constructing strains and plasmids:
the E.coli DH 5. Alpha. Strain is used as a host for plasmid construction and amplification, and the identification is performed by using E.coli Nissle1917 (EcN). The plasmids and strains used are shown in tables 2 and 3, all constructed with pDawn-AG43 as vector:
(1) In order to construct the pDAWN-GFP gene, the GFP coding region was PCR amplified using primers GFP-F and GFP-R, and then fused using fusion PCR;
(2) Deleting the blue light induction module by using the primers pR-F and pR-R;
(3) Self-ligating the linear fragments to obtain plasmid pR-GFP;
(4) To construct PDAWN-mIL-10, the coding region of mIL-10 was amplified by PCR using the primers mIL-10-F and mIL-10-R and fused by fusion PCR. The blue light inducing module was then deleted with primers pR-F and pR-R. The linear fragment was self-ligated to give plasmid pR-mIL-10.
The nucleotide sequence of the coding region of mIL-10 is shown as SEQ ID NO.6, and specifically comprises the following steps:
atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaactga。
TABLE 2 plasmid types
TABLE 3 Escherichia coli Strain types
Note that: reference 1.Cui, m., et al Optotheranostic Nanosystem with Phone VisualDiagnosis and Optogenetic Microbial Therapy for Ulcerative Colitis At-Home care. Acs Nano,2021.15 (4): p.7040-7052.
TABLE 4 primer sequences
Note that: the primers pDAWN-F and pDAWN-R were used for the amplification of pDAWN-series plasmids.
2) Hydrogel microspheres were used as carriers to facilitate the precise release and colonization of (upconverting material microgels) UCMs and EcN-mIL-10 in the intestinal tract (FIG. 10). Fig. 10 shows that: the blue UCR is uniformly doped in the hydrogel;
the up-conversion material oil-soluble NaYF4 is adopted: yb was used as an alternative intracavity light source to activate the light responsive EcN strain to convert near infrared light having a depth of penetration deeper than blue light into blue light (475 nm) in vivo. If the UCR is delivered to the intestine to be cytotoxic and result in excessive dispersion in UCRs, the intensity of light may be reduced. Biocompatible microgel-encapsulated UCMs effectively avoid these problems. The UCMs emit at 475nm under 980nm laser excitation, exhibiting a uniform size, resembling spheres of about 350 μm diameter. The surface and the interior of UCMs are characterized by using a scanning electron microscope, and the internal structure is compact and dense.
3) Natural Calcium Alginate (CA) and chitosan polysaccharide (SC) have good biocompatibility, and we choose them as the encapsulating material for the outer layer assembly. Determination of the water carrying capacity and bacterial count of MGPs containing 90wt% water and 3.8X10 7 Individual colony forming units. In Simulated Gastric Fluid (SGF), CA and SC form a tight bond by electrostatic adsorption; in Simulated Intestinal Fluid (SIF), SCs are separated from SAs by electrostatic repulsion and EcN/UCMs are released in ECN@CA loaded UCMs to achieve spatial co-localization. UCMs show high stability and low permeability in 20h under different pH values, and in order to facilitate storage and transportation, the freeze-drying GMPS protection fresh-keeping technology is adopted, so that the biological activity can last for more than 21 days, and the survival rate of the hydrogel containing microorganisms before and after storage is superior to that of liquid culture. EcN growth was significantly inhibited in SGF or SIF (fig. 11). FIG. 11 shows the growth curve of ECN cultured in SGIF at 37 ℃ (ECN cultured in SGF for 4h followed by SIF);
to evaluate their biosafety and stability in vivo, we assessed leakage of MGPs and contents at gastric or intestinal pH in raw broth (LB). MGPs were cultured in SGF environment to evaluate the protective effect of MGPs on EcN. Bacterial viability was quantified as shown by counts at different time points (fig. 11). Bare EcN was inactive after 0.5h incubation in SGF for a prolonged period of 1-2 h, MGPs were released, ecN survival decreased from 4.71% to 1.71%, and only a small portion EcN survived successfully after 12h exposure to SGF. Thus, the pH of the environment has a direct and large impact on the activity of EcN. In contrast, ecN release was not observed when MGPs were incubated in SIF for 4h, indicating a significant enhancement in tolerance of MGPs to intestinal conditions. Bile salts are contacted when passing through the gut and they can dissolve the cell wall and membrane of the lesion EcN, inactivating EcN, thus further assessing resistance of MGPs to bile acids is needed: after 12h incubation in bile acid, the survival rate of naked EcN was reduced to 11.14%. In addition, even after bile salt exposure, the survival rate of EcN produced by MGPs remained at a high level of 47.14% throughout the incubation period extended to 6 hours.
Furthermore, the intestinal mucosa ELISA results showed that the MIL-10 content of MGPs-EcN-MIL-10+NIR or MGPs-EcN-MIL-10-HIS+NIR groups was significantly higher than that of the other groups in vivo (FIG. 12). These results indicate that EcN released by MGPs can accurately produce mll-10 in the intestinal mucosa in the up-conversion optogenetic system. Finally, MGPs have great stability and safety against both microorganisms and mammalian cells. In conclusion, the protection of MGPs on EcN ensures the bioavailability of recombinant probiotics and also meets the requirement of oral administration.
Example 4 photothermal kit for real-time monitoring and long-term alleviation of acute UC
On the basis of the intelligent mobile phone optical sensing module and the light genetic engineering bacterial treatment system, the invention researches the photo-thermal kit in an acute rat colitis model of the ingestion DSS. DSS is an orally administrable chemical substance that induces damage to the colon epithelium and impaired barrier function in mice. Mice continue to ingest DSS for 7 days, MGPs for 7 days, and resume for 5 days, during which time the course of disease was monitored using the developed photothermal kit. Fig. 13 shows the results of analyzing UC mouse fecal biomarker CALP by the smartphone fluorescence sensing platform. Then, to obtain the average fluorescence intensity of each MBMs, MBMs were whitened and background blackened, and the results of ELISA were compared with the sensitivity of the smartphone-based naked eye detection device, showing that the fecal CALP concentration of dss+mgps-EcNMIL-10 group was significantly correlated with the severity of colitis, and the results were consistent with ELISA (serum/fecal). The CALP concentration is related to the severity of the DSS-induced colitis, the optical sensing module of the photothermal instrument is utilized to track the course of each group of UC on the basis of a smart phone, and the result shows that the platform has good stability and real-time monitoring capability on mice UC.
More importantly, ecN persists in the intestinal tract for at least 5 days after gastric lavage, and successful colonization of EcNmIL-10 provides a basis for subsequent treatment of acute UC by the photogenetically engineered bacteria treatment system. The effect of EcN-produced photoreactive mIL-10 on the development of colitis was evaluated by oral/intravenous injection of MGPs+ EcN-mIL-10, ecNmIL-10, MGPs-EcN, SASP and anti-TNF- α. The body weight of the DSS group was consistently reduced compared to the control group, and the body weight of the MGPs+ EcN-MIL-10 group was significantly recovered on day 8, with similar changes in the relative body weight curves of the mice in the DSS+ EcN-mIL-10, DSS+MGPs-EcN, DSS+SASP or DSS+anti-TNF- α groups, all being progressively higher after body weight reduction. The result shows that the DSS+MGPs-EcN-MIL-10 group has no obvious difference from the control group in colon length. DSS, DSS+ EcN-mIL-10, DSS+MGPs-EcN, DSS+SASP, and DSS+anti-TNF- α groups all had shorter colon. However, other indicators (hematochezia, diarrhea, and Disease Activity Index (DAI) scores) did not differ significantly between dss+mgps-EcN-MILs-10, dss+ EcN-MILs-10, dss+mgps-EcN, dss+sasp, and dss+anti-TNF- α DSS groups. In conclusion, MGPs + EcN-mIL-10 improved the clinical signs of acute UC.
We also assessed the ability of the photogenetically engineered bacterial treatment system to restore abnormal intestinal inflammatory environments and lesions by immunohistochemical staining (IHC). DSS groups manifest as loss of primary crypt structure of the colon, loss of goblet cells, and edema of tissue surrounding the colon. In contrast, MGPs-EcN-mIL-10 group had significant improvement: less edema, better preservation of the crypt structure and more complete epithelium; ecN-mIL-10 treatment also significantly reduced neutrophil expression compared to the DSS group.
The IL-6 levels of mice intestinal tissue homogenates were determined by ELISA as shown in fig. 14, the protein form presented a concentration per 100mg tissue, n=5 per group, mean ± SD, <0.05, <0.01, <0.001. As can be seen from FIG. 14, the concentration of IL-1, IL-6, IL-12, myeloperoxidase (MPO) and TNF- α was significantly increased in the DSS group compared to the control group, reflecting the increased neutrophil infiltration in the colon tissue. The results showed that the MPO concentration was significantly lower in the DSS+MGPs-EcN-mIL10 group than in the DSS group, reflecting a reduction in neutrophil infiltration. This further demonstrates the feasibility of the proposed photogenetically engineered bacterial treatment system in vivo. To investigate the effect of EcN on the inflammatory response and intestinal permeability of the DSS group, eNOs and IFN- α mRNA levels were quantitated. Cytokine studies showed that MGPs-EcN-mIL-10 treated groups showed greater inhibition of colon eNOs and IFN- α mRNA expression than DSS groups. By combining the data, the kit achieves the effects of self diagnosis and real-time intervention in home care of acute UC.
Example 5 photothermal kit for real-time monitoring and long-term remission of chronic UC model
In order to better simulate the process of chronic colitis, we tried to investigate the diagnosis and efficacy of photothermal kits on chronic colitis. Mice were treated with MGPs-EcN-mIL-10 orally after three repetitions of the procedure, where mice were induced with 2% (w/v) DSS in drinking water for 5 days, followed by 5 days of water. In the whole process, the intelligent mobile phone optical sensing technology is used in the photothermal instrument to pre-display the UC process, and the change of the CALP concentration along with time is shown in figure 15. FIG. 15 shows that the fecal CALP concentration of the DSS+MGPs-EcN-mIL-10 group correlated with the severity of colitis, and the results were consistent with ELISA (serum/fecal). Thus, the smartphone optical sensing module of the photo-thermal kit contains a simple, inexpensive and objective tool to predict UC.
According to the good curative effect of MGPs-EcN-mIL-10 in the treatment of acute colitis, the dosage of chronic colitis is regulated, namely, the oral MGPs-EcN-mIL-10 is used for treating the 1 st day and the 4 th day, and the EcN can last at least 10 days in the intestinal tract. The DSS+MGPs-EcN-mIL-10 group had a significant promoting effect on weight gain on day 32 compared to the DSS group. The relative body weight curves of DSS+ EcN-mIL-10, DSS+MGPs-EcN, DSS+SASP or DSS+anti-TNF- α treated mice varied similarly. Notably, there was an increase in the maximal life of mice in the MGPs-EcN-mIL-10 treated group. The colon length of DSS+MGPs-EcN-MIL-10/DSS+anti-TNF-alpha group is not obviously different from that of the control group. In contrast, the colon of the DSS+MGPs-EcN group and the DSS+SASP group contracted. However, other measurements (hematochezia, diarrhea, and DAI scores) did not differ significantly between dss+ecnmil-10, dss+mgps-EcN, dss+sasp, and dss+anti-TNF- α groups. All the results demonstrate that in situ production of mIL-10 alleviates the symptoms of chronic colitis. We also used IHC to assess the effect of the photogenetically engineered bacterial system on intestinal histopathology. DSS groups showed complete disappearance of goblet cells, disappearance of crypt structures, edema of tissues surrounding the colon. In contrast, MGPs-EcN-mIL-10 group had significantly improved, better crypt preservation, less inflammatory cell infiltration, less edema, and more complete epithelium.
The pro-inflammatory mediators TNF- α and interleukins involved in inflammatory processes and intestinal epithelial cell disorders are detected directly or indirectly. The results show experimentally measured IL-1, IL-6, IL-12, MPO and TNF- α concentrations in colon tissue samples from mice from different treatment groups, which were significantly increased in the DSS group and inhibited by MGPs-EcN-MIL-10 treatment. The MPO concentration in colon tissue of the MGPsEcN-mIL-10 treated mice is significantly lower than that of DSS/anti-TNF-alpha treated mice. This further demonstrates that the photogenetically engineered bacterial system has the effect of reducing MPO activity and modulating inflammatory cytokine secretion in vivo. To investigate the effect of EcN on inflammatory response in the DSS group and intestinal permeability, eNOs and IFN- α mRNA levels were quantitated. DSS group IFN- α and eNOs mRNA expression was increased and MGPs-EcN-mIL-10 was partially reversed. By combining the data, the kit achieves the effects of self diagnosis and real-time intervention in home care of chronic UC.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for preparing hydrogel microbeads, comprising the following steps:
providing a microfluidic chip, wherein the microfluidic chip comprises an internal phase pipeline, an external phase pipeline and a main pipeline converging the internal phase pipeline and the external phase pipeline; the internal phase pipeline is provided with an internal phase material inlet, the external phase pipeline is provided with an external phase material inlet, and the main pipeline is provided with a nano hydrogel outlet;
injecting an internal phase material into the internal phase material inlet, injecting an external phase material into the external phase pipeline inlet, mixing the external phase material and the internal phase material in a main pipeline, irradiating the main pipeline by using an ultraviolet light source, and obtaining hydrogel microbeads at the nano hydrogel outlet;
the internal phase material comprises polyethylene glycol diacrylate, polyethylene glycol, an ultraviolet initiator and functional nano particles, wherein the functional nano particles comprise a core and a shell layer, and the core is carboxyl modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan; the up-conversion material comprises Yb 3+ 、Ho 3+ 、Er 3+ And Tm 3+ One or more of the following;
the external phase material comprises dimethyl siloxane.
2. The method according to claim 1, wherein the volume ratio of polyethylene glycol diacrylate to polyethylene glycol is 5.8-6:1-1.2;
The volume ratio of the polyethylene glycol diacrylate to the photoinitiator is 5.8-6:1-1.2;
the mass ratio of the polyethylene glycol diacrylate to the functional nano particles is 2.7-3:1-1.4.
3. The preparation method according to claim 1 or 2, wherein the injection rate of the internal phase material is 72 to 80 μΙ.min -1 The method comprises the steps of carrying out a first treatment on the surface of the The injection rate of the external phase material is 3150-3200 mu L min -1 。
4. The hydrogel microbead prepared by the preparation method of any one of claims 1-3, which comprises a photo-cured hydrogel and functional nanoparticles distributed on the surface and inside of the photo-cured hydrogel, wherein the functional nanoparticles comprise an inner core and a shell layer, and the inner core is carboxyl-modified Fe 3 O 4 Or an upper conversion material, wherein the shell layer is a cross-linked product of calcium alginate and chitosan; the photo-curing hydrogel is obtained by photo-curing polyethylene glycol diacrylate, polyethylene glycol and dimethyl siloxane.
5. The use of the hydrogel microbead of claim 4 in the preparation of a reagent for detecting colitis or in the preparation of a probiotic.
6. A kit for detecting colitis based on optical sensing of a smart phone, comprising the magnetic hydrogel microbead of claim 4, further comprising a monoclonal antibody to fecal calprotectin and a secondary antibody to phycoerythrin-labeled fecal calprotectin.
7. The probiotic bacteria preparation is characterized by being obtained by wrapping probiotics with hydrogel microbeads according to claim 4, wherein the inner cores of functional nano particles in the hydrogel microbeads are upper conversion materials.
8. The probiotic bacterial agent according to claim 7, characterized in that the probiotics comprise recombinant probiotics; the original bacteria of the recombinant probiotics comprise escherichia coli Nissle1917.
9. The probiotic bacterial agent according to claim 7, characterized in that the recombinant probiotic bacteria comprise recombinant plasmids; the recombinant plasmid takes pDawn as an original vector; the recombinant plasmids comprise a first recombinant plasmid and/or a second recombinant plasmid; the coding gene of IL-10 is inserted into the first recombinant plasmid, and insertion sites are SalI and HindIII; the nucleotide sequence of the coding gene of the IL-10 is shown as SEQ ID NO. 3; the second recombinant plasmid is inserted with a reporter gene, and insertion sites are SalI and HindIII; the nucleotide sequence of the reporter gene is shown as SEQ ID NO. 4.
10. Use of the probiotic bacterial agent of any one of claims 7 to 9 for the preparation of a food, a health product or a pharmaceutical product containing probiotics; the dosage form of the medicine comprises an oral preparation.
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