CN115521893A - Artificial receptor capable of selectively identifying microorganisms and application thereof - Google Patents
Artificial receptor capable of selectively identifying microorganisms and application thereof Download PDFInfo
- Publication number
- CN115521893A CN115521893A CN202211072502.6A CN202211072502A CN115521893A CN 115521893 A CN115521893 A CN 115521893A CN 202211072502 A CN202211072502 A CN 202211072502A CN 115521893 A CN115521893 A CN 115521893A
- Authority
- CN
- China
- Prior art keywords
- artificial receptor
- microorganisms
- antigen
- polymer
- affinity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010031480 Artificial Receptors Proteins 0.000 title claims abstract description 82
- 244000005700 microbiome Species 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 230000000694 effects Effects 0.000 claims abstract description 22
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 7
- 229920000642 polymer Polymers 0.000 claims description 35
- 230000000813 microbial effect Effects 0.000 claims description 31
- 239000002105 nanoparticle Substances 0.000 claims description 29
- 210000004027 cell Anatomy 0.000 claims description 28
- 239000002158 endotoxin Substances 0.000 claims description 19
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 19
- 239000000203 mixture Substances 0.000 claims description 19
- 239000000178 monomer Substances 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000427 antigen Substances 0.000 claims description 18
- 108091007433 antigens Proteins 0.000 claims description 18
- 102000036639 antigens Human genes 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical group [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000006116 polymerization reaction Methods 0.000 claims description 15
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 claims description 13
- 108010013639 Peptidoglycan Proteins 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 13
- 238000005516 engineering process Methods 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 239000004005 microsphere Substances 0.000 claims description 11
- XFHJDMUEHUHAJW-UHFFFAOYSA-N n-tert-butylprop-2-enamide Chemical compound CC(C)(C)NC(=O)C=C XFHJDMUEHUHAJW-UHFFFAOYSA-N 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 101710160107 Outer membrane protein A Proteins 0.000 claims description 9
- 239000003999 initiator Substances 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 239000002122 magnetic nanoparticle Substances 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 7
- 239000004094 surface-active agent Substances 0.000 claims description 7
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 6
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 6
- JESXATFQYMPTNL-UHFFFAOYSA-N 2-ethenylphenol Chemical compound OC1=CC=CC=C1C=C JESXATFQYMPTNL-UHFFFAOYSA-N 0.000 claims description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 6
- 239000003431 cross linking reagent Substances 0.000 claims description 6
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 6
- BPCNEKWROYSOLT-UHFFFAOYSA-N n-phenylprop-2-enamide Chemical compound C=CC(=O)NC1=CC=CC=C1 BPCNEKWROYSOLT-UHFFFAOYSA-N 0.000 claims description 6
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- QWMJEUJXWVZSAG-UHFFFAOYSA-N (4-ethenylphenyl)boronic acid Chemical compound OB(O)C1=CC=C(C=C)C=C1 QWMJEUJXWVZSAG-UHFFFAOYSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- OSSNTDFYBPYIEC-UHFFFAOYSA-N 1-ethenylimidazole Chemical compound C=CN1C=CN=C1 OSSNTDFYBPYIEC-UHFFFAOYSA-N 0.000 claims description 4
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 239000011246 composite particle Substances 0.000 claims description 4
- 230000001276 controlling effect Effects 0.000 claims description 4
- 238000000502 dialysis Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- JAHNSTQSQJOJLO-UHFFFAOYSA-N 2-(3-fluorophenyl)-1h-imidazole Chemical compound FC1=CC=CC(C=2NC=CN=2)=C1 JAHNSTQSQJOJLO-UHFFFAOYSA-N 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 125000002147 dimethylamino group Chemical group [H]C([H])([H])N(*)C([H])([H])[H] 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical compound CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 claims description 3
- LVHBHZANLOWSRM-UHFFFAOYSA-N methylenebutanedioic acid Natural products OC(=O)CC(=C)C(O)=O LVHBHZANLOWSRM-UHFFFAOYSA-N 0.000 claims description 3
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 3
- 239000000376 reactant Substances 0.000 claims description 3
- BWYYYTVSBPRQCN-UHFFFAOYSA-M sodium;ethenesulfonate Chemical compound [Na+].[O-]S(=O)(=O)C=C BWYYYTVSBPRQCN-UHFFFAOYSA-M 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 claims description 2
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 claims description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 2
- 239000000022 bacteriostatic agent Substances 0.000 claims description 2
- 239000006185 dispersion Substances 0.000 claims description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 2
- 238000011065 in-situ storage Methods 0.000 claims description 2
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 2
- HXJGFDIZSMWOGY-UHFFFAOYSA-N n-(2-azaniumylethyl)prop-2-enimidate Chemical compound NCCNC(=O)C=C HXJGFDIZSMWOGY-UHFFFAOYSA-N 0.000 claims description 2
- XHIRWEVPYCTARV-UHFFFAOYSA-N n-(3-aminopropyl)-2-methylprop-2-enamide;hydrochloride Chemical compound Cl.CC(=C)C(=O)NCCCN XHIRWEVPYCTARV-UHFFFAOYSA-N 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- OEIXGLMQZVLOQX-UHFFFAOYSA-N trimethyl-[3-(prop-2-enoylamino)propyl]azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCCNC(=O)C=C OEIXGLMQZVLOQX-UHFFFAOYSA-N 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 9
- 108020003175 receptors Proteins 0.000 abstract description 6
- 230000033228 biological regulation Effects 0.000 abstract description 4
- 238000013537 high throughput screening Methods 0.000 abstract description 4
- 238000007885 magnetic separation Methods 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 230000001954 sterilising effect Effects 0.000 abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 3
- 230000007547 defect Effects 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 description 36
- 241000588724 Escherichia coli Species 0.000 description 21
- 238000009472 formulation Methods 0.000 description 13
- 230000027455 binding Effects 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 230000018612 quorum sensing Effects 0.000 description 12
- 230000012010 growth Effects 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 10
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 8
- 239000010931 gold Substances 0.000 description 8
- 229910052737 gold Inorganic materials 0.000 description 8
- 244000063299 Bacillus subtilis Species 0.000 description 7
- 241000192125 Firmicutes Species 0.000 description 7
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 7
- 238000007789 sealing Methods 0.000 description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 244000000010 microbial pathogen Species 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 101710145505 Fiber protein Proteins 0.000 description 5
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000005090 green fluorescent protein Substances 0.000 description 5
- 238000005286 illumination Methods 0.000 description 5
- 150000002460 imidazoles Chemical class 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 241001515965 unidentified phage Species 0.000 description 5
- 230000008859 change Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000002207 metabolite Substances 0.000 description 4
- 238000003380 quartz crystal microbalance Methods 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 3
- 241001411320 Eriogonum inflatum Species 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 229910004298 SiO 2 Inorganic materials 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 108091006047 fluorescent proteins Proteins 0.000 description 3
- 102000034287 fluorescent proteins Human genes 0.000 description 3
- 238000010907 mechanical stirring Methods 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000012673 precipitation polymerization Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 101100524589 Arabidopsis thaliana RH16 gene Proteins 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 241000186660 Lactobacillus Species 0.000 description 2
- 241000232299 Ralstonia Species 0.000 description 2
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 2
- 238000001878 scanning electron micrograph Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 235000012239 silicon dioxide Nutrition 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- UYTRITJAZOPLCZ-SCSAIBSYSA-N (4r)-4,5-dihydroxypentane-2,3-dione Chemical compound CC(=O)C(=O)[C@H](O)CO UYTRITJAZOPLCZ-SCSAIBSYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- NJSVDVPGINTNGX-UHFFFAOYSA-N [dimethoxy(propyl)silyl]oxymethanamine Chemical compound CCC[Si](OC)(OC)OCN NJSVDVPGINTNGX-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- OALAWUCDYURMPU-UHFFFAOYSA-N boric acid;styrene Chemical compound OB(O)O.C=CC1=CC=CC=C1 OALAWUCDYURMPU-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000012688 inverse emulsion polymerization Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229920000344 molecularly imprinted polymer Polymers 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an artificial receptor capable of selectively identifying microorganisms and application thereof. The invention utilizes a high-throughput screening method to simply and quickly carry outArtificial receptors with high affinity and selectivity to different microorganisms are designed and screened, and the application of the artificial receptors in selective magnetic separation of microorganisms, photo-thermal sterilization and regulation of flora effects is researched. The artificial receptor obtained by the invention has higher affinity and excellent selectivity to target microorganisms, and the affinity constant reaches 2.90 multiplied by 10 ‑12 M, can replace a natural receptor to realize the enrichment and detection of microorganisms. The artificial receptor is prepared by a chemical method, has higher stability, longer service life and stronger capability of resisting severe environment, overcomes the defects of high cost, low preparation efficiency, long screening period, changeability, difficult preservation and the like of the traditional natural receptor, and can be repeatedly used.
Description
Technical Field
The invention belongs to the technical field of biological identification, and particularly relates to an artificial receptor capable of selectively identifying microorganisms and application thereof.
Background
Microorganisms typically include bacteria, fungi, viruses, microalgae, and the like. The size of the microorganism is micron level, the shape is different, and the individual is tiny, and is closely related to human. Although most are harmless, many species have proven pathogenic in clinical trials and can cause serious infections, which in some cases can be fatal. Contamination by pathogenic microorganisms is a serious and permanent risk in most fields, and developing methods for identifying and detecting pathogenic microorganisms is of great significance and urgency for preventing outbreaks of major diseases. Conventional diagnostic techniques for microorganisms are often very slow and expensive, and have limited specificity and sensitivity. Therefore, the development of products and methods for specifically identifying microorganisms is of great research significance for the detection and diagnosis of microorganisms.
The currently common biological detection methods mainly include biochemical detection technology, immunological technology, metabolic detection technology, molecular biology technology and the like.
For example, in the citation: mo Yi, fang Linyi, etc. in Chinese patent, a multi-color fluorescent magnetic chemical nasal sensor for rapid detection of pathogenic microorganisms (CN 109115741A). The method is a method for preparing a chemical microorganism sensor, and is used for screening antibiotics acting on microorganisms, wherein the 'chemical nose' sensor comprises multicolor fluorescent protein and quaternary ammonium salinized magnetic nanoparticles. Based on the chemical nose sensor, quenching the multicolor fluorescent protein signals, then competitively combining with pathogenic microorganisms to be detected, dissociating the multicolor fluorescent protein to emit fluorescent signals, and then obtaining response signals of different microorganisms according to the change difference of the fluorescent signals; furthermore, the 'chemical nose' sensor competes with the microorganism to be tested under the action of the antibiotic for binding, and the antibiotic is screened. However, in this method, quaternary ammonium salts generally have strong toxicity, and the use of quaternary ammonium salts may cause contamination and may have limitations in the application of detection.
In the citation document, the application of pseudomonas aeruginosa bacteriophage fiber protein (CN 109136196A) in preparing a bacteria detection reagent discloses the application of pseudomonas aeruginosa bacteriophage fiber protein in preparing a bacteria detection reagent, belonging to the technical field of microorganism detection. The pseudomonas aeruginosa bacteriophage with the preservation number of CGMCC No.15569. The pseudomonas aeruginosa bacteriophage fiber protein P069 is used for preparing a bacterial detection reagent. The invention has the beneficial effects that: provides a fiber protein P069 capable of specifically recognizing pseudomonas aeruginosa, which is used as a molecular recognition reagent to specifically capture the pseudomonas aeruginosa and then is combined with other analysis technologies to detect the whole cells of the pseudomonas aeruginosa. However, in the invention, the cost is increased to a certain extent for the used pseudomonas aeruginosa bacteriophage fiber protein, and the protein is difficult to obtain and is fragile, so that the protein has certain difficulty in storage.
In addition, microorganisms utilize a series of chemical reactions to regulate their behavior and react to the environment. Quorum sensing is a method by which microorganisms utilize signal molecules to regulate their physiological metabolic behavior, so that regulation of quorum sensing can affect the growth and metabolism of microorganisms. In the research of pathogenic microorganisms, the inhibition of quorum sensing can inhibit the growth of pathogenic microorganisms. In the biological processing process, the microbial cell factory can be used for synthesizing fuels and chemicals, and the yield can be improved by enhancing the quorum sensing in the multi-bacterium cooperative adaptation work. In current studies, quorum sensing effects are often enhanced by the addition of quorum sensing molecules or synthetic quorum sensing signal molecule promoters. However, quorum-sensing molecules and promoters are expensive and are easily quenched and inactivated. Therefore, the research and development of new technology and new method for regulating and controlling the effect of microbial cell flora are of great research significance.
Disclosure of Invention
The invention aims to simply and quickly design and screen artificial receptors with high affinity and selectivity on different microorganisms by utilizing a high-throughput screening method, and research the application of the artificial receptors in selective magnetic separation of microorganisms, photo-thermal sterilization and flora regulation and control effects.
The preparation method of the artificial receptor capable of selectively identifying the microorganisms comprises the following steps:
(1) Dissolving N-tert-butyl acrylamide (TBAm) or N-Phenyl Acrylamide (PAM) in ethanol, dissolving N-isopropyl acrylamide (NIPAm), a functional monomer, a cross-linking agent and an initiator in water, mixing the reactants in different proportions, adding the reactants into a detection pool, removing oxygen in the mixed solution by introducing nitrogen for 5-10min by ultrasonic, and carrying out in-situ polymerization reaction at the bottom of the detection pool to obtain a polymer thin layer; washing the polymer thin layer with water for 3-5 times after reaction to remove unreacted monomers;
(2) Adding microbial cell surface antigen into the detection pool for incubation, wherein the antigen is adsorbed on the polymer thin layer at the bottom of the detection pool; determining the affinity of the polymer to the antigen by detecting the amount of antigen adsorbed by the polymer thin layer or detecting the amount of residual antigen in the supernatant, and screening out a polymer synthesis formula with high affinity to the antigen;
(3) Preparing raw materials according to the formula screened in the step (2), adding a surfactant and a template agent, removing oxygen in the mixed solution by introducing nitrogen for 5-10min through ultrasound, stirring for polymerization reaction to obtain polymer nanoparticles, eluting the template, and purifying by dialysis; then measuring the affinity constant K of the polymer nano particles and the antigen by a biomembrane interference technology D And screening out polymer nanoparticles with high affinity and selectivity to microbial cells, namely the finally constructed artificial receptor.
The functional monomer is selected from one or more of (3-acrylamidopropyl) trimethyl ammonium chloride (ATC), N- (3-aminopropyl) methacrylamide hydrochloride (APM), 1-vinyl Imidazole (IM), N- [ (3- (dimethylamino) propyl ] methacrylamide (DMAPAA), N- (2-aminoethyl) Acrylamide (AEM), acrylamide (AAm), acrylic acid (AAc), 2-hydroxyethyl methacrylate (HEMA), itaconic Acid (IA), sodium Vinyl Sulfonate (SVS), methacrylic acid (MAA), 4-vinyl benzene boric acid (VPBA) and Vinyl Phenol (VPL).
The cross-linking agent is one or more of N, N '-methylene bisacrylamide, N' -vinyl bisacrylamide and ethylene glycol dimethacrylate.
The initiator is ammonium persulfate, azobisisobutyronitrile or tetramethylethylenediamine. The addition amount of the initiator is 0.3-2mg/mL.
The mol percentage of the N-tert-butyl acrylamide or the N-phenyl acrylamide is 0 to 40 percent, the mol percentage of the N-isopropyl acrylamide is 15 to 96 percent, and the proportion of the functional monomer is 2 to 40 percent.
The dosage of the cross-linking agent is 2-10%.
The total monomer concentration is 10-1000mM.
The polymerization reaction in the step (1) is carried out in a sealed or nitrogen atmosphere, the temperature of the polymerization reaction is 25-80 ℃, and the time of the polymerization reaction is 2-72h.
The microbial cell surface antigen is lipopolysaccharide and peptidoglycan on the surface of a microbial cell.
The microbial cell surface antigen is lipopolysaccharide on the cell surface of gram-negative bacteria or peptidoglycan on the cell surface of gram-positive bacteria.
The template agent is microbial cell surface antigen or a silicon dioxide microsphere, a ferroferric oxide magnetic sphere or a glass bead microsphere modified on the surface of the microbial cell surface antigen. The dosage of the template agent is 0.05-0.2mg/mL calculated by the content of the microbial cell surface antigen.
The surfactant is sodium dodecyl sulfate or cetyl trimethyl ammonium bromide, and the dosage of the surfactant is 0.2-0.4mg/mL.
The method for eluting the template in the step (3) comprises the following steps: the template agent is eluted by raising the temperature to 50-90 ℃, lowering the temperature to 0-10 ℃, adding 1-5M NaCl aqueous solution, stirring for reaction for 30min, adding the same volume of methanol and acetic acid mixed solution (the volume ratio of methanol to acetic acid is 9:1), stirring for 30min, or adding the same volume of 30% acetonitrile aqueous solution, and stirring for 30min.
The polymerization reaction in the step (3) is carried out in a sealed or nitrogen atmosphere, the polymerization reaction temperature is 25-80 ℃, and the polymerization reaction time is 2-72h.
The rotating speed of the stirring in the step (3) is 300-1000rpm.
A method for regulating the effect of microbial flora: 0.3-4.8mg/mL of an artificial receptor dispersion capable of selectively recognizing a microorganism is added to the corresponding microorganism in a volume ratio of 1.5-8. After the artificial receptor is added, the bacteria can be specifically targeted, the aggregation of the bacteria is promoted, the signal molecules of the bacteria are limited in diffusion, the concentration of the signal molecules near the bacteria is increased, the quorum sensing of the bacteria is promoted, the growth of the bacteria is promoted, and the yield of metabolites is increased.
A preparation method of a magnetic artificial receptor capable of selectively identifying and separating microbial cells comprises the step of adding magnetic nanoparticles into reaction liquid in the step (3) of preparing the artificial receptor capable of selectively identifying and separating microbes, and finally synthesizing composite particles wrapping the magnetic nanoparticles, namely the magnetic artificial receptor capable of selectively identifying and separating the microbial cells.
The magnetic nanoparticles have a particle size of 10-500nm and a concentration of 50-500 μ g/mL
A preparation method of a photothermal bacteriostatic agent comprises the following steps: and (3) preparing an artificial receptor capable of selectively identifying microorganisms, and adding nanogold into the reaction solution in the step (3) to finally synthesize nanogold-coated composite particles, namely the photothermal bacteriostat. Under the illumination condition, the photothermal bacteriostat generates photothermal effect, the temperature is increased, and the effect of inhibiting bacteria is achieved.
The size of the nano gold is 10-300nm, and the concentration is 0.1-5mg/mL.
The excitation wavelength of the illumination is 650-1100nm.
The artificial receptor obtained by the invention has higher affinity and excellent selectivity to target microorganisms, and the affinity constant reaches 2.90 multiplied by 10 -12 M, can replace the natural acceptor, realize the enrichment and detection of the microorganism; the artificial receptor is prepared by a chemical method, has higher stability, longer service life and stronger capability of resisting severe environment, and overcomes the defects of high cost and preparation of the traditional natural receptorLow efficiency, long screening period, easy degeneration, difficult preservation and the like. According to the invention, the magnetic artificial receptor synthesized by wrapping the magnetic nanoparticles with the artificial receptor can identify and separate 10 microbial cells mixed in every 100 ten thousand blood cells (about 1mL of blood) within 20min, and the capture rate of the microbial cells is as high as 97%; the artificial receptor coated nano gold particles can generate a photo-thermal effect within 5min under 808nm illumination, and have a good sterilization effect; the artificial receptor of the invention selectively adsorbs microorganisms through specificity, promotes the aggregation of the microorganisms, forms a cell mass, limits the diffusion of signal molecules generated by the microorganisms, and keeps higher concentration in the cell mass, thereby enhancing the quorum sensing of microbial cells, regulating and controlling the flora effect of the microorganisms, further promoting the growth and metabolism of the microbial cells, and finally improving the yield of metabolites by about 20%. The artificial receptor prepared by the invention can be repeatedly used, and the synthesis process and the regeneration process are simple, so that the cost is greatly reduced.
Drawings
FIG. 1 is a scanning electron microscope image of a molecularly imprinted artificial receptor selectively targeted to recognize gram-negative bacteria (formulation: NP 40).
FIG. 2 is a scanning electron micrograph of a molecularly imprinted artificial receptor selectively targeting and recognizing gram-positive bacteria (formula: NP 94).
FIG. 3 scanning electron micrograph of FITC-labeled artificial receptor nanoparticles (formulation: NP 40).
FIG. 4 flow cytometry is used to determine the binding of the artificial receptor nanoparticles to E.coli, ralstonia H16 (RH 16), bacillus subtilis, yeast cells (formulation: NP 40).
FIG. 5 flow cytometry is used to determine the binding of the artificial receptor nanoparticles to Bacillus subtilis, lactobacillus, yeast cells, E.coli, ralstonia H16 (RH 16) (formulation: NP 94).
FIG. 6 results of frequency and mass change of nanoparticles binding to E.coli measured by Quartz Crystal Microbalance (QCM) method (formulation: NP 40).
FIG. 7 shows the result of the regulation of the flora effect of the artificial receptor on E.coli expressing green fluorescent protein (the final metabolite, green fluorescent protein, was increased by about 20%) (formula: NP 40).
FIG. 8 magnetic artificial receptor Escherichia coli expressing green fluorescent protein (formulation: NP 40) was selectively isolated from whole blood.
FIG. 9 is the scanning electron microscope image of the molecular imprinting artificial receptor wrapping the nano-gold rod (formula: NP 40).
FIG. 10 photo-thermal diagram of the molecularly imprinted artificial receptor encapsulating the nanogold rod (formulation: NP 40).
FIG. 11 is a diagram showing the bacteriostatic effect of the molecularly imprinted artificial receptor with nanogold rods wrapped (formulation: NP 40).
Detailed Description
To better understand the application of the artificial receptor, the following description will be made in conjunction with examples.
Example 1: monomer formula for primarily obtaining artificial receptor capable of selectively identifying microbial cells through high-throughput screening
(1) Dissolving hydrophobic monomer N-tert-butyl acrylamide (TBAm) or N-Phenyl Acrylamide (PAM) in ethanol, dissolving basic monomer N-isopropyl acrylamide (NIPAm), other functional monomers, a cross-linking agent and an initiator in water, preparing all monomers into 200mM stock solution, adding the stock solutions of the monomers according to the molar ratio of table 1 and table 2 to obtain mixed solution with the total volume of 200 mu L, finally adding 3mg/mL initiator Ammonium Persulfate (APS) solution of 50 mu L, respectively placing the mixed solution in each hole of a 96-hole plate, ultrasonically introducing nitrogen for 10min to remove oxygen in the mixed solution, sealing the 96-hole plate by using a seal sticker, then placing the 96-hole plate in an oven at 80 ℃ for reaction for 3 hours, obtaining a polymer thin layer at the bottom of each hole of the 96-hole plate, and washing the polymer thin layer for 3 times by using pure water.
(2) 150 mu L of lipopolysaccharide of the fluorescence modified bacterial surface antigen gram-negative bacteria and peptidoglycan (100 mu g/mL) of the gram-positive bacteria surface are respectively added into each hole of the 96-hole plate, the cells are incubated for 3h on a shaking table, the antigen is adsorbed on a polymer thin layer at the bottom of the 96-hole plate, the supernatant is sucked out, the fluorescence value of the supernatant is detected, and the fluorescence value reflects the affinity of the functional monomer for the antigen. Lower fluorescence of the supernatant indicates stronger affinity; the weaker the affinity, in contrast.
The best functional monomers targeting gram-negative bacteria are 1-vinylimidazole (IM) and N- [ (3- (dimethylamino) propyl ] methacrylamide (DMAPAA), N-tert-butylacrylamide (TBAm) and the amount of functional monomers used are 40% and 18%, respectively.
The functional monomers with better effect of targeting gram-positive bacteria are 1-vinyl Imidazole (IM), acrylic Acid (AAC) and 4-vinyl phenylboronic acid (VPBA); the amount of N-tert-butylacrylamide (TBAm) was 30%, the amount of IM was 2%, the amount of AAC was 10%, and the amount of VPBA was 5%.
TABLE 1 Synthesis formulation of gram-negative bacteria-targeting nanoartificial receptor polymers
TABLE 2 Synthesis formulation of gram-positive bacteria-targeting nanoartificial receptor polymers
Example 2: the affinity and selectivity of the screened artificial receptor to the microbial cells are further improved by utilizing the molecular imprinting technology and the biomembrane interference technology
After the mixture ratio with high affinity to lipopolysaccharide or peptidoglycan obtained by high throughput screening according to example 1 is selected, lipopolysaccharide on the surface of gram-negative bacteria escherichia coli or peptidoglycan on the surface of gram-positive bacteria bacillus subtilis are respectively used as template molecules, 50mL of reaction mixed liquid is prepared according to the optimal mixture ratio obtained in example 1, then 2.5mg of lipopolysaccharide or peptidoglycan is respectively added as template molecules, 10mg of surfactant Sodium Dodecyl Sulfate (SDS) is added, the above solutions are mixed in a round-bottomed flask, then a bottle stopper and a sealing film are used for sealing the round-bottomed flask and introducing nitrogen for 30min, magnetic stirring or mechanical stirring is assisted, and the mixture reacts at 65 ℃ for 3h and 6h respectively through precipitation polymerization to synthesize the polymer nanoparticles. Adding NaCl solution (NaCl final concentration is 2M) into the nanoparticles, stirring for 30min, transferring into a 7000Da dialysis bag for dialysis for 7 days, eluting to remove the template, and finally obtaining the molecularly imprinted artificial receptor, wherein the appearance of the molecularly imprinted artificial receptor is shown in a scanning electron microscope image in figure 1 and figure 2.
Or fixing lipopolysaccharide or peptidoglycan template molecules on a material for imprinting, firstly preparing a solid phase support carrier, which can be microspheres such as silicon dioxide microspheres, ferroferric oxide microspheres, glass beads and the like, modifying amino groups, carboxyl groups and other chemical bonds on the surfaces of the microspheres, and then fixing lipopolysaccharide or peptidoglycan on the surfaces of the solid phase support carrier. Taking the fixation of lipopolysaccharide on ferroferric oxide microspheres as an example, the specific operation process is as follows: (1) synthesizing magnetic ferroferric oxide microspheres MagNP: 1.3g FeCl was weighed 3 ·6H 2 O, 0.62g of cetyltrimethylammonium bromide and 2.6g of anhydrous sodium acetate were put in a round-bottomed flask, 40mL of ethylene glycol was added, and stirred at 80 ℃ for one hour until completely dissolved, and then transferred to a reaction vessel. Reacting at 200 ℃ for 10h, washing with water and ethanol, and drying at 60 ℃ for later use. (2) Synthesizing SiO on the surface of magnetic ferroferric oxide microsphere 2 Thin layer (MagNP @ SiO) 2 ): 100mg of Mag NP was uniformly dispersed in 87.1mL of 80% (v/v) ethanol solution, 1.4mL of 25% NH was added 3 ·H 2 O, performing ultrasonic treatment for 1min; then 11.5mL tetraethyl orthosilicate TEOS was added and the reaction was stirred mechanically at 600-700rpm in a water bath at 30 ℃ for 6h. After reaction, washing with water until the pH value is neutral, washing with ethanol, and drying at 60 ℃ for later use. (3) MagNP @ SiO 2 Surface-modified amino group: will 300mg MagNPs@SiO 2 Dispersed in 180mL ethanol solution (ethanol: water =3/1,v/v) and sonicated for 30 minutes at room temperature. Introducing nitrogen for 30min, placing in 40 deg.C water bath, injecting 4mL 3- (aminopropyl) trimethoxy silane (APTMS) into flask, mechanically stirring for reaction overnight to obtain MagNP @ SiO (amino) with surface bonded with amino 2 -NH 2 Washing with water and ethanol, and oven drying at 60 deg.C. (4) immobilization of template molecules: taking MagNP @ SiO 2 -NH 2 Dissolving 50mg of the aldehyde group in 30mL of water (containing 5mL of glutaraldehyde), performing ultrasonic reaction for 10min at the speed of 700rpm, mechanically stirring at room temperature, and reacting for 3h to bond the aldehyde group on a magnetic ball; washing with PBS (0.01 mol/L, pH 7.2-7.4) for 3 times with a magnet to remove unreacted glutaraldehyde; incubating the aldehyde-modified magnetic spheres in 6mL of PBS (containing 1mg of lipopolysaccharide) for 2h; the particles were separated by magnetic collection and washed with PBS (0.1M, pH 7.2-7.4) to obtain a solid phase template, which was stored at 4 ℃. Adding 50mL of the reaction mixed solution prepared according to the optimal proportion obtained in the example 1, stirring with an auxiliary machine, reacting for 12 hours at 65 ℃ through precipitation polymerization or inverse emulsion polymerization, and eluting the solid-phase template molecule by raising the temperature to 65 ℃ or lowering the temperature to 4 ℃ after the polymerization is finished to obtain the molecular imprinting artificial receptor.
Measuring the affinity between the molecular engram artificial receptor and lipopolysaccharide or peptidoglycan by using a biological membrane interference technology (BLI), wherein the affinity is the binding affinity constant K between the molecular engram artificial receptor obtained by an experiment and the lipopolysaccharide or the peptidoglycan D As a result, the prepared molecularly imprinted artificial receptor has high affinity for lipopolysaccharide or peptidoglycan, K D The value reaches 2.90 multiplied by 10 -12 M, K compared to non-imprinted polymers D The difference of the values is 2-3 orders of magnitude, which indicates that the molecular imprinting technology greatly improves the affinity of the polymer to the surface antigen of the microbial cell and selectively identifies the microbial cell in a targeted way.
TABLE 3 determination of affinity between molecularly imprinted artificial receptor polymers, non-imprinted polymers and lipopolysaccharides by biofilm interference technique (BLI)
TABLE 4 determination of affinity between molecularly imprinted artificial receptor polymers, non-imprinted polymers and peptidoglycans by biofilm interference technique (BLI)
Example 3: specific binding of fluorescent labeled molecular imprinting artificial receptor nanoparticles and bacteria by flow cytometry
Different bacteria were grown to the same OD 600 Measuring the OD of the bacteria by using an ultraviolet spectrophotometer 600 And taking out when the temperature is 0.8. The bacteria were centrifuged at 3000r/min for 10min, the supernatant was discarded, the bacteria were resuspended in 0.01M PBS buffer (pH = 7.2-7.4), the centrifugation and resuspension procedure was repeated 3 times, the fluorescently labeled molecularly imprinted polymer (fig. 3) was added, the cells were incubated at 37 ℃ for 2h, and the unbound nanoparticles were removed by washing 3 times with the PBS buffer according to the centrifugation and resuspension procedure described above.
The fluorescence was measured by flow cytometry, and the results are shown in tables 5 to 6 and FIGS. 4 to 5. For the molecular imprinting artificial receptor capable of selectively identifying gram-negative bacteria, as lipopolysaccharide on the surface of escherichia coli is used as a template molecule in the molecular imprinting process, the finally obtained molecular imprinting artificial receptor nano-particle has excellent specificity and selectivity on the escherichia coli, the binding rate is as high as 96.8%, and the binding rate of a non-imprinted polymer on the escherichia coli is only 41.4%. Meanwhile, the surface of the gram-negative bacteria RH16 also has lipopolysaccharide antigen, so the molecular imprinting artificial receptor (formula: NP 40) also has certain selective recognition effect on RH16, and the binding rate is 76.9%. But has no selectivity to gram-positive bacteria such as bacillus subtilis and lactobacillus and eukaryotic yeast cells. Meanwhile, the combination condition of the molecular imprinting artificial receptor (formula: NP 40) and the non-imprinting polymer to the escherichia coli is measured by using a Quartz Crystal Microbalance (QCM) (figure 6), and the high specificity and selectivity of the molecular imprinting artificial receptor to the escherichia coli are further proved.
For a molecular imprinting artificial receptor (NP 94) capable of selectively recognizing gram-positive bacteria, because peptidoglycan on the surface of bacillus subtilis is used as a template molecule in the molecular imprinting process, the finally obtained molecular imprinting artificial receptor nanoparticle has excellent specificity and selectivity on the bacillus subtilis, the binding rate is up to 89.3%, and the binding rate of a non-imprinted polymer on the bacillus subtilis is only 38.1%. It has no selectivity to gram-negative bacteria Escherichia coli and eukaryotic yeast cells.
TABLE 5 binding rates of molecularly imprinted artificial receptor nanoparticles and non-imprinted nanoparticles, which can selectively recognize gram-negative bacteria, to different microbial cells, respectively, as measured by flow cytometry (formulation: NP 40)
TABLE 6 binding rates of molecularly imprinted artificial receptor nanoparticles and non-imprinted nanoparticles for selectively recognizing gram-positive bacteria, respectively, to different microbial cells as determined by flow cytometry (formulation: NP 94)
Example 4: molecular imprinting artificial receptor for regulating and controlling flora effect of escherichia coli
A molecularly imprinted nano artificial receptor, a molecularly imprinted artificial receptor using IM and DMAPAA as functional monomers, and non-imprinted nanoparticles (formulation: NP 40) were synthesized according to the method in example 1.
Coli producing green fluorescent protein (EGFP) was cultured to logarithmic growth phase, nanoparticles of different concentrations and a blank control (pure water) were added, and the effect of the change of nanoparticles with time on the bacteria was tested. When the bacteria reach the logarithmic phase of growth, taking a proper amount of bacterial liquid, adding equal volume of nanoparticles with different concentrations (0.3 mg/mL, 1.2mg/mL and 4.8mg/mL respectively) for co-incubation, and detecting the growth condition of the bacteria.
Sampling every 2h from the addition of the nanoparticles, and measuring the fluorescence and absorbance of the bacterial liquid by using an enzyme-labeling instrument, namely the fluorescence value and OD of the EGFP 600 The value is obtained. This represents the metabolic and growth of the bacteria, respectively, and the results are shown in FIG. 7.
The signal molecule DPD detection method is to detect (R) -4,5 dihydroxy-2,3-pentanedione (DPD) by using a gas phase mass spectrometry method. The DPD detection method comprises the following steps: coli by high speed centrifugation, the reaction conditions are 8000rpm, and the centrifugation time is 30min. 4mL of supernatant can be obtained from 5mL of bacterial liquid, and 1,2-phenylenediamine is added for derivatization. 1,2-phenylenediamine was dissolved in PBS to prepare 1,2-phenylenediamine 4mg/mL. The derivative mixture was placed in an oven at 37 ℃ and incubated for 2h in the dark. Extracting with dichloromethane, adding 4mL of dichloromethane into each sample, standing for layering, sucking the upper half liquid, discarding, adding anhydrous calcium chloride into the rest liquid, and drying. The extract was concentrated under nitrogen atmosphere, and N-methyl-N- (trimethylsilane) trifluoroacetamide was added in an amount of 150. Mu.L per sample, the mixture was heated at 60 ℃ for 30min, and 1mL of dichloromethane was added to the remaining liquid to dissolve it, and GC-MS measurement was performed.
When the bacteria reach the logarithmic phase of growth, a proper amount of bacteria liquid is taken, an isometric nanoparticle solution and a blank control are added, samples are taken every 2 hours, the signal molecule concentration at different times is measured according to the DPD detection method, the change condition of the signal molecule concentration after the nanoparticles are added is researched, and the result is shown in figure 7.
It can be seen that appropriate amount of molecularly imprinted nanoartificial receptors, which have influence on both escherichia coli growth and metabolism, were added to EGFP-producing escherichia coli. Research results show that the artificial receptor can promote escherichia coli to gather, thereby promoting bacterial quorum sensing, promoting bacterial growth and improving the yield of escherichia coli metabolite EGFP by about 20%. The GC-MS method is used for determining that the yield of signal molecules in the escherichia coli liquid added with the molecular imprinting nano-particles is obviously increased, and the promotion effect of the artificial receptor on the quorum sensing of escherichia coli is proved.
Example 5: selective recognition and separation of escherichia coli from whole blood by magnetic molecular imprinting artificial receptor
Synthesis of magnetic molecular imprinting artificial receptor: preparing 30mL of reaction mixed solution according to the formula of NP40, then respectively adding 1.5mg of lipopolysaccharide as a template molecule, then adding 6mg of surfactant Sodium Dodecyl Sulfate (SDS), mixing the above solutions in a round-bottomed flask, sealing the round-bottomed flask by using a bottle stopper and a sealing film, introducing nitrogen for 30min, and assisting mechanical stirring. After 20mg of initiator Ammonium Persulfate (APS) is added, 25 mu L of magnetic spheres (the mass volume ratio is 10-25%) are added, and the artificial receptor wrapping the magnetic spheres is synthesized by precipitation polymerization at 65 ℃ for 1 h. The magnetic molecularly imprinted artificial receptor was obtained by magnetic collection and washing with PBS (0.1M, pH 7.2-7.4).
1mL of whole blood, and using erythrocyte lysate to lyse erythrocytes for later use; taking 1mL of Escherichia coli liquid expressing green fluorescent protein (the count of a flow counter is about 10) 5 mL), centrifuged (3000rpm, 10min), resuspended with the treated whole blood solution, and the number of bacteria was counted as 1; respectively taking magnetic molecular imprinting artificial receptors (1 mg/mL,125-2000 μ L) with different volumes, separating for 5min by using a magnetic separation frame, and adding the bacterial liquid; incubating for 20-60min (shaking table, 37 deg.C, 200 rpm), adsorbing with magnet for 5min, and counting the number of bacteria as 2; the adsorption rate = (number 1-number 2)/number 1 × 100%, and the capture rate of the magnetic molecular imprinting artificial receptor to the bacteria is as high as 97%. After the bacteria are captured by the magnetic balls, the bacteria are dispersed by a small amount of water and transferred to a MUG culture medium for shake cultivation for 24 hours. All showed fluorescence (365 nm). The measurement results are shown in FIG. 8.
Example 6: nano artificial receptor wrapping Jin Gan photothermal effect for inhibiting bacterial growth
Preparing 30mL of reaction mixed solution according to the formula of NP40, then respectively adding 1.5mg of lipopolysaccharide as a template molecule, then adding 6mg of Sodium Dodecyl Sulfate (SDS) as a surfactant, mixing the above solutions in a round-bottomed flask, sealing the round-bottomed flask by using a bottle stopper and a sealing film, introducing nitrogen for 30min, and assisting mechanical stirring. After 20mg of initiator Ammonium Persulfate (APS) is added, 1mL of nano Jin Gan (0.5 mg/mL) is added to synthesize the artificial receptor wrapping the nano gold rod. The apparent morphology was characterized by scanning electron microscopy, as shown in fig. 9.
And (3) carrying out full-wavelength scanning on each sample, determining that the sample has a maximum absorption peak near 800nm, and exciting by adopting laser with the wavelength of 808nm in an experiment.
Carrying out photo-thermal performance test on the artificial receptor wrapped by the nano gold rod, wherein the illumination condition is as follows: 808nm of laser and 2.0W/cm of power 2 Irradiation for 5min, dosage: 1mL of the artificial receptor wrapping the nano-gold rod is 1mg/mL, and the result proves that the artificial receptor has a good photo-thermal effect, as shown in FIG. 10. The temperature can be raised to 45 ℃ within 5min, and the photo-thermal effect is good.
Coli was cultured overnight (37 ℃,180 rpm), 1mL of the bacterial liquid was taken, centrifuged (3000rpm, 10min), resuspended in pure water, and 1mL of different materials were added: blank control-water, molecular imprinting artificial receptor (1 mg/mL), non-imprinting nano-particles, molecular imprinting artificial receptor wrapping nano-gold rods, non-imprinting polymer wrapping nano-gold rods (1 mg/mL,1 mL) were incubated for 2h, and then respectively irradiated by light (808nm, 2.0W/cm) 2 ) 5min and keeping away from light, taking a proper amount of treated bacteria to be flatly laid on a solid culture medium, culturing for 24h, and obtaining a final result as shown in figure 11, wherein the existence of the bacteria cannot be basically observed in the molecular imprinting artificial receptor group wrapping the nanogold rods, the bacteriostatic effect is very good, and the non-imprinting polymer group wrapping the nanogold rods also has certain recognition and aggregation effects on the bacteria, so that under the condition of illumination, the effect is slightly poor compared with the molecular imprinting artificial receptor wrapping the nanogold rods.
Claims (10)
1. A preparation method of an artificial receptor capable of selectively identifying microorganisms is characterized by comprising the following specific steps:
(1) Dissolving N-tert-butyl acrylamide or N-phenyl acrylamide in ethanol, dissolving N-isopropyl acrylamide, a functional monomer, a cross-linking agent and an initiator in water, mixing the reactants in different proportions, adding the mixture into a detection pool, removing oxygen in the mixed solution by introducing nitrogen for 5-10min through ultrasound, and carrying out in-situ polymerization reaction at the bottom of the detection pool to obtain a polymer thin layer; washing the polymer thin layer with water for 3-5 times after reaction to remove unreacted monomers;
(2) Adding microbial cell surface antigen into the detection pool for incubation, wherein the antigen is adsorbed on the polymer thin layer at the bottom of the detection pool; determining the affinity of the polymer to the antigen by detecting the amount of antigen adsorbed by the polymer thin layer or detecting the amount of residual antigen in the supernatant, and screening out a polymer synthesis formula with high affinity to the antigen;
(3) Preparing raw materials according to the formula screened in the step (2), adding a surfactant and a template agent, removing oxygen in the mixed solution by introducing nitrogen for 5-10min through ultrasound, stirring for polymerization reaction to obtain polymer nanoparticles, eluting the template, and purifying by dialysis; then measuring the affinity constant K of the polymer nano particles and the antigen by a biomembrane interference technology D And screening out polymer nanoparticles with high affinity and selectivity to microbial cells, namely the finally constructed artificial receptor.
2. The preparation method of claim 1, wherein the functional monomer is selected from one or more of (3-acrylamidopropyl) trimethyl ammonium chloride, N- (3-aminopropyl) methacrylamide hydrochloride, 1-vinylimidazole, N- [ (3- (dimethylamino) propyl ] methacrylamide, N- (2-aminoethyl) acrylamide, acrylic acid, 2-hydroxyethyl methacrylate, itaconic acid, sodium vinylsulfonate, methacrylic acid, 4-vinylphenylboronic acid, and vinylphenol, the crosslinking agent is one or more of N, N '-methylenebisacrylamide, N' -vinylbisacrylamide, and ethylene glycol dimethacrylate, and the initiator is ammonium persulfate, azobisisobutyronitrile, or tetramethylethylenediamine.
3. The method according to claim 1, wherein the polymerization reaction in step (1) is carried out in a sealed or nitrogen atmosphere at a temperature of 25 ℃ to 80 ℃ for a period of 2 to 72 hours.
4. The method according to claim 1, wherein the antigen on the surface of the microbial cell is lipopolysaccharide or peptidoglycan on the surface of the microbial cell.
5. The preparation method according to claim 1, wherein the template agent is a microbial cell surface antigen or a microbial cell surface antigen surface-modified silica microsphere, a ferroferric oxide magnetic sphere or a glass bead microsphere.
6. The method for preparing a template according to claim 1, wherein the method for eluting the template in step (3) comprises: eluting the template agent by raising the temperature to 50-90 ℃, reducing the temperature to 0-10 ℃, adding 1-5M NaCl aqueous solution, stirring and reacting for 30min, adding the same volume of methanol and acetic acid mixed solution, stirring for 30min, or adding the same volume of acetonitrile aqueous solution with the volume concentration of 30%, stirring for 30min.
7. The method of claim 1, wherein the polymerization reaction of step (3) is carried out in a sealed or nitrogen atmosphere, the polymerization reaction temperature is 25 ℃ to 80 ℃, and the polymerization reaction time is 2 to 72 hours.
8. A method for regulating and controlling microbial community effect is characterized by comprising the following specific operations: 0.3-4.8mg/mL of an artificial receptor dispersion capable of selectively recognizing a microorganism is added to the corresponding microorganism in a volume ratio of 1.5-8.
9. A preparation method of a magnetic artificial receptor capable of selectively identifying and separating microbial cells is characterized by comprising the following specific operations: the method of claim 1, wherein magnetic nanoparticles are further added to the reaction solution in the step (3) of preparing the artificial receptor capable of selectively recognizing and separating microorganisms, and finally composite particles coated with the magnetic nanoparticles are synthesized, namely the magnetic artificial receptor capable of selectively recognizing and separating microorganism cells.
10. The preparation method of the photothermal bacteriostat is characterized by comprising the following specific operations: the method of claim 1, wherein nanogold is further added to the reaction solution of step (3) for preparing the artificial receptor capable of selectively recognizing microorganisms, and finally, nanogold-coated composite particles, namely the photothermal bacteriostatic agent, are synthesized.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211072502.6A CN115521893A (en) | 2022-09-02 | 2022-09-02 | Artificial receptor capable of selectively identifying microorganisms and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211072502.6A CN115521893A (en) | 2022-09-02 | 2022-09-02 | Artificial receptor capable of selectively identifying microorganisms and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115521893A true CN115521893A (en) | 2022-12-27 |
Family
ID=84697640
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211072502.6A Pending CN115521893A (en) | 2022-09-02 | 2022-09-02 | Artificial receptor capable of selectively identifying microorganisms and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115521893A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101802213A (en) * | 2007-08-01 | 2010-08-11 | 日立化成工业株式会社 | Pathogen detection in the large-volume particulate samples |
| US20120263922A1 (en) * | 2010-07-13 | 2012-10-18 | The University Of Houston System | Sensors and separation based on molecular recognition via electropolymerization and colloidal layer templates |
| CN103992252A (en) * | 2014-05-23 | 2014-08-20 | 华中科技大学 | Dopamine derivative, molecular imprinted polymer and preparation methods and application of dopamine derivative and molecular imprinted polymer |
| US20190077850A1 (en) * | 2017-08-10 | 2019-03-14 | President And Fellows Of Harvard College | Pathogen binding methods and compositions |
| CN110483683A (en) * | 2019-07-21 | 2019-11-22 | 北京化工大学 | A kind of preparation method and purposes of target tumor nano artificial antibody |
-
2022
- 2022-09-02 CN CN202211072502.6A patent/CN115521893A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101802213A (en) * | 2007-08-01 | 2010-08-11 | 日立化成工业株式会社 | Pathogen detection in the large-volume particulate samples |
| US20120263922A1 (en) * | 2010-07-13 | 2012-10-18 | The University Of Houston System | Sensors and separation based on molecular recognition via electropolymerization and colloidal layer templates |
| CN103992252A (en) * | 2014-05-23 | 2014-08-20 | 华中科技大学 | Dopamine derivative, molecular imprinted polymer and preparation methods and application of dopamine derivative and molecular imprinted polymer |
| US20190077850A1 (en) * | 2017-08-10 | 2019-03-14 | President And Fellows Of Harvard College | Pathogen binding methods and compositions |
| CN110483683A (en) * | 2019-07-21 | 2019-11-22 | 北京化工大学 | A kind of preparation method and purposes of target tumor nano artificial antibody |
Non-Patent Citations (2)
| Title |
|---|
| DIETER BAURECHT等: "Raman and scanning probe microscopy for differentiating surface imprints of E. coli and B. cereus", 《JOURNAL OF MATERIALS CHEMISTRY B》, vol. 10, no. 35, 13 April 2022 (2022-04-13), pages 6758 - 6767 * |
| Y. LONG等: "Novel polymeric nanoparticles targeting the lipopolysaccharides of Pseudomonas aeruginosa", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》, vol. 502, no. 1, 17 February 2016 (2016-02-17), pages 232 - 241, XP029449406, DOI: 10.1016/j.ijpharm.2016.02.021 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5678565B2 (en) | Magnetic fine particles and method for producing the same | |
| CN113351182B (en) | Magnetic microsphere with surface modified by zwitterionic polymer and preparation method and application thereof | |
| CN111235233A (en) | Staphylococcus aureus colorimetric sensing detection method based on aptamer recognition-HCR reaction and application thereof | |
| CN107746841B (en) | Zwitterion magnetic composite hydrogel immobilized enzyme carrier and preparation method thereof | |
| Ye et al. | An electrochemical immunoassay for Escherichia coli O157: H7 using double functionalized Au@ Pt/SiO2 nanocomposites and immune magnetic nanoparticles | |
| CN101037676A (en) | New function and usage of magnetic nano material | |
| KR20110033575A (en) | Mesoporous silica conjugate integrating magnetic nano particles having peroxidase activity and enzymes and method for manufacturing the same | |
| CN109655609B (en) | Platinum-nanoflower and preparation method and application thereof | |
| Bone et al. | Physisorption and chemisorption of T4 bacteriophages on amino functionalized silica particles | |
| Li et al. | A novel low-field NMR biosensor based on dendritic superparamagnetic iron oxide nanoparticles for the rapid detection of Salmonella in milk | |
| CN104749365A (en) | Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria | |
| EP2972370B1 (en) | Method of capturing bacteria on polylysine-coated microspheres | |
| CN107271410B (en) | Method for rapidly detecting activity of bacteria or fungi | |
| CN111100840A (en) | Magnetic nano-composite for specifically capturing and effectively releasing circulating tumor cells and preparation method thereof | |
| CN104928275A (en) | Preparation method and application of bacterial spore functional microsphere | |
| CN115521893A (en) | Artificial receptor capable of selectively identifying microorganisms and application thereof | |
| Wang et al. | A mild and safe gas-responsive molecularly imprinted sensor for highly specific recognition of hepatitis B virus | |
| Dušak et al. | Application of magneto‐responsive Oenococcus oeni for the malolactic fermentation in wine | |
| CN107132206A (en) | Virus activity quick determination method | |
| CN111269366A (en) | Preparation method of high-selectivity ceftriaxone sodium magnetic molecularly imprinted polymer | |
| CN115595146A (en) | Colorimetric fluorescent double-signal nano-microsphere and preparation method and application thereof | |
| Nekounam et al. | Application of functional magnetic nanoparticles for separation of target materials: a review | |
| De Liu et al. | Light scattering sensing detection of pathogens based on the molecular recognition of immunoglobulin with cell wall-associated protein A | |
| CN109337894B (en) | Preparation method of yeast functional microspheres and application of yeast functional microspheres in immunoassay | |
| Ji et al. | Functionalized magnetic nanobeads for SERS-based detection of Staphylococcus aureus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |