The Detection Of Glutathione And Escherichia Coli O157:H7 Based On Polyoxometalate Nanozymes

Objective:By screening the mimic enzyme activity of polyoxometalates?POMs?,a new POMs nanozyme P₂W₁₈Fe₄/PDA was designed and synthesized.The new method of detection of glutathione?GSH?and foodborne pathogenic bacteria O157:H7?E.coli O157:H7?was constructed.The methods were evaluated and provided new technologies and new methods for the detection of human health-related factors and food safety.Methods:1.The POMs were characterized by IR and UV-vis spectrum.The enzyme mimetic activities of POMs were investigated with the reaction of chromogenic substrates 3,3',5,5'-tetramethylbenzidine?TMB?and o-phenylenediamine?OPD?catalyzed by POMs.The catalytic conditions of polyoxometalate were optimized by varing the pH,concentration of substrate and polyoxometalate and buffer composition.The Km,Vmax and Kcat were determined by Michaelis and LineweaverBurk equation and compared with HRP to screen the target nanoenzyme.2.The iron-substituted tungstophosphorate polydopamine nanozyme P₂W₁₈Fe₄/PDA was successfully prepared by hydrothermal method and characterized with IR,UV-vis,TEM,SEM and Zeta.Through the color reaction of H₂O₂ and OPD,the peroxidase-like activity of P₂W₁₈Fe₄/PDA was determined by UVs.The catalytic conditions of POMs were optimized by varing the pH,concentration of substrate and POMs and buffer composition.The Km,Vmax and Kcat were determined by Michaelis and LineweaverBurk equation to investigated the catalytic kinetics mechanism.3.A new method of fluorescence and ultraviolet dual-mode detection of GSH based on P₂W₁₈Fe₄/PDA nanozyme was constructed using ultraviolet visible spectrum and fluorescence spectrum.The method was evaluated by linear curve,linear correlation coefficient,detection limit,actual sample spiked recovery,relative standard and interference test to justify the practicability.4.The E.coli O157:H7 polyclonal antibody was conjugated on the surface of P₂W₁₈Fe₄/PDA by covalent bond to form enzyme-labeled antibody.A new ELISA method for detection of food-borne pathogenic bacteria E.coli O157:H7 was established.The method was evaluated by linear curve,linear correlation coefficient,detection limit,relative standard,specificity and interference test.Results:1.Among the eighteen POMs,H₃PW₁₂O₄₀,H₄SiW₁₂O₄₀,H₄GeW₁₂O₄₀,?-?NH₄?₆P₂W₁₈O₆₂,?-K₆P₂W₁₈O₆₂·14H₂O,Na₈H[?-PW₉O₃₄],Na₁₀[?-SiW₉O₃₄],Na₁₀[?-GeW₉O₃₄],K₈[?-SiW₁₀O₃₆],K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈ and Na₁₀H₂W₁₂O₄₂ had the peroxidase activities.Eu₃PMo₁₂O₄₀,H₃PMo₁₂O₄₀,H₄SiMo₁₂O₄₀,?-1,2,3-K₆H[SiW₉V₃O₃₄],H₆P₂Mo₁₈O₆₂ and H₅PMo₁₀V₂O₄₀ showed the oxidase-like activities.K₄GeW₁₂O₄₀ did not show the peroxidase and oxidase activities.The Na₈H[?-PW₉O₃₄],Na₁₀[?-SiW₉O₃₄]and Na₁₀[?-GeW₉O₃₄]showed intrinsic enzyme activities at alkaline conditions,which were different from other type of POMs.The sandwich-type K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈ displayed the strongest peroxidase activity,which is similar to natural horseradish peroxidase.The Kcat of K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈ was 3.80×10^-33 S^-1.2.The iron-substituted tungstophosphorate polydopamine nanomimetic enzyme P₂W₁₈Fe₄/PDA was successfully prepared.The P₂W₁₈Fe₄/PDA had better peroxidase activity than K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈.The K_M and Vmax of P₂W₁₈Fe₄/PDA nanozyme with H₂O₂ as substrate were 0.51mM and 6?M·min^-1.The K_M and Vmax of P₂W₁₈Fe₄/PDA with OPD as substrate were 0.84?M and 0.6?M·min^-1.The K_M of P₂W₁₈Fe₄/PDA with H₂O₂ as substrate was in the same order of magnitude with P₂W₁₈Fe₄.But the K_M of P₂W₁₈Fe₄/PDA with OPD as substrate was about two orders of magnitude lower than P₂W₁₈Fe₄ which illustrated that polydopamine significantly improved the affinity of K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈ for substrate OPD.3.The fluorescence and colorimetric dual-mode detection of GSH was established with the peroxidase activity of P₂W₁₈Fe₄/PDA.This method can realize the sensitive and specific detection of GSH under the conditions of simultaneous interference of vitamin C,amino acid and ions.As for GSH colorimetric detection,the lower limit of detection of was found to be 0.18mM?3?/S?in the detection range from 2 to 8 mM.As for GSH fluorescence detection,the lower limit of detection was4.02?M with linear range from 15.63 to 250?M.The linear relationship was good and the correlation coefficients were above 0.99.The fluorescence method was used to detect the glutathione concentration in SH-SY5Y cell lysate.The recovery was97.86¹01.72%with the relative standard deviation from 1.13 to 4.39%.The results were accurate and precise.4.The ELISA method for the detection of E.coli O157:H7 was established with the peroxidase activity of P₂W₁₈Fe₄/PDA.The lower detection limit was determined to be 4.2×?10²?cfu/mL with linear range from 10³ to 10⁶ cfu/mL.The linear relationship was good and the correlation coefficients were above 0.99.Staphylococus aureus,Pseudomonas and Listeria monocytogenes did not influence the assay of E.coli O157:H7 at the concentration of 10⁶ cfu/mL.Conclusion:1.POMs may have peroxidase activity and oxidase activity,and their enzyme activity was related to their structural composition.For the first time,it was found that the POMs with absent-Keggin structure has peroxidase activity under alkaline conditions.The sandwiched polyacid K₁₀P₂W₁₈Fe₄?H₂O?₂O₆₈ had the highest peroxide activity among the POMs screened.2.The iron-substituted tungstophosphorate polydopamine nanozyme P₂W₁₈Fe₄/PDA was successfully prepared.The P₂W₁₈Fe₄/PDA was uniform in size and morphology and had peroxidase-like activities.3.The colorimetric and fluorescence dual-mode method based P₂W₁₈Fe₄/PDA nanozyme for the detection of GSH was established.This method was simple,fast,low detection limit,wide linear range and had well anti-interference and accuracy.4.The ELISA method based P₂W₁₈Fe₄/PDA for the detection of foodborne pathogens was established and the detection of E.coli O157:H7 was initially achieved.This method had high specificity and wide linear range.