Application Of Nanozymes In Mimicing Polyphenol Oxidase Activity In Food Analysis

Nanozyme refers to nanomaterials with enzymatic activity.At present,it has attracted the attention of researchers.It aims to solve the limitations of natural enzymes and traditional artificial enzymes.And with the development of nanotechnology,biotechnology and catalytic science has made outstanding achievements.Polyphenol oxidase is often found in plants and animals and is also known as catechol oxidase,tyrosinase,or laccase.Polyphenol oxidase is a copper-containing oxidoreductase that can catalyze the hydroxylation of monophenols to bisphenols and catalyze the oxidation of bisphenols to quinones.It is often used to remove aromatic pollutants in pollutants.In recent years,there have been few studies on nanozymes that mimic polyphenol oxidase.Therefore,this article aims to synthesize nanozymes that mimic polyphenol oxidase and apply them to food analysis.The main research contents include:1.With adenosine triphosphate?ATP?,adenosine diphosphate?ADP?,adenosine monophosphate?AMP?and uridine monophosphate?GMP?for organic ligands,and copper???coordinate bond formation with polyphenol oxidase activity of nanonzymes?ATP-Cu,ADP-Cu,AMP-Cu and GMP-Cu?.Such nanozymes also have fluorescence characteristics.Due to the small excitation wavelength,the fluorescence generated by nanozymes are not easy to be observed by the naked eye.Therefore,Pdots with strong fluorescence is doped into AMP-Cu nanozymes to synthesize a new fluorescent nanocomposite?Pdots@AMP-Cu?.We will also dimethyl imidazole and copper???complex two-dimensional copper fund belongs to 2D metal organic framework?2D Cu-MOF?,and respectively from the same quality of and into the nanozyme concentrations,stability and kinetic parameters are compared.2.Through the comparison of nanozymes,we found 2D Cu-MOF has excellent polyphenol oxidase activity and stability,2D Cu-MOF oxidizes the colorless catechol to a light yellow color,resulting in a color signal,and therefore based on 2D Cu-MOF established a simple and rapid colorimetric detection method of catechol.The absorption intensity and concentration of catechol linearly related,the detection limit?LOD?is 3.5?g/mL,the method is used for analysis of catechol in food water.3.Adenosine triphosphate?ALP?activity was analyzed by fluorescence and colorimetry based on nucleotide-Cu nanozymes.By catalyzing the chromogenic reaction of 2,4-dichlorophenol?2,4-DP?and 4-aminoantipyrine?4-AP?,the three nanozymes?ATP-Cu,ADP-Cu and AMP-Cu?all showed the activity of polyphenol oxidase.However,there were significant differences in polyphenol oxidase activity and fluorescence intensity among the three nanozymes produced by the same concentration of nucleotides(keeping the concentration of Cu²⁺at 5 mM).When the concentration of nucleotide was 3 mM,the catalytic activity of ADP-Cu and AMP-Cu were significantly higher than that of ATP-Cu.In addition,when ATP hydrolyzed into ADP and AMP by ALP,more nanozymes were produced,and the catalytic activity and fluorescence intensity of the system increase,resulting in significantly enhanced colorimetry and fluorescence signals.Within the range of 0-30 U/L,the colorimetric signal strength was proportional to the ALP concentration,and the detection limit of ALP detected by colorimetric method was 0.3 U/L.In addition,the fluorescence intensity of the prepared nanozyme was also proportional to the ALP concentrations within the range of 1-30U/L,and the fluorescence detection limit was 0.45 U/L.Therefore,an ALP detection method based on fluorescence and colorimetry was established.Compared with protein,amino acid and other interfering components,this method has higher selectivity for alkaline phosphatase activity.In addition,this method was also used to detect ALP activity in milk samples,showing great practical value.4.We developed the fluorescent nanozymes Pdots@AMP-Cu for sensitive and selective detection of dopamine?DA?.The results showed that DA could be oxidized by Pdots@AMP-Cu to produce eumelanin,and the fluorescence of Pdots@AMP-Cu was effectively quenched by eumelanin due to electron transfer.Therefore,a new DA detection method was established.A good linear relationship was observed between fluorescence quenching intensity and DA in the concentration range of 10-400?M,and the detection limit was 4?M.The method of detecting DA based on Pdots@AMP-Cu established by us can also be used to detect DA in human serum.Since this detection principle is based on the phenolic hydroxyl structure of DA,we speculate that phenolic compounds with a DA-like structure,such as epinephrine,levodopa,2,6-dimethoxyphenol,hydroquinone,and ractopamine,can also be sensitively detected in principle by fluorescent nanozymes.Therefore,this method can be used not only in clinical diagnosis,biosensing,environmental monitoring,but also in food quality and safety testing,etc.,and has a good application prospect.