Application Of Manganese Dioxide Nanosheets Mediated Hypoxanthine Biosensor For Aquatic Products’ Freshness Evaluation

Aquatic products are nutritious and flavorful,are rich in protein,fat,vitamins,and minerals needed by the human body,and have become an essential dietary component on the table.Due to its rich water content and soft muscle tissue,it is easily affected by endogenous enzymes,exogenous microorganisms,and environmental factors in transportation,storage,and processing.Compared with other meat products,it is more susceptible to spoilage,posing a severe threat to food safety and human diet health.Recently,hypoxanthine（Hx）has been regarded as an essential index for aquatic products’freshness evaluation,especially suitable for quality monitoring at the early stage of spoilage.Hx can react with the dissolved oxygen under the catalysis of xanthine oxidase（XOD）to yield hydrogen peroxide（H₂O₂）and uric acid（UA）.Existing Hx biosensors are based on detecting H₂O₂ alone,and deficiencies still need improvements,such as low sensitivity,complex operation,or dependence on catalase.This thesis proposes two kinds of manganese dioxide（MnO₂）nanosheets mediated Hx biosensors to solve or improve these problems.The detection mechanism relies on Hx’s catalytic products;H₂O₂ and UA can decompose MnO₂ nanosheets.Subsequently,the change of MnO₂ nanosheets’oxidation and oxidase-like activity can regulate the fluorescence and multi-color colorimetric signals to achieve the highly sensitive and convenient detection of Hx.The main research contents are as follows:1.The fluorescence sensing system mediated by MnO₂ nanosheets was developed for highly sensitive and rapid detection of Hx.Calcein,also known as fluorescein,whose solution is yellow with green fluoresces,has been widely used in thermostatic amplification reactions.Studies have shown that divalent manganese ion(Mn²⁺)can coordinate with calcein and quench its fluorescence.Therefore,when the system contains Hx,H₂O₂ and UA generated under the catalysis of XOD will decompose MnO₂ nanosheets into Mn²⁺,which can reduce the fluorescence intensity of calcein.Because the fluorescence intensity of calcein decreases with the increased concentration of Hx,a highly sensitive and convenient fluorescence sensing system was constructed to detect Hx quantitatively.It was successfully applied to the freshness evaluation of aquatic products.According to the experiment results,this method displayed good linear response from 1 to 800μM with a detection limit of 0.396μM;the detection time was only 12 min.The freshness evaluation results of crucian and shrimp samples confirmed that the system performed well in monitoring the spoilage process,and the recoveries ranged from 94.09%to 115.05%.This method is expected to serve as a potential,rapid and sensitive method for the freshness evaluation of aquatic products and food safety.2.Smart phone-assisted multi-color Hx sensing method was proposed mediated by MnO₂ nanosheets for highly sensitive,on-site evaluation of aquatic freshness.Gold nanorods（Au NRs）have unique local surface plasmon resonance（LSPR）properties,which show rainbow color changes by adjusting aspect ratio and are ideal"color developing substrates"for multi-color systems.MnO₂ nanosheets could oxidize colorless 3,3’,5,5’-tetramethylbenzidine（TMB）to yellow TMB²⁺due to the oxidase-like activity.TMB²⁺can effectively etch the surfactant-coated Au NRs through oxidation,reducing the aspect ratio and showing a color change from blue-green to blue to purple to pink.Therefore,when the system contains Hx,H₂O₂ and UA generated under the catalysis of XOD could decompose MnO₂ nanosheets and weaken their oxidase-like activity,thus inhibiting the formation of TMB²⁺and the following etching process.With the increased Hx’concentration,the sensing system presented a color change from pink to purple to blue to blue-green.The constructed palm freshness evaluation small program named"RGB analysis&Freshness"based on RGB（red/green/blue）analysis could give corresponding freshness suggestions by taking photos,selecting pictures,and RGB analysis,which achieved visual and online freshness monitoring of aquatic products.This method displayed good linear response from 1 to 100μM with a detection limit of 0.378μM;the detection time was only 14min.The constructed palm freshness evaluation program displayed good linear response from 1 to 100μM with a detection limit of 0.8906μM.Additionally,the proposed strategy performed well in real-time monitoring of crucian and shrimp spoilage under different storage conditions with temperatures of 37℃,25℃,and 4℃,respectively.This method is expected to serve as a high-sensitivity and visual on-line freshness evaluation method in real-time monitoring of aquatic product freshness.In summary,this study first achieves the quantitative analysis of Hx and aquatic products’freshness evaluation through simultaneous detection of Hx catalytic products H₂O₂ and UA.Based on MnO₂ nanosheets’oxidation and oxidation-like activity and the degradation of MnO₂ nanosheets caused by H₂O₂ and UA,MnO₂nanosheets mediated fluorescence and multi-color colorimetric biosensors were constructed,respectively,and successfully applied to the freshness evaluation of aquatic products.The proposed method is simple,sensitive,and short in detection time,which provides a new direction for aquatic products’freshness evaluation.