Establishment Of Visualized Evaluation Method For Aquatic Product Freshness Based On Hypoxanthine Detection

Aquatic products are nutritious and rich in high-quality protein,lipid,minerals.They usually have soft texture and high moisture content,and are easily affected by microorganisms and endogenous proteases in the process of storage,transportation and marketing process.Therefore,the freshness is one of the most important indicators to evaluate the quality of aquatic products.There are many methods for evaluating the freshness of aquatic products,but these methods possess a series of disadvantages,such as complex detection instruments,cumbersome sample pretreatment and high testing cost.Biosensor methods have the advantage of simple operation and possess high specificity,and thus has been widely applied to the freshness evaluation of aquatic pruducts in recent years.Hypoxanthine,a metabolite of ATP after the death of aquatic product,accumulates right after the fish is slaughtered and has the potential to determine the early stages of fish spoilage.In this paper,two hypoxanthine biosensing methods were established and visual freshness evaluation of aquatic product by converting the biochemical signal into color signal was realized.The specific research contents and results are as follows:1.A colorimetric enzyme biosensor was developed for one-step detection of hypoxanthine(Hx)based on the peroxidase activity of xanthine oxidase Grade I ammonium sulfate suspension(XOD-ASS).It could oxidize different peroxidase substrates,including3,3',5,5'-tetramethylbenzidine(TMB)in the presence of H2O2,producing visible color reactions.Further experiment indicated that XOD-ASS displayed effective peroxidase activity and could be used for H2O2 detection.Based on this,a one-step Hx detection method was established using only XOD-ASS as the catalyst.The method displayed a good linear relationship in the range from 20 ?M to 100 ?M with a detection limit of 6.93 ?M.Additionally,we successfully applied this method in testing Hx accumulation in sea bass fish samples of different storage time.The method was applied to detect hypoxanthine content in sea bass samples and the recoveries were ranged from 97.44% to 102.56%.This method successfully evaluate the freshness of sea bass samples.2.A paper-based enzyme biosensor for Hx was developed,which could realize direct,visual and one-step detection of fish freshness without any pretreatment.Xanthine oxidase(XOD)and horseradish peroxidase(HRP)were immobilized on nitrocellulose membrane combined with TMB to output color signal.We demonstrated that polysaccharide modified nitrocellulose membrane could promote the enzymatic reaction and produce focused colorimetric signal.The developed paper chip produces a linear response from 0.01-0.16 m M with a detection limit of 8.22 ?M.The biosensor has high selectivity towards hypoxanthine and showed 96.13 %-103.11 % recovery for fish meat sample.This paper biosensor can be directly attached on the surface of fish sample without any pre-treatment process and the whole detection procedure can be finished within 3 min.The color signal can be preliminary judged by naked eyes to discriminate fresh and spoilage fish samples and further analyzed by a smart phone for quantitative analysis.The developed biosensor is simple,low-cost and equipment-free,showing great potential in on-site aquatic product freshness evaluation.This study established a one-step colorimetric detection system and a paper-based enzyme biosensor system to detect hypoxanthine.The colorimetric detection method realized facile,one-step and colorimetric detection of hypoxanthine by using only XOD-ASS as the catalyst,providing a robust advantage on economical reaction system and short time consumption.The paper-based enzyme biosensor system could reduce the effect of complex biochemical characteristics of the matrix composition,and realizes the direct judgment of the freshness quality by naked eyes.The establishment of testing methods can meet the major needs of pre-judgment of the quality of aquatic raw materials,which is of great significance for ensuring the quality of raw aquatic products.