Fabrication A Novel Nano-biosensor For Efficient Colorimetric Determination Of Uric Acid

Uric acid（C₅H₄N₄O₃,UA）is the main end product of purine（such as adenine,guanine）metabolism in the human system.It mainly exists in human blood serum and urine.Generally,uric acid levels in biological samples are within a fixed range and abnormal uric acid level often means the occurrence of some diseases.For example,high levels of UA lead to diseases such as gout and hyperuricemia,while low levels of UA lead to multiple sclerosis.So,uric acid can be used as a predictor or marker for these diseases and developing a method to detect uric acid is particularly important.There are several techniques available to measure uric acid in biological materials,such as high-performance liquid chromatography（HPLC）,gas chromatography,capillary electrophoresis（CE）and chemiluminescence analysis.However,these methods often have complex equipment,high cost,complex operation and long time consuming.In contrast,the colorimetric method is simpler which can directly observe the change of color by naked eyes.This method has higher sensitivity,low price,strong practicability and fast detection.In this research,we have prepared a novel nano-biosensor and used it to identify UA.Our work mainly included the following two aspects:Firstly,uricase was used as plasticizer to shape copper nanozyme,and a novel nano-biosensor was obtained by mild preparation in this study.The effects of uricase concentration,preparation time,preparation temperature and p H on the formation of the nanoparticles were studied in detail.The optimal preparatory circumstances were attained:60μL uricase solution（1 mg/m L）and 20μL copper sulfate solution（120 m M）was added to 3 m L PBS buffer solution（5 m M,p H 7.4）.The mixed solution was gently shaken and placed at 25℃for 72 hours to obtain the best shape of the nano-biosensor.After that,scanning electron microscopy（SEM）、energy dispersive X-ray spectroscopy（EDX）and Fourier Transform infrared spectrometer（FT-IR）were characterized the nano-biosensor.This study confirmed that the nano-biosensor contains Cu²⁺,PO₄^3-of copper nanozyme and amide bond I,II of uricase.These results suggest that uricase has been successfully embedded into the copper nanozyme.Furthermore,we used TMB and ABTS as substrates to verify that the nano-biosensor had good peroxidase-like activity.We further studied the steady-state kinetics of peroxide-like enzymes.It was found that the K_m value and V_max value of the nano-biosensor were both greater than HRP,indicating that the nano-biosensor has low affinity with the substrate,but high catalytic efficiency.The nano-biosensor can be used as a substitute for natural HRP.Based on the previously mentioned work,the prepared nano-biosensor has been applied to uric acid colorimetric detection.The detection process and related influencing factors were studied in depth.The detection environment consisted 20 m M4-AAP and 25 m M phenol.They reacted by coupling to produce red indole dye which the reactant became from colorless to red.A series of experiments were carried out to determine the best uric acid detection conditions.The optimal uric acid detection conditions were as follows:The optimum of catalyst content was 1.6 mg,the optimum of temperature was 50℃,the optimum of reaction time was 10 min,and the optimum of p H was 8.5.Then the absorbance at 505 nm was measured by UV-spectrophotometer.The results demonstrated that our designed nano-biosensor had a good linearity within1-50μM and a detection limit of 0.6μM.At the same time,the experimental results displayed that the prepared nano-biosensor has a high selectivity for UA detection.Subsequently,to verify the practicality of the nano-biosensor,uric acid levels in real human samples have been measured.The results revealed that the recoveries of uric acid in urine was 93.25%-102.50%,and the relative standard deviation（RSD,n=3）was less than 3.0%.Moreover,our experimental results also showed that the nano-biosensor has good storage stability and reusability.In summary,a novel nano-biosensor has been prepared and successfully applied in one-step uric acid detection,which greatly simplifying the detection process.Concurrently,its accuracy,reliability and selectivity make the nano-biosensor has great application prospect in clinical detection.