Construction And Application Of A Portable Biosensor For Detection Of Acetylcholinesterase In Patients With Myasthenia Gravis During Perioperative Period

Myasthenia gravis is a chronic autoimmune disease characterized by skeletal muscle weakness and abnormal fatigue.Muscles innervated by cranial nerves are especially affected.Myasthenia usually occurs in the muscles of the neck,trunk and limbs,and even affects respiratory muscles in severe cases.Acetylcholinesterase activity is closely related to the condition of myasthenia gravis patients,and it is often used as a biomarker to help judge the condition.When cholinesterase inhibitor is overdosed,AChE activity decreases excessively,which may lead to cholinergic crisis in patients.Therefore,it is of guiding significance for perioperative management of myasthenia gravis to obtain information of AChE activity immediately.Traditional methods for detecting cholinesterase activity mainly include liquid chromatography,mass spectrometry,liquid chromatography-mass spectrometry and ferric chloride spectrophotometry,etc.However,due to expensive equipment,high maintenance cost and long detection time,real-time monitoring cannot be realized.In this paper,the intrinsic optical properties of nanomaterials and the spatial structure of hydrogels were used,and a portable biosensor was developed by combining smart phones and 3-D printing technology,which was applied to the real-time detection of AChE activity.The main research contents of this paper are as follows.1.By simulating the active center structure(central atom and amino acid microenvironment)of laccase,a new functional nano-enzyme His-Cys-Cu was developed.It can catalyze 2,4-dichlorophenol(2,4-DP)and 4-aminoantipyrine(4-AP)to produce 4-N-(1,4-benzoquinone imine)antipyrine(4-NBP),which causes the reaction system to change from colorless to red and has a strong characteristic absorbance at 510 nm.AChE-catalyzed thiocholine(ATCh)can competitively bind to the active site of His-Cys-Cu,thus destroying the catalytic performance of nanoenzyme and regulating the color development effect.Therefore,the detection of AChE was realized by measuring the absorbance of the system.Further use portable optical imaging system to collect high-definition color pictures.The gray value is analyzed by Image J software,and finally the purpose of detecting AChE is achieved,with the detection range from 1.25 to 25 U L-1.2.In order to shield the interference of serum matrix,AuNCs/Co OOH NFs sensing system based on red luminescence was established.Combined with SA hydrogel and3-D printing technology,a low-cost,simple and easy hydrogel sensing platform was made,and the on-site quantitative analysis of AChE was realized.The fluorescent hydrogel was prepared by encapsulating gold nanoclusters(AuNCs)indicator into sodium alginate(SA)hydrogel.The fluorescence of AuNCs was effectively quenched by F?rster resonance energy transfer(FRET)effect when Co OOH NFs was added.AChE can catalyze the generated TCh,further etch Co OOH NFs to generate Co2+,and regulate the quenching efficiency of Co OOH NFs.The analysis and detection of AChE can be realized according to the change of fluorescence intensity of the system.Combined with SA hydrogel and 3-D printing optical imaging accessories,a hydrogel detection kit was made,and the on-site quantitative analysis of AChE was realized.The detection range was 0.5-5000 U L-1.The prepared hydrogel kit provides a promising method for on-site detection of AChE,and has potential value in clinic.3.In order to improve the stability of the sensor,a sensor system based on AuNCs@ZIF-8 is constructed.The space confinement effect of ZIF-8 was used to encapsulate AuNCs,which not only effectively improved the luminous intensity,but also enhanced the stability of AuNCs in salt solution and organic solvent environment.Based on this,a fluorescent hydrogel sheet with aggregation induced emission(AIE)effect was designed,and the on-site detection of AChE was realized.Encapsulating AuNCs with zeolite imidazole ester metal-organic framework material(ZIF-8)can not only enhance the fluorescence intensity through AIE effect,but also improve the stability of AuNCs.AuNCs@ZIF-8 hydrogel sheet combines the AIE effect of auncs with the plasticity and high toughness of double-network hydrogel,which significantly improves the sensing characteristics.AChE catalytic product TCh combined with Zn2+competitively,which led to the collapse of ZIF-8 skeleton structure and the leakage of AuNCs,resulting in the weakening of AIE effect and fluorescence intensity of AuNCs.Based on this,a portable biosensor for detecting AChE was constructed by combining fluorescent hydrogel with smart phones and 3-D printing accessories.The detection range was 100-3000 U L-1.In order to test the practicability of the sensor,the developed AuNCs@ZIF-8fluorescent hydrogel biosensor was applied to detect the AChE activity of red blood cells in patients with myasthenia gravis during perioperative period.Ellman standard method was used to detect erythrocyte AChE at the same time,and the results showed that there was a high correlation,which further verified the accuracy of the constructed AChE sensor.The portable biosensor was successfully used to detect the activity of AChE in patients with myasthenia gravis during perioperative period.