Multifunctional Electrochemical Biosensing Platform Construction And Its Application In Vitro Diagnosis

In last decades,electrochemical biosensor has become a research hotspot in the field of in vitro diagnosis,for its advantages of simple operation,portability,rapid detection,low cost,easy miniaturization,multi-functional integration and functional diversification.The development of disposable and portable creatinine sensors for in vitro monitoring in multiple scenarios without going to the hospital are worth the expectation of the general public,especially for diagnosis of chronic kidney disease at home.In the construction of microfluidic electrochemical platform based on multienzyme system,not only sample transmission,sample pretreatment and creatinine detection of target analyte are performed,but also POCT can be realized by integrating with a mobile phone.Novel coronary pneumonia(COVID-19)presents similar symptoms with influenza infection,which increases the difficulty of diagnosis and control of this kind of infectious disease.Rapid and convenient detection of severe acute respiratory syndrome coronavirus 2(SARS-COV-2)is of great significance for timely and effective control of this respiratory epidemic disease.Multichannel electrochemical sensing platform can detect multiple samples of SARS-COV-2 or influenza virus at one time.Therefore,it can not only integrate multiple functions together and realize onestep detection of samples on the electrochemical platform,but also detect different target analytes and multiple samples at the same time.The main research results were summarized as follows:(1)Herein,we proposed a novel nano-integrated portable microfluidic electrochemical biochip based on enzyme cascade reaction for single-step point-of-care testing(POCT)of creatinine without process.The microfluidic biochip could automatically eliminate plenty of interferences from practical biological samples and enzymatic products,such as ascorbic acid,uric acid,creatine,sarcosine,acetaminophen,glucose,glycine and urea,using a multi-enzyme-integrated sample-pretreated structure.Gold nanostructure-and carbon nanotube-based screen-printed carbon electrodes(SPCE)were integrated to microfluidic structures to improve detection performance for creatinine.The microfluidic electrochemical biochip showed rapid response(within 10s),exhibiting linear range from 0.01 m M to 1 m M creatinine,with a limit of detection(LOD)of 0.5 μM.Significantly,the whole testing process can be completed within only5 minutes.The design of such a nano-and micro-integrated biochip could integrate sample transportation,processing and detection functions together and benefit the development of various microfluidic devices for POCT diagnosis of various disease biomarkers in plenty of clinical scenes.(2)Herein,a multi-channel electrochemical immunoassay(MEIA)platform was developed based disposable screen-printed carbon electrode(SPCE)array for the onsite detection of SARS-CoV-2 and A(H1N1)virus.The developed MEIA was constructed with eight channels and allowed rapid detection of multiple samples on a single array.On the SPCE surface,monoclonal antibodies against A(H1N1)haemagglutinin(HA)protein and SARS-CoV-2 spike protein were coated to capture the target antigens,which then interact with horseradish peroxidase(HRP)labeled detection antibody to form immuno-sandwich complex.The MEIA approach exhibits good performance with the linear range of 4～64 unit/m L and limit of detection of 1.12unit/m L for A(H1N1),and with the linear range of 0.15～100 ng/m L(10 times wider than that of ELISA)and 0.15 ng/m L for SARS-CoV-2 spike protein.The proposed immunoassay method also presents comparable or even higher specificity in comparison with commercial ELISA kit.For 79 clinical samples including A(H1N1),the MEIA showed 100% positive rate to positive samples,in accordance with ELISA;but it presented 5.4% to negative sample,much lower than 40.5% false positive rate in ELISA,suggesting its higher specificity.As for different concentrations of SARS-CoV-2 pseudo-virus,the response signal of the MEIA method was equivalent to that of the ELISA.The results suggest that the proposed MEIA could meet urgent needs in pointof-care testing of SARS-CoV-2 and A(H1N1).Therefore,the multi-channel electrochemical sensing platform provides a potential means for on-site diagnosis of infection,which would significantly benefit the efficient control and prevent the spread of these infectious diseases in community or resource limited region in future.In conclusion,a new portable microfluidic electrochemical biochip with multifunction and multi-enzyme system was constructed.The biochip could automatically eliminate plenty of interferences from practical biological samples and enzymatic intermediate products,which may carry out sample transmission,sample processing and detection.The microfluidic electrochemical biochip holds promise to become a practical device for medical diagnosis,especially POCT.Meanwhile,in order to meet the needs of the society,a disposable MEIA platform based on SPCE was constructed,which can be used for the on-site detection of A(H1N1)and SARS-CoV-2 and realize the rapid detection of multiple samples on a single array.Multifunctional electrochemical sensing system is a simple,portable,easy to use,cost-effective and high accuracy detection platform,which will be widely used in in vitro diagnosis of various diseases.