Engineering Of Electrochemical Biosensor And Its Application In Indirect Toxicity Monitoring

The use of chemicals has become increasingly widespread,and traditional animal model toxicity assessment methods for chemicals have failed to meet the dramatic growth in exposure to hazardous chemicals and the need for evaluation of many new compounds.As an alternative method for traditional experimental animals,the in vitro living cell model can highly sensitively perceive changes in the external environment of the cells and directly feedback through changes in various cellular parameters.Therefore,monitoring the changes of cellular physiological parameters such as metabolism and impedance under the chemicals,drugs,environmental toxins and other external stimulus can indirectly analyze cell toxicity of substances.Based on the microfluidic technology and electrochemical biosensor technology,this paper developed a chemical cytotoxicity assessment platform for early warning of chemical toxicity,which can speed up the research process of drug toxicity and mechanism in the development of new drugs.The main work is as follows:In the part of theory and design,the basic principles of cell impedance,cell culture,cell metabolism,and microfluidic systems related to indirect toxicity monitoring platform were highlighted.The structure of the two core sensor chips,the bioreactor and the cell metabolism monitoring chip,in the indirect toxicity monitoring platform for chemicals was designed.Two microchips were connected by the microchannel and the external pipeline under the control of a syringe pump system.The bioreactor was a detachable design that enables cell culture,real-time monitoring of cell impedance and rapid and easy cleaning of the chip during use.The whole bioreactor is made of transparent or translucent materials which help to observe the growth state of the cells during measurement by optical microscopy.The detachable design of bioreactor can realize cell culture,detection of cell impedance,and,providing a new low-cost way for chemical cytotoxicity monitoring.The cell metabolism monitoring chip is a sandwich detachable structure in which the detection electrode is a screen-printed electrode(SPE)modified with glucose oxidase and lactate oxidase for the simultaneous on-line detection of glucose and lactic acid in the extracellular environment.In the section of platform preparation and test,based on the above scheme,the chip designed by CAD mapping software was fabricated using micro-machining processes such as sputtering,photolithography,and computer numerical control(CNC)machining,and the morphology and performance of the chips were investigated.The bioreactor consists of three parts: PDMS cell culture chamber,interdigitated electrode layer and PMMA fixtures as support finger electrode layer and fixed PDMS layer.The electrode strips in the interdigitated electrode layer were complete,the electrode width was 15 ?m and the electrode spacing was 30 ?m.The cell culture chamber is biocompatible with the interdigital electrode layer without dead space.Cell metabolism monitoring chip was made up of PMMA fixtures,PDMS channel layers,and screen-printed electrodes.The prepared SPE yielded a wide linear response of 0~25 mM and 0~20 mM for glucose and lactate,respectively.Before sample measurement,standard solution calibration,air purge,and PBS stabilization can be performed using a microfluidic system,and this progress took 10 min.Theindirect toxicity monitoring platform for chemicals has a sampling interval of 10 s for cell impedance and10 min for cell metabolism monitoring.Cell impedance and cell metabolism during the growth of Hep G2 cells was monitored by using the chemical toxicity indirect monitoring platform.Furthermore,five prototypic reference drugs(D-sorbitol,acetaminophen,cyclophosphamide,amiodarone and rotenone)known to be hepatotoxic were investigated with respect to their toxicological potential on HepG2 cells revealed distinct parameters of cellular impedance and glucose uptake and lactate production.And it identified the possibility of platform for toxicity assessment of chemicals.In addition,a risk assessment strategy was proposed.This as-developed platform makes it possible to better explain and distinguish the mechanism of different chemicals on cells.It is expected to be applied in the field of biomedicine such as cell metabolism physiology,mitochondrial respiratory function determination,and cell culture condition optimization.What's more,it also provides methodological basis for the establishment of relevant chemicals and ecological risk assessment systems.Finally,it has far-reaching significance in the aspects of the safety of people's lives and property and the construction of ecological civilization.