Biosensing Techniques For Cell Apoptosis Assay

Apoptosis is an indispensable process in a variety of biological activities, asimportant as the proliferation and differentiation of cells. The detection of apoptosisof related cell lines is of great importance for the diagnosis and treatment of thesediseases induced by the irregularity of apoptosis. Caspases-3is one of the extremelyimportant proteases in the apoptotic process, and cell apoptosis can be detectedthrough evaluating the activity of caspases-3. In addition, the resonant frequency andresonant resistance responses of a quartz crystal microbalance (QCM) reflect thechange of mass and viscoelasticity on the electrode surface, thus QCM technology canbe applied to the real-time monitoring of cell attachment, proliferation and apoptosis.For the fabrication of cell-based biosensors, the construction of cytocompatiblesurface to improve cell growth is the primary and essential procedure. Based on theabove considerations, we developed some biosensing techniques for cell apoptosisassay. The main points of this thesis are summarized as follows:1.The application of electropolymerized poly-o-phenylenediamine (PPD)insulating thin film to support mammalian cells adhesion and proliferation for thefabrication of living cell-based quartz crystal microbalance (QCM) biosensor wasreported for the first time. PPD film was formed on indium tin oxide (ITO) glasses bycyclic voltammetry, and several different cell lines (MCF-7, MG-63and NIH3T3)were cultured on it. The experimental results showed that cells seeded on PPD filmcould attach and spread more quickly than on ITO and tissue culture polystyrene(TCPS), and that the viability of cells cultured on PPD film was higher than on TCPS,suggesting the excellent cytocompatibility of PPD film. PPD film was used to modifythe gold electrode surface of a quartz crystal to support the growth of MCF-7cells forthe fabrication of a cell-based QCM biosensor. And QCM technique was successfullyapplied to monitor the growth and apoptosis process of MCF-7cells in real time,using camptothecin as a model apoptosis inducer. The QCM results were generally inaccordance with the fluorescence microscopic observation. The PPD film can beremoved completely and easily from gold surface, greatly improving the regenerationof the quartz crystal resonators. 2.A simple, rapid, low-cost and effective method to improve thecytocompatibility of chitosan film was proposed. Based on its excellent compatibility,graphene oxides were self-assembled on chitosan film. The graphene oxide-modifiedchitosan film (GO/CS) was characterized by scanning electron microscopy andcontact angle measurements, and the results indicated that the self assembly of GOpromotes the roughness and hydrophilicity of chitosan. The adhesion, spreading andproliferation of rat fibroblast cell line NIH-3T3were studied using the invertedoptical microscope and MTT assay. It was found that NIH-3T3hardly adhered onchitosan film but spread well on GO/CS film, presenting the normal cell morphologyof spindle. Effects of self-assembly time and grapheme oxide concentration were alsoinvestigated.3.A label-free electrochemical method for caspase-3assay was developed basedon the electrochemical activity of tyrosine. A CY9peptide containing DEVDsequence (the recognition sites of caspase-3) and tyrosine was used. Differential pulsevolametry (DPV) was applied to study the electro-oxidation of pepetideself-assembled on gold electrode. The effects of self-assembly time and pH value ofthe detection solution on DPV signal were also investigated. The results showed thatpH value exerted tremendous influence on tyrosine electro-oxidation. With theincrease of pH, the tyrosine proned to electrochemical oxidation, the peakpotential shifted negatively and the peak current increased. The activity of caspase-3was detected through evaluating the decrease of DPV peak current. This simple andconvenient method creates a new idea for label-free detection of apoptosis.