Chronoamperometric characterization of secreted alkaline phosphatase from single-cell entrapped in a poly(dimethylsiloxisane) microwell

Single-cell analysis has become a powerful method to construct a new type of whole cell sensor integrating with individual cellular responses all together However sensitivity of electrochemical detection system in the present stage is insufficient to discuss in detail about the results obtained from a large number of data set because the individual single-cell responses are largely fluctuated To improve sensitivity chronoamperometry (CA) and electric charge analysis were performed for a single-cell entrapped within a poly(dimethylsiloxisane) (PDMS) microwell HeLa cells transfected with vectors encoding SEAP (secreted alkaline phosphatase) have been seeded in the cylindrical PDMS microwell arrays with and without groove A microelectrode tip located the top of the PDMS microwell to isolate the volume of the measuring solution containing p-aminophosphate (PAPP) as an enzymatic substrate After a certain time period (t(accu)) to promote the SEAP reaction the tip potential was stepped from 00 to 0 3 V and the CA response for oxidation of p-aminophenol (PAP) was converted to the electric charge by time integration of the current The electrochemical response from HeLa cells transfected with SEAP (HeLa-SEAP) was significantly larger than that from wild-type HeLa for both the PDMS microwell with and without groove For the PDMS microwell without groove accumulation of PAP was evidently observed with increasing t(accu) For the PDMS microwell with a groove of 5 mu m width and 5 mu m depth the PAP oxidation current was large but the accumulation of PAP was not evident with increasing t(accu) due to the promoted mass transfer of PAP and electric connection according to the groove (C) 2010 Elsevier Ltd All rights reserved