Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), Or kappa B (binding site for NF kappa B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 mu m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL(-1) dexamethasone, 10 ng mL(-1) forskolin,or 100 ng mL(-1) TNF-alpha (tumor necrosis factor alpha) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring Solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAR, pCRE-SEAP, and pNF kappa B-SEAP patterned oil the same solid Support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 101 cells per well. (c) 2009 Elsevier B.V. All rights reserved.