Electrochemical chip integrating scalable ring-ring electrode array to detect secreted alkaline phosphatase.

An electrochemical platform for parallel monitoring of secreted alkaline phosphatase (SEAP) has been microfabricated on a device with a mammalian-cell array chip. A 4 × 4 ring-ring electrode array was designed at the rim of the round cellular pattern with a diameter of 270 µm. Electrochemical characterization was carried out, and it was found that the collection efficiency was about 50% in dual mode when the inner-ring and the outer-ring electrodes were selected as the collector and generator electrodes, respectively. The current amplification ratio for the dual mode normal to single mode was 2.84. SEAP expressing from the cells was parallelly monitored by using a multiplexer switching system at the 16 round cellular spots. The reduction current for HeLa cells transfected with plasmid encoding SEAP observed at the collector outer ring electrode was found to be significantly higher than that for wild-type HeLa. Finally, the top of the microwell with the round cellular pattern was covered with a poly(dimethylsiloxane) block for 5 min to accumulate the secreted enzyme and the product of the enzyme reaction so that further signal enhancement could be observed.

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 kappaB (binding site for NFkappaB (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4x4 array of circles of diameter 300 microm 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-SEAP, pCRE-SEAP, and pNFkappaB-SEAP patterned on 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 5x10(4) cells per well.

Electrochemical gene-function analysis for single cells with addressable microelectrode/microwell arrays.

To each his own: An addressable electrochemical device consisting of orthogonally arranged rows and columns of electrodes has been constructed to monitor protein expression in genetically engineered cells at the single-cell level. The response based on redox cycling reflected the different expression levels of the enzyme from individual HeLa cells transfected with a plasmid vector including secreted alkaline phosphatase.