Viruses are one of four classes of biothreat agents, and bacteriophage MS2 has been used as a simulant for biothreat viruses, such as smallpox. A paramagnetic bead-based electrochemical immunoassay has been developed for detecting bacteriophage MS2. The immunoassay sandwich was made by attaching a biotinylated rabbit anti-MS2 IgG to a streptavidin-coated bead, capturing the virus, and then attaching a rabbit anti-MS2 IgG-beta-galactosidase conjugate to another site on the virus. beta-Galactosidase converts p-aminophenyl galactopyranoside (PAPG) to p-aminophenol (PAP). PAPG is electroinactive at the potential at which PAP is oxidized to p-quinone imine (PQI), so the current resulting from the oxidation of PAP to PQI is directly proportional to the concentration of antigen in the sample. The immunoassay was detected with rotating disk electrode (RDE) amperometry and an interdigitated array (IDA) electrode. With an applied potential of +290 mV vs Ag/AgCl and a rotation rate of 3000 rpm, the detection limit was 200 ng/mL MS2 or 3.2 x 10(10) viral particles/mL with RDE amperometry. A trench IDA electrode was incorporated into a poly(dimethyl siloxane) channel, within which beads were collected, incubated with PAPG, and PAP generation was detected. The two working electrodes were held at +290 and -300 mV vs Ag/AgCl, and electrochemical recycling of the PAP/PQI couple by the IDA electrode lowered the limit of detection to 90 ng/mL MS2, or 1.5 x 10(10) MS2 particles/mL.
A microfluidic electroporator operating under a continuous low dc voltage (7 to approximately 15 V) is reported. The proposed electroporation microchip exploits the ionic conductivity of polyelectrolytic gel electrodes to precisely control the electric field that is applied to cells without bubble generation in the microchannel. In this study, pDADMAC (poly diallyldimethylammonium chloride) was used to efficiently apply the electric potential difference to the cells in the microchannels. Impedance analysis showed that the pDADMAC plugs could work as ionic conductors with a conductivity of approximately 16 S m(-1). In accordance with the calculation using CFD-ACE, an input voltage of only 10 V could generate an electric field of 0.9 kV cm(-1) across the microchannel; this meets the requirements for electropermeation. The electropermeation of K562 human chronic leukemia cells was observed in the microchip from 7 V, and the efficiency increased up to 60% upon the application of an input voltage of 15 V with a viability of 80%. An amount of 10(5) cells could be transfected every minute under a constant potential difference. The transfection and expression of DNA plasmids were also successfully demonstrated in the suspension cell line.
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