(196c) Merging Electrical and Centripetal Forces With An Enzyme Cascade On a Compact Disc for the Ultimate in Analytical Performance in Molecular Diagnostics

In this contribution we introduce the merging of three platform technologies to achieve optimal lower limits of detection at a low cost for point of care molecular diagnostics. The first platform technology entails inexpensive Carbon-MEMS based Interdigitated Electrode Arrays (IDEAs) that achieve a redox amplification factor of more than 30. Detection limits of attomols are within reach with this technology. The 3D carbon IDEAs are fabricated by first nano-imprinting 3D patterns on a polymer precursor followed by pyrolysis of the polymer in an inert atmosphere. The second platform technology is based on specific DNA detection using an enzymatic cascade of a set of kinetically well-balanced enzymes: DNA polymerase, ATP sulfurylase, flavin adenine dinucleotide (FAD) synthetase and an apo-enzyme (apo-glucose oxidase). PPi generated by isothermal DNA amplification is converted into ATP by the ATP sulfurylase that is used by the FAD synthetase to produce FAD. FAD diffuses to an enzymatic electrode where apo-glucose oxidase has been entrapped in a hydrogel. Apo-glucose oxidase is a glucose oxidase without its prosthetic factor FAD. In the presence of FAD produced by the cascade, the reconstituted glucose oxidase, produces thousand of electron per second in presence of glucose. The third platform is the CD microfluidic platform, that not only automates and multiplexes the enzyme cascade assay but further increases the lower limit of detection because of the flow of the analyte over the redox amplifying electrodes. In the case demonstrated a reciprocating system fluidic system on the CD, using a micro-balloon system, allows for flow measurements at relatively low rpm and with very small amounts of sample.