Picogram levels of antibodies against the protective antigen (PA) of Bacillus anthracis were detected in an automated electrochemical sandwich-type enzyme-linked immunosorbant assay. The antibodies were captured and detected using an 8 x 3 array of 50-microm-diameter cavities. The reagent and sample volumes were as low as 200 nL in a less than 25-min assay from capture to signal generation. The electrochemical detection of the antibodies was demonstrated at 0.05-10 microg/mL containing only 10-5,000 pg antibodies. The limit of detection is 10 fg for a 200-nL sample. Detection of anti-PA immunoglobulin G performed in spiked normal human serum and fresh whole human blood did not show a significant difference from detection in a buffer. The initial automation of the assay involved the use of a digital syringe pump for the delivery of reagents to the capture surface.

We report the results of our preliminary studies toward the development of assays for the detection of whole organism using a 1cm 2 Au-coated silicon wafer chip for capture. Signal generation involved a VEG4x1™ chip that contains an array of 16-picoliter geometric volume microwells with self-contained electrochemistry. The 50-µm diameter microwells contain a gold disk electrode at the bottom that serves to seal one end of the well, a 50 nm thick gold electrode embedded along its wall and the top gold layer that also serves as the rim of the well.The self-contained microelectrochemical cell-nature of the microcavity needs only very small volume for signal generation. Initial research on the application of our technology for the detection of C. parvum, G. lamblia, E. coli and B. globigii spores are discussed.

Studies toward the development of automated, rapid, sensitive, low-cost, and reliable biomedical and environmental assays using arrays of microcavities with three individually addressable electrodes is reported. Each microcavity in the array contains a bottom electrode called recessed microdisk electrode (or RMD), a middle layer electrode found along the wall called tubular nanoband electrode (or TNB), and a rim/top electrode. The microelectrode TNB was used to detect the signal from a microwell assay for as low as 100 pg of Plasmodium falciparum CSP gene over 5-min enzyme substrate incubation. A selfcontained microelectrochemical assay for Cryptosporidium parvum was generated using the RMD for capture, the TNB as the detecting electrode, and the rim/top as the pseudoreference/auxiliary electrodes. The 4-µm distance of the capture surface to the TNB detecting electrode provided close proximity to the assay components which allowed for a rapid response where the first signal was recorded within 30 s of enzyme substrate incubation. The self-contained nature of the microcavity provided low cost assays with the use of reagents down to 200 nL. The self-contained microelectrochemical microarrays were compatible with automation that was demonstrated in the detection of 25 pg Bacillus anthracis anti-PA IgG in < 25 min total assay time.