Y-009. Aptamer-Based Electrochemical Biowarfare Sensor

J. S. Gulledge1, A. Bogomolova2, M. Aldissi2, E. Komarova2, K. Reber2;
1Univ. of South Florida Ctr. for Biological Defense, Tampa, FL, 2Fractal Systems,Inc., Safety Harbor, FL.

The intentional release of Bacillus anthracis (BA) initiated the development of a simple, rapid device highly sensitive for identifying biological agents in response to a terrorist attack or outbreak. Fractal Systems, Inc. has designed an aptamer-based electrochemical sensor for this purpose. Oligonucleotide aptamers ensure higher environmental stability than antibody based sensors. The disposable array has 8 electrodes to allow simultaneous modification with aptamers of different specificities. Thiolated aptamers are immobilized on gold electrodes by self-assembly. Prior to this, the electrodes are electrochemically cleaned and blocked with weakly-bound cytosine molecules, resulting in optimal presentation of aptamer probes. DNA and RNA aptamers specific to Ricin and DNA aptamers for Shiga toxin, BA and Bacillus thuringiensis (BT) spores were tested. The array/chamber is fitted with a reference electrode; and the 8 working electrodes are connected sequentially to the CH Instruments Potentiostat. Total measurement time is under 15 minutes. The following agents (or simulants) were tested: BA Sterne and BT spores, Ricin, Ricin A Chain (RTA), and Shiga toxin. Binding of the agent changes the AC Impedance spectra, recorded in the presence of redox agent before and after 1-hour exposure to the analyte. The binding efficiency was calculated in relative units, (percentage of impedance increase relative to initial probe impedance). Shiga toxin showed binding to anti-shiga aptamer probe at 1μg/ml, so did RTA, which was binding anti-ricin RNA aptamer-modified electrode at 1μg/ml or higher. Ricin did not reveal any binding. Significant cross-reactivity was present between anti-BA aptamer-modified electrode reacting with BT spores in the range 103-106/ml; while BA spores had stronger preference to anti-BA aptamer probe. Nonspecific binding to irrelevant aptamer probes on the same array was noticeable, although clearly lower then specific binding. The method is suitable for rapid qualitative field detection. Further work is required to improve sensitivity of detection and obtain quantitative response.