There exists an unmet need for rapid, sensitive, and field-stable assays for pathogen detection. Bacillus anthracis is the causative agent of anthrax poisoning. This Gram-positive bacterium secretes a tripartite toxin including a cell-binding protective antigen (PA), and the delivered toxins edema factor (EF) and lethal factor (LF). Anthrax poisoning has high mortality and, when delivered in the form of B. anthracis spores, has a very high environmental stability.

These attributes make the detection of anthrax a priority with regards to issues ranging from food safety to bioterrorism. PA is an important target for detection, so it is not surprising that various biosensor platforms for the detection of PA have been reported, with peptide binders coupled to graphene-, carbon nanotube-, and ZnO-based electrodes or surface-enhanced Raman spectrometer (SERS) substrates achieving limits of detection under 20 pg/mL. These systems are, by and large, not appropriate for field use due to their experimental complexity and/or unproven capacity to detect PA out of complex media such as blood serum.

Of the five fatalities in the 2001 bioterrorism attacks against the United States involving anthrax, the causative agent was only identified in one individual before death. This points to the need for high-performance assays for the analysis of complex samples under such demanding physical conditions.

This work focuses on the development of a stable, synthetic peptide-based capture agent against PA that, when coupled with a specially designed electrochemical assay, permits the detection of PA with a limit of detection (LoD) of 170 pg/mL (2.1 pM), with little sensitivity loss in diluted human serum. The powdered capture agent may be safely stored in a sealed environment at up to 65 °C for several days. The gold standard reagents for protein detection are monoclonal antibodies (mAbs). Although they can exhibit impressive affinity and specificity, as biological reagents, they can also be plagued by high cost, batch-to-batch variability and poor stability. Alternative capture agent technologies address some of these problems, although achieving high target selectivity is often challenging.

The technique of iterative in-situ click chemistry was developed for the production of protein catalyzed capture agents (PCC Agents). The technique uses the protein target itself as a highly selective catalytic scaffold for promoting the reaction between two substrates (one presents an azide, the other an acetylene group) to form a covalent triazole linkage. It uses, as one of the substrates, a peptide that was identified via bacterial display screening techniques. That peptide was chemically altered to present an acetylene group, plus a biotin handle linked through a polyethylene glycol oligomer.

A gold-black nanostructured substrate was used as the working electrode to significantly increase the available electrode surface area, and thus amplify the electrochemical signal used for PA detection. A drawback of electrochemical ELISAs is that the layers of immobilization and recognition species that cover the electrode surface can inhibit diffusion- limited electron transfer from the redox-active substrate to the electrode.

A DNA-encoded antibody library technique was used to prepare a dense DNA scaffold for a high-density display of the PCC Agent. An advantage of this scaffold is that electron transfer to the redox-active reporter substrate is facilitated by the DNA duplex monolayer. The detection limit of the electrochemical assay was sharply decreased relative to an otherwise equivalent optical ELISA assay. Electrochemistry also provided a straightforward approach toward detecting PA in optically dense or turgid samples, such as 5% human serum, and exhibited excellent physical stability.

This work was done by Blake Farrow, Sung A Hong, Errika Romero, Bert Lai, Matthew B. Coppock, Kaycie M. Deyle, Amethist S. Finch, Dimitra N. Stratis-Cullum, Heather Agnew, Sung Yang, and James R. Heath of the Army Research Laboratory. ARL-0172