Current technologies for frontline monitoring of Bacillus anthracis spores, the causative agent of anthrax, suffer from a tradeoff between analysis time and accuracy.  We are interested in developing highly reliable fluorescent-based sensors for real-time monitoring of B. anthracis spores.  Central to the success of this detection scheme is the availability of short peptides that interact rapidly and selectively with the spore surface of B. anthracis, thereby minimizing the occurrence of false positives / negatives.  We have examined various conjugation strategies to attach the peptides to fluorescent systems.  Furthermore, the quality of the fluorescent probe will dictate sensor shelf life, sensitivity, and reusability.  To this end we look to harness the desirable optical properties of semiconductor quantum dots (QDs).

        As a proof-of-principle experiment and adapting a method from literature¹, a 10-mer peptide (ATYPLPIRGGGC) was conjugated to the surface of a highly fluorescent protein, R-phycoerythrin (R-PE), and used to discriminate between closely related spore types (B. anthracis Sterne and B. cereus).  Shown in Figure 1 are confocal microscopy images and Fluorescence Activated Cell Sorting (FACS) analysis that characterize the binding results.  R-PE is easily photobleached, and we have started investigating the attachment of the peptide to QDs.  In addition to size-dependant absorption / emission maxima, QDs are extremely bright nanoparticles that feature long fluorescent lifetimes and a high resistance to photobleaching.  These properties make QDs an ideal choice for many biological applications.  We are currently working on optimizing the QD conjugation protocol to provide the highest degree of binding to B. anthracis spores.

Figure 1.  The left differential interference contrast (DIC) image shows B. anthracis Sterne spores.  The corresponding middle fluorescence image shows R-PE-Peptide that binds with the surface of the B. anthracis Sterne spores, forming ring-like fluorescence. The FACS data shown on the right illustrates the selectivity of the peptide for B. anthracis Sternecereus against the closely related B cereus. (D. D. Williams, O. Benedek, C. L. Turnbough, Jr. Applied and Environmental Microbiology. 69(10) (2003) 6288-6293.)

1.  W. C. Schumacher, P. K. Dutta, A. J. Phipps, “Highly Selective Quantum Dot Based Fluorescence Sensor for Real-Time Monitoring of Bacillus Anthracis Spores” March 2-3, 2005. The Ohio Nanotechnology Summit Poster Presentation, WPAFB, Ohio.

2.  W. C. Schumacher, P. K. Dutta, A. J. Phipps, “Fluorescence-Based Sensors for Real-Time Monitoring of Bacillus Anthracis Spores” December 9, 2005. IGERT Symposium, The Ohio State University.