John R. Waldeisen

Disassembly of a Core–Satellite Nanoassembled Substrate for Colorimetric Biomolecular Detection

The disassembly of a core-satellite nanostructured substrate is presented as a colorimetric biosensor observable under dark-field illumination. The fabrication method described herein utilizes thiol-mediated adsorption and streptavidin-biotin binding to self-assemble core-satellite nanostructures with a sacrificial linking peptide. Biosensing functionality is demonstrated with the protease trypsin, and the optical properties of the nanoassemblies are characterized. A figure of merit is presented to determine the optimal core and satellite size for visual detection. Nanoassemblies with 50 nm cores and 30 or 50 nm satellites are superior as these structures achieve an orange to green color shift greater than 70 nm that is easily discernible by the naked eye. This colorimetric substrate may prove to be a favorable alternative to liquid-based colloidal sensors and a useful visual readout mechanism for point-of-care microfluidic diagnostic assays.

Strategies for nanoplasmonic core-satellite biomolecular sensors: Theory-based Design

We present a systematic theoretical study of core-satellite gold nanoparticle assemblies using the Generalized Multiparticle Mie formalism. We consider the importance of satellite number, satellite radius, the core radius, and the satellite distance, and we present approaches to optimize spectral shift due to satellite attachment or release. This provides clear strategies for improving the sensitivity and signal-to-noise ratio for molecular detection, enabling simple colorimetric assays. We quantify the performance of these strategies by introducing a figure of merit. In addition, we provide an improved understanding of the nanoplasmonic interactions that govern the optical response of core-satellite nanoassemblies.

A Real-Time PCR Antibiogram for Drug-Resistant Sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔCt<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 Gram-negative and 2 Gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.