Improved Performance in Plasmonic Biosensors for Bacterial Detection Through Dielectric Conditioning and Dielectrophoretic Enhancement

Abstract

The need for rapid and specific pathogen detection is of utmost importance. Current culture based methods are time consuming, and more efficient technologies would benefit wideranging fields including food safety, biomedicine, homeland security, and environmental monitoring. Plasmonic biosensing, such as surface-enhanced Raman scattering (SERS) or surface plasmon resonance (SPR), can afford real-time interrogation of the unique biochemical composition of pathogenic bacteria. However, plasmonic measurements, void of complex labels or long integration times, are typically conducted with concentrated samples (108 CFU/mL) of pure cultures that are far outside the clinically relevant range. Real-world samples are often dilute (< 103 CFU/mL), and present in bacterial mixtures and/or complex media. The objective of this work is to develop SERS and SPR-based approaches to enable realtime detection of dilute and/or mixed bacterial samples. To combat these issues, we have developed a two-pronged plasmonic-based approach using: (1) long-range SERS (LR-SERS) devices to extend the effective sensing volume, and (2) SERS and SPR microfluidic devices integrated with dielectrophoresis (DEP) for the concentration and selective detection of bacterial targets. In the former, SERS-active nanohole arrays (NHAs) were embedded in refractive indexmatched environments, and resulted in extension of the effective sensing region. Finitedifference time-domain simulations were conducted to investigate the plasmonic response of NHAs in symmetric and asymmetric dielectric environments. The optimal structures were fabricated, and the SERS signals of surface bound analytes dramatically increased when placed in the refractive index-matched environment. Furthermore, SERS signals were observed at a distance of 10 nm from the nanohole array surface. The increased penetration depth could enable examination of the unique bacterial composition between the peripheral cell wall and cytoplasm. In the latter, DEP was incorporated into the SERS biosensor by dual-function, nanostructured electrodes. “Point-and-plate” and “interdigitated” electrode configurations were studied with respect to SERS detection performance. Generally, bacteria localize on the sensing surface in regions with high or low electric field gradients depending on the unique cellular dielectric properties. The effect of the applied AC frequency on the capture efficiency and SERS signals of bacteria was investigated for the point-and-plate configuration. Application of DEP afforded the successful detection of 105 CFU/mL E. coli solutions, and the applied electric fields did not alter the SERS spectra of Gram-positive and Gram-negative bacteria. Additionally, DEP-active SPR chips containing interdigitated electrodes enable sensitive, rapid, and selective E. coli detection. The DEP-SPR strategy enabled sensitive detection of E. coli, with a limit of detection of 3 x 102 CFU/mL. Integration of secondary antibody amplification led to selective detection of E. coli in the presence of concentrated (108 CFU/mL) non-target bacteria in ~2 h.