(134c) Integration of Surface-Enhanced Raman Scattering and Dielectrophoresis for Rapid Separation and Detection of Bacteria in Real-Time

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 wide-ranging fields including food safety, biomedicine, homeland security, and environmental monitoring. Surface-enhanced Raman scattering (SERS) can afford real-time interrogation of the unique biochemical composition of pathogenic bacteria. Species-level differentiation is enabled by the differences present in the bacterial cell wall. However, SERS measurements 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 was to develop SERS-based approach to enable real-time detection of dilute and/or mixed bacterial samples. To combat these issues, we have developed a two-pronged SERS approach using: (1) long range SERS (LR-SERS) devices to acquire SERS characteristics of the subsurface cellular makeup and (2) SERS devices integrated with dielectrophoresis (DEP) for the concentration and separation of bacterial cells.

We conducted finite-difference time-domain (FDTD) electromagnetic simulations to design SERS-active substrates with extended strong electric fields (i.e., hot spots) to dielectric medium. To achieve this, SERS-active Au nanohole arrays (NHAs) were embedded in refractive index-matched environments. FDTD simulations were also conducted to investigate the plasmonic response of NHAs in symmetric and asymmetric dielectric environments. The optimal structures were determined for both circular and x-shaped NHAs and fabricated via nanofabrication methods. 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.

We incorporated DEP 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. Interdigitated electrodes contain periodic regions with high and low electric field gradients at the edge and center of the electrode, respectively, and can therefore be used to separate bacterial mixtures at either the edge or center of the electrodes. The concentrated and separated bacterial mixtures can be directly detected using SERS.

The SERS devices developed here, used in conjunction with portable Raman spectrometers, could offer frontline diagnostic solutions to simultaneously separate, concentrate, and differentiate dilute bacterial mixtures in real-time.