(Invited) Dielectrophoretic Capture and Detection of Microbial Pathogens Using Nanoelectrode Arrays

Abstract

Rapid and highly sensitive detection of pathogenic microbes is critical for food safety and environmental protection. To achieve this, it requires combining novel sample preparation techniques and extremely sensitive detection methods. In this presentation, I summarize a series of studies in my lab on developing a nanoscale dielectrophoresis (DEP) device for effective capturing and concentrating pathogenic particles such as viral and bacterial particles in a fluidic chip. This technique was then integrated with various detection methods such as fluorescence imaging, surface enhanced Raman spectroscopy (SERS) and real-time impedance measurements for quantitative assessment of the pathogen concentration. Due to the small size (~1 micron for bacterial cells and ~100 nm for viral particles), it is challenging to capture and concentrating these pathogenic particles from dilute solutions. Here we demonstrate that the highly focused electric field at the tip of an embedded nanoelectrode array can be utilized to generate a large DEP force to effectively manipulate pathogenic particles. The nanoelectrode array was made of vertically aligned carbon nanofibers (VACNFs), about 5 micron in length and 100 nm in diameter, and embedded in a SiO2 film. It was placed at the bottom of a microfluidic channel against a macroscopic indium tin oxide (ITO) top electrode, forming a DEP device in “points-and-lid” configuration. We have shown that E. coli DHα5 cells flowing through the channel can be effectively captured and concentrated at the desired microscale areas inside the channel using a small AC voltage (~6-10 Vpp). This device is particularly effective in capturing viral particles (such as bacteriophage T4r) due to the comparable size between the viral particles and the exposed VACNF tip. To achieve specific detection, the DEP chip has been integrated with a Raman probe for SERS measurements of nanotag-labelled E. coli DHα5 cells. The SERS nanotags are based on iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of ~50 nm in size, which are coated with QSY21 Raman reporters and attached to E. coli cells through specific antibodies. The combination of the greatly enhanced Raman signal by the SERS nanotags and the effective DEP concentration significantly improves the detection limit and speed. The SERS signal has been fully validated with fluorescence microscopy measurements under all DEP conditions. A concentration detection limit as low as 210 cfu/mL using a portable Raman system has been obtained with a DEP capture time of only ~50 s. The detection has been validated in purified solutions as well as complex samples including chicken broth, soil solution and apple juice. Real-time impedance measurements has also been demonstrated as a feedback during DEP capture of vaccinia viral particles. By controlling the proper AC voltage, the nucleic acid contents in the viral particles can be released by opening the envelope with electroporation under the high electric field at the VACNF tips. These results demonstrate the potential to develop a compact portable system for rapid and highly sensitive detection of specific pathogens. Furthermore, this technique is based on physical method and the pathogenic particles are fully released after the AC voltage is turned off, making it a robust device that can be repeatedly used.