Dielectrophoresis-Assisted Pathogen Detection on Vertically Aligned Carbon Nanofibers Arrays in a Microfluidic Device

In this chapter, we focus on utilizing nanoelectrode arrays fabricated with vertically carbon nanofibers (VACNFs) for pathogen detection based on a “point-and-lid” dielectrophoretic device in a microfluidic channel. This technique is utilized to concentrate particles from the bulk flow and detect pathogens based on fluorescence, surface-enhanced Raman spectroscopy (SERS) and impedance measurements. The advantage of VACNFs is their ultrasmall diameter (~100 nm) and the high aspect ratio (50:1). When coupled with a macroscopic indium tin oxide (ITO) electrode, it produces a large electric field gradient (∇E2 = ~1019 − 1020 V2 m−3) which is harnessed for pathogen detection based on dielectrophoresis. Several noninfectious pathogens including bacteria Escherichia coli DHα5, inactivated vaccinia virus (species: Copenhagen strain, VC-2), and Bacteriophage T4r were utilized as model species to study the size effect and kinetics of dielectrophoretic capture in this study. The comparable size of the nanoelectrode produced strong interaction with virus particles, generating striking lightning capture patterns and high detection sensitivity. The dielectrophoretic capture at the nanoelectrode arrays is successfully integrated with a portable Raman probe as a microfluidic chip for ultrasensitive detection of bacteria E. coli DHα5 using SERS-tagged gold nanoparticles co-functionalized with specific antibodies

Nanoelectrode based devices for rapid pathogen detection and identification

Doctor of PhilosophyDepartment of ChemistryJun LiDeveloping new and rapid methods for pathogen detection with enhanced sensitivity and temporal resolution is critical for protecting general public health and implementing the food and water safety standards. In this research vertically aligned carbon nanofiber nanoelectrode arrays (VACNF NEAs) have been explored as a sample manipulation tool and coupled with fluorescence, surface enhanced Raman scattering (SERS) and impedance techniques for pathogen detection and identification. The key objective for employing a nanoelectrode array is that the nano-Dielectrophoresis (nano-DEP) at the tip of a carbon nanofiber (CNF) acts as a potential trap to capture pathogens. A microfluidic device was fabricated where nanofibers (~ 100 nm in diameter) were placed at the bottom of a fluidic channel to serve as a ‘point array’ while an indium tin oxide coated glass slide acted as a macroscale counter electrode. The electric field gradient was highly enhanced at the tips of the CNFs when an AC voltage was applied. The first study focused on the capture of the viral particles (Bacteriophage T4r) by employing a frequency of 10.0 kHz, a flow velocity of 0.73 mm/sec, and a voltage of 10.0 Vpp. A Lithenburg type of phenomenon was observed, that were drastically different from the isolated spots of bacteria captured on VACNF tips in previous study. At the lowest employed virus concentration (1 × 10[superscript]4 pfu/mL), a capture efficiency of 60% was observed with a fluorescence microscope. The motivation of the second study was to incorporate the SERS detection for specific pathogen identification. Gold-coated iron-oxide nanoovals labeled with Raman Tags (QSY 21), and antibodies that specifically bound with E.coli cells were utilized. The optimum capture was observed at a frequency of 100.0 kHz, a flow velocity of 0.40 mm/sec, and a voltage of 10.0 Vpp. The detection limit was ~210 CFU/mL for a portable Raman system with a capture time of 50 seconds. In the final study, a real-time impedance method was employed to detect Vaccinia virus (human virus) in the nano-DEP device at 1.0 kHz and 8.0 Vpp giving a detection limit of 2.51 × 10[superscript]3 pfu/mL

Integration of a nanostructured dielectrophoretic device and a surface-enhanced Raman probe for highly sensitive rapid bacteria detection

This work reports a synergistic approach to the concentration, detection and kinetic monitoring of pathogens through the integration of nanostructured dielectrophoresis (DEP) with nanotag-labelled Surface Enhanced Raman Spectroscopy (SERS). A nanoelectrode array made of embedded Vertically Aligned Carbon Nanofibers (VACNFs) at the bottom of a microfluidic chip was used to effectively capture and concentrate nanotag-labelled E. coli DHα5 cells into a 200 μm × 200 μm area on which a Raman laser probe was focused. The SERS nanotags were based on iron oxide-gold (IO-Au) core-shell nanoovals (NOVs) of ∼50 nm size, which were coated with a QSY21 Raman reporter and attached to E. coli through specific immunochemistry. The combination of the greatly enhanced Raman signal by the SERS nanotags and the effective DEP concentration significantly improved the detection limit and speed. The SERS signal was measured with both a confocal Raman microscope and a portable Raman probe during DEP capture, and was fully validated with fluorescence microscopy measurements under all DEP conditions. The SERS measurements were sensitive enough to detect a single bacterium. A concentration detection limit as low as 210 cfu ml-1 using a portable Raman system was obtained with a DEP capture time of only ∼50 s. These results demonstrate the potential to develop a compact portable system for rapid and highly sensitive detection of specific pathogens. This system is reusable, requires minimum sample preparation, and is amenable to field applications. This journal i