Nanoelectrode Based Biosensors for Pathogen Detection and Identification

Nanoelectrode Based Biosensors for Rapid Pathogen Detection and Identification

Foram R. Madiyar,¹ Saheel Bhana,³ Sherry Basset,² Luxi Swisher,¹ Christopher T. Culbertson,¹ Stefan Rothenburg,³ Xiaohua Huang,² and Jun Li¹*

¹Department of Chemistry, Kansas State University, Manhattan, KS 66506

²Department of Biology, Kansas State University, Manhattan, KS 66506

³Department of Chemistry, The University of Memphis, Memphis, TN 38152

Abstract

Reduction in electrode size down to nanometers dramatically enhances the detection sensitivity and temporal resolution. Well-separated nanoelectrode arrays (NEAs) or ensembles (NEEs) are of particular interest for highly sensitive electroanalysis, the study of fast electrochemical kinetics, and biosensing. Development in this area, however, has been limited by the lack of reliable fabrication methods. Vertically aligned carbon nanofibers (VACNFs) of diameter ~100 nm were grown on Ni and Cr-coated Si substrate using DC-biased plasma enhanced chemical vapor deposition  (PECVD). Embedded  carbon nanofiber nanoelectrode arrays (CNF NEAs) were then fabricated using tetraethylorthosilicate (TEOS) chemical vapor deposition  (CVD) for silicon dioxide (SiO₂) encapsulation followed by mechanical polishing and reactive ion etching (RIE) to expose the CNF tips. Thus obtained embedded NEA (Figure 1A) was integrated into the microfluidic device (Figure 1B) and employed for development of new and rapid methods for pathogen detection to protect general public health and improve the food and water safety standards. Vertically aligned carbon nanofiber nanoelectrode array (VACNF NEA) have been explored as a sample manipulation tool for pathogen detection in couple with fluorescence, surface enhanced Raman scattering (SERS) and impedance signals. 

The key motivation behind using nanoelectrode is that nano-Dielectrophoresis (DEP) occurring at the tip of a carbon nanofiber (CNF)  acts as a potential trap to capture pathogen particles.  To make this possible, a microfluidic device has been fabricated, where nanofibers (~ 100 nm) placed at the bottom of fluidic channel serve as a ‘point array' in a window of 200 μm x 200 μm exposed using photolithography methods. An indium tin oxide (ITO) coated glass slide serves as a macroscale counter electrode. Electric field gradient is highly enhanced at the tips of the CNF when an AC voltage is applied. The first study is focused on the capture of the viral particles (Bacteriophage T4r) by employing the optimum condition with a frequency of 10.0 kHz, a flow velocity of 0.73 mm/sec, and a voltage 10.0 V_pp. Figure 1C shows the distribution of the labeled Bacteriophage T4r over the 200 μm x 200 μm.  A lightning streaks is formed; that is drastically different from the isolated spots of bacteria captured on VACNF tips. The lowest concentration measured has been found to 1×10⁴ pfu/mL with a capture efficiency of 60%.

The motivation of the second study is to incorporate the SERS detection for specific pathogen identification. Gold-coated iron-oxide nanoovals labeled with Raman Tags (QSY 21) and antibodies that specifically bind with E.coli cells are utilized. The optimum capture is obtained when dielctrophoretic force  (F_DEP) is greater than hydrodynamic drag force (F_DRAG) at a frequency of 100.0 kHz, a flow velocity 0.40 mm/sec, and a voltage 10.0 V_pp (Figure 1D). The detection limit reaches ~210 CFU/mL with a portable Raman system with a capture time of 50 sec. 

Lastly, real-time impedance measurement method is employed to detect Vaccinia virus (human virus) in solution at 1.0 kHz at 8.0 V_pp with a detection limit of 630 pfu/mL.

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                        Figure 1: Overview for pathogen detection on nanoelectrode array (NEA)

(A)             A scanning electron microscope image of the embedded vertically aligned carbon nanofibers nanoelectrode array (VACNF NEA). Each of the bright spots are the exposed carbon nanofiber tips. (B) Schematic of the device assembly, the VACNF NEA as the point array electrode and indium tin oxide coated glass slide as the counter electrode along with glass connectors and microbore tubing for inlet and outlet. (C) An optical microscopic image (magnification 4 X) showing the distribution of the labeled bacteriophage T4r as they flow over the exposed 200 μm x 200 μm NEA area. (D) Schematic image of the particles captured on the exposed tips of VACNF by the dielectrophoretic force (F_DEP).