(700c) 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.

.

                        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).