Abstract
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.
Keywords
- dielectrophoresis
- pathogen detection
- vertically aligned carbon nanofibers (VACNFs)
- nanoelectrode array (NEA)
- indium tin oxide (ITO)
- microfluidic device
- electroporation
- plaque-forming units (pfu)
- bacteria E. coli Dhα5
- Bacteriophage T4r
- vaccinia virus
- iron-oxide gold nanoovals (IO-Au NOVs)
- surface-enhanced Raman spectroscopy (SERS)
- impedance
- fluorescence
1. Introduction
The need for rapid and reliable pathogen monitoring and detection is imperative in the food industry, biodefense, drug discovery, animal healthcare, clinical diagnosis, water, and environmental quality control. Among these, the food industry is the area where most attention has been focused on due to public health implications. In 2015, the World Health Organization (WHO) estimated that 77 million people every year fall victim to contaminated food and about 9000 deaths annually. The WHO has identified 31 agents of foodborne diseases including bacteria, virus, parasites, toxins, and chemicals, among which 95% are caused by
The conventional pathogen identification methods are standard microbiological techniques and involve necessary steps such as preenrichment, selective enrichment, biochemical screening, and serological confirmation [2]. The traditional methods take up to 72 h to obtain confirmed results which are based on the morphological evaluation, culture growth in various media under various conditions, and enumerating colonies of the bacteria [3, 4]. However, the development of polymerase chain reaction (PCR)-based molecular analysis techniques [5, 6, 7], the conventional biochemical methods such as enzyme-linked immunosorbent assays (ELISAs), and blot assays have led scientists to target genes, proteins, and carbohydrate moieties instead of the whole microorganisms [8] to obtain molecular fingerprints of the pathogens. These techniques despite being highly sensitive and selective require experienced personnel, expensive equipment, reagents, and long readout time, thus making the process costly and difficult for on-site applications and causing a delay in the pathogen detection, preventing immediate medical action toward infected patients. There is a keen interest in developing new rapid point-of-care biosensing systems for early detection of pathogens with high sensitivity and specificity.
Recent developments in micro- and nanotechnology offer many technological advances in fabricating devices that incorporate nanoscale features to enhance sensitivity, reduce detection time, and enable multiplexing capability [9, 10, 11, 12]. Most important, the properties of nanomaterials can be tailored by changing the size, shape, and composition, modifying the nanomaterial surface with appropriate functionalization, and conjugation with affinity ligands, antibodies, epitopes, and aptamers [13, 14]. Representative nanomaterials utilized for pathogen detection include metal nanoparticles [15, 16, 17], nanotubes and nanofibers [18], quantum dots [19], and magnetic nanoparticles [20]. These nanomaterials are used in conjugation with signal transduction techniques [21] such as fluorescence [22], bioluminescence [23], flow cytometry [24], colorimetry [25], electrochemistry [26, 27, 28, 29], piezoelectrics [30], surface plasmon resonance (SPR) [31], quartz crystal microbalance [32], chemiluminescence [33], optical waveguides [34], and surface-enhanced Raman spectroscopy (SERS) [17, 25, 35, 36, 37, 38, 39].
In this chapter, we summarize an innovative pathogen capture and detection system based on dielectrophoresis (DEP). The device is a unique assembly of nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs) and a transparent macroscopic indium tin oxide (ITO) glass electrode in a “point-and-lid” geometry in which pathogens are introduced using microfluidic channels. The study of capture kinetics was accomplished using fluorescence, SERS, and impedance measurement techniques. The test pathogens utilized in this study were bacteria such as
2. Principles, design, and fabrication
The phenomenon of dielectrophoresis (DEP) is renowned as a particle manipulation technique based on the uneven electrical force on the opposite sides of polarized particles in an electric field with a high gradient produced by the electrodes. The larger the electric field gradient, the stronger the DEP force acts on the particle. This phenomenon was first described by Pohl in 1951 [42] and has been widely used in biological science to separate live and dead bacteria [43, 44], viruses [45, 46, 47], cells [48, 49, 50, 51, 52], yeast cells [53, 54], and DNA [55, 56, 57]. When we consider radius of the particle
where:
The use of physical fields for the separation of cells takes advantage of the heterogeneity of physical parameters for Eq. (2), such as
In the microfluidic device, a particle experiences two forces orthogonal to each other, i.e., DEP force (
where
3. DEP device fabrication and setup for pathogenic particles
Figure 1a is the image of the device produced in the lab at Kansas State University. The detailed procedure of device fabrication is given in reference [58]. Figure 1a shows that the size of the devices is comparable to a US penny and illustrates the “points-and-lid” design. Figure 1b shows that the NEA comprises randomly distributed VACNFs (diameter ~100–120 nm, the density of ~2 × 107 exposed CNFs/cm2) embedded in silicon dioxide (SiO2) matrix (tip exposed) with an average spacing of ~1–2 μ. The active area exposed on NEA is 200 × 200 μm2, and the rest is covered with a 2-μm-thick photoresist film to shield the effect of the rest of exposed tips. The ITO glass slide containing a photolithographically fabricated 500-μm-wide microfluidic channel in an 18-μm-thick photoresist film is permanently vacuum bonded.
In the experimental setup, DEP device was placed under an upright fluorescence optical microscope (Axioskop II, Carl Zeiss) using 50 X objective lens. The microorganisms such as
4. Detection of viruses: Bacteriophages and vaccinia virus using fluorescence and impedance method
4.1. DEP capture and kinetics of Bacteriophage T4r using fluorescence method
Figure 2 depicts the increase in an integrated fluorescence intensity to a saturation level in less than 10.0 s as a 10 Vpp AC bias when applied to the DEP device while flowing 5 × 109 pfu/mL
The DEP kinetics dramatically changed with concentration (Figure 2e) when two diluted concentrations, i.e., 5.5 × 108 and 2.5 × 107 pfu/ml, were used. The viruses could be individually counted (40 out of 67 particles) at an extremely low concentration of
4.2. DEP capture and electroporation of vaccinia virus coupled with real-time impedance detection
Electrochemical sensors based on impedimetric measurements have emerged as an attractive low-cost portable technique for the rapid detection of pathogenic microbes and other microorganisms. In this capture study, vaccinia virus was a probe to study the impedance kinetics and electroporation of the viruses due to high electrical field gradient generated at VACNFs tips.
The details of the growth and enumeration by conventional techniques are given in a previous report [60]. Briefly, in-house stocks of vaccinia virus (
The details of the fluorescence experiment setup and videos are described in Section 3.0. The frequency (
The integrated fluorescence intensity was measured at the end of capture period (54.0 s) and compared to the percentage change of the final impedance signal (ZF) relative to the initial impedance signal (Zo), i.e., %(ZF − Zo)/Zo. The optimum flow velocity for vaccinia virus was 0.40 mm/s at the frequency of 50.0 Hz and the voltage of 8.0 Vpp as shown in Figure 3a. The optical image is shown in Figure 3c which indicates the
Finally, to investigate the electroporation of lipophilic membrane due to the high electric field on tips of VACNF NEAs, PI dye was added to the mannitol solution containing 3.0 × 106 particles/mL of DiO dye-labeled vaccinia virus and observed in Neubauer chamber. The absence of the red fluorescence indicated there was no structural damage of virus due to UV inactivation [62]. For electroporation experiment in a microfluidic device, the frequency of 50.0 Hz was used. The voltage of 8.0 Vpp was turned on for 65 s for maximal DEP capture with the flow velocity set at 0.05 mm/s (for maximum capture and interaction of dye and DNA). Figure 3f shows the schematic figure of electroporation of lipophilic membrane of vaccinia virus in the presence of high electric field at the VACNF tips. It is observed that the electroporation made the membrane more permeable and the DNA is likely extracted out of the membrane to interact with PI dye in the mannitol solution which increases the PI dye fluorescence intensity. There is evidence that, after the AC voltage is turned off, some PI-intercalated ds-DNAs are physically adsorbed on the VACNF tip or the NEA chip surface [61, 63].
5. Detection of bacteria: DEP capture and identification of E. coli strain DHα5 by surface-enhanced Raman spectroscopy
DEP capture of bacterial cells was demonstrated with nontoxic
To demonstrate the potential of this method, both confocal (DXR, Thermo Fisher Scientific) and portable systems (ProRaman L, Enwave Optronics. Inc) were used. The similar studies were carried out with the two spectrophotometers at varied flow velocity and frequency. Figure 4d shows the full Raman spectrum of QSY21 at different AC frequencies during the capture of bacteria. The highest peak in the full spectra, 1496 cm−1, was used in the further calculation, and the higher capture was seen at the AC frequency of 100.0 kHz. The results between these two Raman systems were very consistent from their fluorescence and Raman intensity plots, with the maximum flow velocity at 0.4 mm/s (0.55 μl/s) (Figure 4e).
To analyze the capture in complex samples, one of the representative data is shown in Figure 4f, i.e., the capture of
Figure 4g summarizes the SERS intensity of the captured NOV-labeled
where RI was the Raman intensity increase after 50 s of DEP capture. For bacteria concentrations below the critical value,
where
6. Discussion and conclusion
The physical phenomenon of DEP was observed on the tips of VACNF NEAs in microfluidic channel design due to high electric field gradient generated by the “point-and-lid” geometry acted as an effective and reversible electronic manipulation technique to rapidly (less than 60 s) concentrate bacteria and viruses into a micro-area from the solution flow. The nanoscale size of the VACNF tips has two critical features: the extremely high electrical field strength at the tip (E = ∼107 V m−1) and the large electric field gradient at the tips of nanoelectrode (giving ∇E2 = 1019 − 1020 V2 m−3) against ITO electrode. The polarizable pathogenic particles in the microfluidic device encounter hydrodynamic drag force along the flow direction and orthogonal (vertical) DEP forces due to the high electric field gradient. Once the pathogens are close to the VACNF tip, the lateral DEP force becomes larger than the hydrodynamic drag force, and the pathogens are captured at the nanoelectrode tip.
According to Eq. (1), the force of DEP highly depends on the volume of the particles (
The second drastic contrast in the capture of viruses is the formation of
The device successfully captured single virus particles observed at isolated spots in the 200 × 200 μm2 active NEA surface at an extremely dilute concentration (8.9 × 104 pfu/ml) in which facilitated studying the impedance kinetics of real-time DEP capture of vaccinia viral particles, yielding a detection limit of 300 particles/ml. VACNF tips have been found to cause electroporation of the lipophilic membrane of the vaccinia virus due to the large electric field produced on the tips. This electroporation phenomenon has allowed extracting the internal nucleic acid contents to the solution.
Finally, highly sensitive detection of
All these studies revealed the exciting interplay between the highly focused electric fields at the nanoelectrode with bioparticles of comparable sizes. The device was successfully integrated with fluorescence, surface-enhanced Raman spectroscopy and electrochemical impedance sensing. All these results are very encouraging and can be further improved by optimizing the DEP design. The combined functions of DEP in concentration, detection, and electroporation make such nano-DEP devices useful to extract intracellular materials, such as DNA or proteins without a lytic agent. It can act as an on-chip portable sample preparation module for potentially capturing pathogenic particles at concentrations approaching 1–10 particles/mL and for future downstream processing and testing of microbial samples.
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