A regenerating ultrasensitive electrochemical impedance immunosensor for the detection of adenovirus
Introduction
The common methods for the detection and identification of viruses, such as cell culture, ELISA and PCR, are time consuming, labor intensive, expensive and are not amenable for on-site use (Caygill et al., 2010, Foudeh et al., 2012, Lazcka et al., 2007). While sensors, particularly immunosensors, offer a route to point of care or on-site detection of pathogens, there remain fundamental technical hurdles to overcome for reliable field use. Sensor durability, regeneration and calibration drift can be overcome in a laboratory environment, but are a challenge for an automated field deployed sensing system. Regeneration of a fresh sensing element is a particular challenge for immunosensors, given the large binding constants involved, and the harsh treatment required to reverse binding, which degrades the antibodies quickly. In order to address some of these issues, we recently utilized an electrochemical reductive desorption technique to repeatedly desorb and reconstruct a gold nanoparticle-loaded self-assembled monolayer (SAM) sensor surface for electrochemical impedance spectroscopy (EIS) detection of oligonucleotides (Mahmoud et al., 2014). EIS biosensors are typically constructed using either SAMs or electrodeposited conducting polymer base-layers to immobilize the bioactive material (antibody, antigen, DNA) on the surface of the electrode (Amano and Cheng, 2005, Caygill et al., 2012, Fang et al., 2010, Hejazi et al., 2010, Jarocka et al., 2014, Jiang and Spencer, 2010, Li et al., 2013, Park et al., 2010, Tang et al., 2004, Cui and Martin, 2003). The long term stability of both approaches has been problematic (Cui and Martin, 2003, Cometto et al., 2012, Cooper and Legget, 1998, Lee et al., 2004, Nishida et al., 1996, Schoenfisch and Pemberton, 1998, Shadnam and Amirfazli, 2005, Yan et al., 2006), as is regeneration of the antibody. While electrochemical reductive desorption of self-assembled alkanethiols from gold electrodes is common (Pensa et al., 2012, Williams and Gorman, 2007, Mandler and Kraus-Ophir, 2011) and proven to be quite effective, studies pertaining to electrochemical removal of large proteins and pathogens covalently immobilized to self-assembled alkanethiols are infrequent. In this report, we show that electrochemical reductive desorption can also be used to develop a reproducible, repeatedly self-assembled, sensitive adenovirus immunosensor based on EIS.
Adenovirus infection in immunocompromised individuals in hospital settings and from contaminated water sources has increased in recent years, causing higher mortality and hospitalization (Chakrabarti et al., 2004, Fowler, 2010, Hansen et al., 2007, Ison, 2006, Jiang, 2006). These ~90 nm diameter, non-enveloped viruses contain linear ds DNA and are classified into 55 serotypes, which are sub-classified into 6 species (Wang et al., 2011). Adenovirus 5 (Ad5) manifests itself primarily as an upper respiratory tract infection, accounting for up to 10% of acute respiratory infections in children (Jiang, 2006), and serious illness in immunocompromised individuals (Ison, 2006). Currently, there are no commercially available sensor devices for the detection of adenovirus, for which the infectious dose is less than 150 plaque forming units (pfu) (http://www.who.int/water_sanitation_health/bathing/recreadischap6.pdf; http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/msds3e-eng.php).
EIS is an analytical technique that is widely used for characterizing the electrical properties (electron-transfer resistance and capacitance) of electrode–electrolyte interfaces (Chang and Park, 2010, Pänke et al., 2008). In this work, we present the fabrication and characterization of a sensitive, reusable label-free impedimetric sensor for the detection of Ad5. There is only one report on EIS detection of Ad5 (Caygill et al., 2012), in which an antibody fragment immobilized on a conducting polymer on a gold electrode gave a detection limit of 103 adenovirus particles/ml. The impedimetric sensor presented here is based on anti-Ad5 covalently bound to gold nanoparticles (Au NPs), self-assembled on a Au electrode loaded with 1,6-hexanedithiol. Recently, construction of EIS biosensors using Au NPs to improve performance has received significant attention (Li et al., 2013, Liu et al., 2011, Bradbury et al., 2008, Zhao et al., 2008, Zhang et al., 2007, Gooding et al., 2014), exploring amplification methods, immunoassays and mechanism. However, reports of EIS using Au NPs for viral particles immunosensing are scarce (Li et al., 2013). The developed sensor is highly sensitive, allowing for the detection of 30 Ad5 particles/ml, and it can be repeatedly regenerated in situ by electrochemical reductive desorption, and thiol self-assembly techniques.
Section snippets
Chemicals and materials
4-Morpholineethanesulfonic acid hydrate (MES), 1,6-hexanedithiol(1,6-HDT), 11-mercaptoundecanoic acid (MUA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), phosphate buffered saline (PBS) tablets, K4[Fe(CN)6], K3[Fe(CN)6], KNO3, gold nanoparticles (Au NPs, dia. 10 nm, 6×1012 particles/ml in 0.1 mM PBS) were purchased from Sigma-Aldrich Canada (Oakville, ON). Anhydrous ethyl alcohol was obtained from Commercial Alcohols Inc. (Brampton, ON). Purified adenovirus
Electrochemical characterization of the immunosensor fabrication steps
The individual steps of electrode modification shown in Scheme 1 were characterized by CV and EIS using as the redox probe. As expected cyclic voltammogram of , with 0.1 M KNO3 as the supporting electrolyte, on a bare gold electrode showed quasi-reversible redox behavior with a peak-to-peak separation potential (ΔEp) of ~100 mV (Fig. 1, curve 1). Bifunctional 1,6-HDT was then assembled, the functionality used to bind to the Au electrode and the Au NPs. After the
Conclusions
The work presented here describes the development of an ultra-sensitive, regenerable impedimetric immunosensor for the detection of adenovirus type 5 based on multi-layered, self-assembled structures that can be repeatedly built on a gold electrode. The architecture of the immunosensor layers provides ease of construction, good impedimetric performance with affinity to Ad5. The immunosensor detects the virus over a broad range of concentrations (10–108 virus particles/ml) with a detection limit
Acknowledgements
The authors would like to acknowledge the support from the National Institute for Nanotechnology and Defence Research and Development Canada – Technology Investment Fund, and the University of Alberta for supporting the NanoFab. We thank Dimitre Karpuzov at the University of Alberta Center for Surface Engineering and Science for collecting XPS spectra.
References (51)
- et al.
Anal. Chim. Acta
(2010) Biosens. Bioelectron.
(2012)Electrchim. Acta
(2012)- et al.
Sens. Actuators A
(2003) Biosens. Bioelectron.
(2010)- et al.
Anal. Biochem.
(2010) Biosens. Bioelectron.
(2014)- et al.
Biosens. Bioelectron.
(2010) - et al.
Biosens. Bioelectron.
(2007) - et al.
Biosens. Bioelectron.
(2011)
Biosens. Bioelectron.
Biosens. Bioelectron.
Biosens. Bioelectron.
Biosens. Bioelectron
Biosens. Bioelectron.
Bioelectrochemistry
Talanta
Analyst
Anal. Bioanal. Chem.
Chem. Phys. Chem.
Anal. Chem.
J. Phys. Chem. C
Lab Chip
Leuk. Lymphoma
Annu. Rev. Anal. Chem.
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