Nanowire-based electrochemical detection of anti-Bovine Viral Diarrhoea (BVD) antibodies in bovine serum

Objectives

Rapid identification of BVD seroconverting individuals is essential in preventing costly outbreaks in naive herds. This is especially true in countries where BVD eradication is underway or complete. There is an unfulfilled need for new diagnostic techniques, suitable for on-farm analysis, that deliver early identification of virally exposed individuals, and yield the opportunity for immediate centralised data collection through remote devices. In this work we present an electrochemical onchip fully integrated nanowire based immunosensor device for detection of BVD antibodies (Ab) in both buffer and serum in 20 minutes.

Materials and Methods

Gold nanowires electrodes, on-chip gold counter and platinum pseudo reference electrodes were fabricated on wafer-scale silicon substrates using hybrid e-beam/optical lithography, metal deposition and lift-off techniques. The on-chip nanowire electrodes were first modified with an electrodeposited polymer, using cyclic voltammetry. The carboxylic terminated polymer was activated using a mixture of EDC/NHS which allows for covalent immobilisation of the BVD capture molecules, i.e. BVD virus. The chip was then immersed in ethanolamine for 30 minutes to block the un-reacted active sites on the nanowire. Finally, the modified electrodes were exposed to known concentrations of Ab solution in phosphate buffered saline (PBS) for 20 minutes to confirm the suitability of the nanowires for label-free immunoassay applications. Each step of the assay was characterized electrochemically, illustrating a layer-by-layer build-up on the nanowire surface. The assay was repeated using serum samples again incubated for 20 min at room temperature. BVD virus and BVD monoclonal Abs specific to the envelop glycoprotein (Erns) of the virus were sourced commercially (APHA Scientific, UK).

Each step of the modifying process was electrochemically characterised using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). These experiments were performed in a 10 mM PBS solution containing 1 mM ferrocene monocarboxylic acid (FcCOOH) as a redox probe.

Results

The layer build-up from the functionalisation process, limits electron transfer of FcCOOH to the electrode. i.e. CV intensity decreased while the nyquist semicircles increased. Following immobilisation of BVD virus, the CV and nyquist spectra remained very similar to the ethanolamine deposition. This showed that the virus immobilised onto the majority of the nanowire surface. On exposure to the BVD Ab solution (PBS), Ab-Ag binding exhibited a strong decrease in the CV and a corresponding increase in the measured impedance. After successful demonstration of BVD antibodies detection in buffer, the BVD virus modified immunosensor was applied to detection of BVD antibody in bovine serum of known status (positive or negative). The same successful results were achieved after the modified wire was exposed to 1% positive serum incubated for 20 minutes. To confirm the bio-functionality and efficacy of the virus capture probe and to demonstrate the specificity of the sensor against BVD antibodies the sensor was exposed to BVD negative serum. We did not see any change in the electrochemical response, indicating that the sensor has the ability to discriminate between BVD seropositive and seronegative bovine samples. These sensors can be applied to the detection of the BVD virus using the same procedure, whereby the BVD antibody is immobilised onto the nanowire and the corresponding virus is detected.

Conclusions

This is the first electrochemical-based on-chip nanowire immunosensor device, using an electrodeposited polymer, to covalently immobilize BVD biomolecules. The nanowire-based BVD virus immunosensor allows specific detection of BVD antibodies in serum in 20 minutes. The ultimate aim is to reduce assay times thereby improve the on-farm applicability of this device.