(250o) Rapid and Sensitive On-site Serodiagnosis of Pseudorabies by AC Electrokinetics-enhanced Capacitive Sensing

Pseudorabies, also called Aujeszky’s disease, is caused by infection of animals with pseudorabies virus (PRV). Since early 1980, pseudorabies spread worldwide and caused significant economic loss to swine industry. In the U.S., the annual cost for eradication of pseudorabies is estimated to be $30 million including $17 million for vaccination efforts. Serological surveys in the U.S. for pseudorabies in feral hog populations revealed up to 61% of tested animals (average 21.5% with 95% confidence interval of 13.4-29.8%) were positive for PRV infection, showing widespread of the disease in the animal populations [1]. Therefore, detection and control of PRV infections both in domestic and wild pigs (feral hogs) are critically important to prevent outbreak of the disease.

Diagnosis of pseudorabies is currently conducted mainly by serology, such as enzyme-linked immunosorbent assay (ELISA), serum neutralization test (SNT) and latex agglutination test (LAT) [2]. Although the serological tests are reliable, they do not differentiate naturally infected animals from vaccinated animals. Also, they cannot detect infection until adaptive immunity is developed enough for the animals to produce antibodies against the virus, which usually takes seven days. Fluorescent antibody tissue section test (FATS) can be used to confirm PRV infection by detecting virus in a matter of hours but requires tissue samples from suspected animals. Virus isolation (VI) also detects virus particles but it requires mammalian cells to propagate the virus. These tests are conducted in diagnostic laboratories and therefore suffer from long turn-around time and high cost. Therefore, there is an urgent need for a rapid, inexpensive, field-deployable diagnostic test for this disease.

Mechanism

This work is based on alternating current electrokinetics (ACEK) capacitive sensing method. The major innovation with this sensing technology is its ability to simultaneously generate microfluidic ACEK effects with the sensing electrodes and to interrogate directly the sensor’s interfacial capacitance. It is based on measuring the interfacial capacitance (C_int) on functionalized microelectrodes with an AC signal capable of inducing ACEK effects. With binding process continuing, the thickness of interfacial layer, d_int, keeps increasing, which leads to a decrease of C_int. By monitoring the change rate of C_int, specific binding of analyte with probe molecules can be detected. The change rate in interfacial capacitance is tracked and used as an indicator of probe-analyte binding.

In order to accelerate binding reaction occurred on the electrodes surface, the AC signal will also induce movement of proteins towards the electrode surface and accelerate the binding to immobilized probe molecules. Based on our prior work [3], electrodes with larger characteristic length have remarkably higher response at the same electric field strength because the electrode geometry is more amenable to ACET convection. The findings have been applied here to achieve faster and more sensitive detection. Both positive dielectrophoresis (pDEP) and AC electrothermal (ACET) effect have been induced to transport target proteins towards the sensor. Positive DEP will caused a particle to move towards high electric field region due to interactions between a particle’s dipole moment and a non-uniform field, while ACET effect will generate vortices-like microflows around an electrode and transport bioparticles by convection. Since DEP force is short-ranged, concomitant generation of ACET flows will significant improve the effective range of macromolecular enrichment.

Results

In this work, a capacitive immunosensor based on ACEK has been developed for detection of pseudorabies virus antibody in serum samples. Interdigitated electrodes with critical dimension of 100 µm have been used as the sensor. Comparing with previous work using 2 µm electrodes [4], the optimal frequency has shifted from 100 kHz to 75 kHz. This is because ACET effect becomes more important relative to DEP with larger electrodes, and capacitive response is larger at a lower frequency with ACET effect, generally speaking. The sensor is very sensitive with a limit of detection of 4.5 fg/mL when testing analytical samples. When testing clinical samples, using 35 serum samples from feral hogs, a diagnostic sensitivity of 91.1-% and a selectivity of 90.9-% are achieved, as confirmed by ELISA results. This sensing method has a response time of 30 seconds and requires minimum pretreatment without washing process, which make it a promising method for on-site disease detection and diagnosis.

References

[1] L. E. Pomeranz, A. E. Reynolds, and C. J. Hengartner, “Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine,” Microbiol. Mol. Biol. Rev., vol. 69, no. 3, pp. 462–500, Sep. 2005.

[2] T. Müller et al., “Pseudorabies virus in wild swine: a global perspective,” Arch. Virol., vol. 156, no. 10, pp. 1691–1705, Oct. 2011.

[3] H. Cui et al., “Rapid and sensitive detection of small biomolecule by capacitive sensing and low field AC electrothermal effect,” Sens. Actuators B Chem., vol. 226, pp. 245–253, Apr. 2016.

[4] S. Li, H. Cui, J. Wu, K. Yang, A. Wadhwa, S. Eda, and H. Jiang, “AC Electrokinetics-Enhanced Capacitive Immunosensor for Point-of-Care Serodiagnosis of Infectious Diseases,” Biosens. Bioelectron., Vol. 51, pp. 437-443, 2014.