Abstract

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 100.13 to 105.13 EID50/0.1 mL, and it had a detection limit of 100.68 EID50/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.

Highlights

  • An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification

  • After Gra was modified with Chi, the folded structure was filled with Chi, and the surface of the Chi-Gra composite became smooth (b)

  • The presence of Chi on Gra was confirmed by energy dispersive spectrometry (EDS) analysis (e)

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Summary

Introduction

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Several methods for detecting NDV, included virus i­solation[3], reverse transcription polymerase chain reaction (RT-PCR)[4], real-time RT-PCR5, immunochromatographic strip (ICS) ­tests[6], and reverse transcription loop-mediated isothermal amplification (RT-LAMP) a­ ssays[7], have been reported. Electrochemical immunosensors are powerful tools that have good specificity, high sensitivity, good precision, and simple instrumentation; give rapid and reliable responses; and are relatively low cost. Chi can immobilize protein molecules and form a film on transducers Due to these properties, nanocomposites consisting of Chi and Gra are an ideal immunosensor material, and our group successfully synthesized a silver nanoparticle-chitosan-graphene composite to construct an electrochemical i­mmunosensor[23]

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