The detection of viruses in complex biological samples is important in the management of infectious disease caused by human viral pathogens. Amplification-free quantitative detection of viral nucleic acid enables simplified diagnostic devices with characteristics appropriate for point-of-care and resource-limited settings. A new nanoparticle assay was developed for detection of a Herpes simplex Virus (HSV) DNA sequence within complex, minimally processed clinical samples. In a sequence-specific manner, viral nucleic acids link magnetic nanoparticles to fluorescent nanoparticle reporter labels. Following washing steps, the number of fluorescent nanoparticles was quantified, leading to a correlated read-out of target molecule concentration.
The assay was first established using commercially available conjugation methods to develop reagents specific for HSV. This format provided a limit of detection in the low picomolar range, with non-specific interactions defining a key aspect of the assay performance. Undesired nonspecific interactions lead to an investigation of particle coating chemistries to reduce non-specific binding. These techniques were based on the living radical polymerisation (LRP) of a copolymer of azide-modified and hydroxyl-terminated oligoethylene glycol methacrylate (OEGMA) polymer brushes from the surface of magnetic nanoparticles. Use of polyethylene oxide based polymers lead to improvements in assay sensitivity by reducing background noise. The azide substituents within the copolymer were conjugated to alkyne-substituted HSV specific capture probes via an orthogonal and efficient copper-catalyzed azide alkyne cycloaddition (CuAAC). LRP and CuAAC were used to synthesise nanoparticle reagents that enabled amplification-free detection of viral DNA in minimally processed clinical samples.
Two microfluidic devices were developed for fluorescent nanoparticle readout, including a static chamber and a continuous-flow magnetophoresis device. The magnetophoresis experimental system provided a method for continuous-flow magnetic particle extraction using an opposed magnet configuration and a passive gravity-actuated system for sample and buffer delivery. Reporter and magnetic nanoparticle complexes were read out using confocal fluorescence microscopy. In a comparison study, the static-chamber device gave improved performance, as determined by a signal-to-noise comparison at 50 pM concentration of DNA. After a final round of buffer component optimisation, including the addition of a critical reagent (proteinase K) and a number of surfactants, final performance of the assay was assessed with the static-chamber readout method. The assay provided equivalent performance of 500 fM (25 attomole) of DNA in buffer samples and neat serum. The study demonstrated the utility of the assay format, surface modifications techniques, buffer components and microfluidic device for DNA assays conducted in minimally processed clinical samples.