Sensitive and quantitative nucleic acid testing from complex biological samples is now an important component of clinical diagnostics. Whereas nucleic acid amplification represents the gold standard, its utility in resource-limited and point-of-care settings can be problematic due to assay interferants, assay time, engineering constraints, and costs associated with both wetware and hardware. In contrast, amplification-free nucleic acid testing can circumvent these limitations by enabling direct target hybridization within complex sample matrices. In this work, we grew random copolymer brushes from the surface of silica-coated magnetic nanoparticles using azide-modified and hydroxyl oligo ethylene glycol methacrylate (OEGMA) monomers. The azide-functionalized polymer brush was first conjugated, via copper-catalyzed azide/alkyne cycloaddition (CuAAC), with herpes simplex virus (HSV)-specific oligonucleotides and then with alkyne-substituted polyethylene glycol to eliminate all residual azide groups. Our methodology enabled control over brush thickness and probe density and enabled multiple consecutive coupling reactions on the particle grafted brush. Brush- and probe-modified particles were then combined in a 20 min hybridization with fluorescent polystyrene nanoparticles modified with HSV-specific reporter probes. Following magnetic capture and washing, the particles were analyzed with an aggregate fluorescence measurement, which yielded a limit of detection of 6 pM in buffer and 60 pM in 50% fetal bovine serum. Adoption of brush- and probe-modified particles into a particle counting assay will result in the development of diagnostic assays with significant improvements in sensitivity.

Amplification-free detection of nucleic acids in complex biological samples is an important technology for clinical diagnostics, especially in the case where the detection is quantitative and highly sensitive. Here we present the detection of a synthetic DNA sequence from Herpes Simplex Virus-1 within swine cerebrospinal fluid (CSF), using a sandwich-like, magnetic nanoparticle pull-down assay. Magnetic nanoparticles and fluorescent polystyrene nanoparticles were both modified with DNA probes, able to hybridise either end of the target DNA, forming the sandwich-like complex which can be captured magnetically and detected by fluorescence. The concentration of the target DNA was determined by counting individual and aggregated fluorescent nanoparticles on a planar glass surface within a fluidic chamber. DNA probe coupling for both nanoparticles was optimized. Polystyrene reporter nanoparticles that had been modified with amine terminated DNA probes were also treated with amine terminated polyethylene glycol, in order to reduce non-specific aggregation and target independent adhesion to the magnetic particles. This way, a limit of detection for the target DNA of 0.8 pM and 1 pM could be achieved for hybridisation buffer and CSF respectively, corresponding to 0.072 and 0.090 femtomoles of target DNA, in a volume of 0.090 mL.

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In this work protein patterning has been demonstrated within a polycarbonate microfluidic device. Channel structures were first coated by plasma polymerization of allylamine (ALAPP) followed by 'cloud point' grafting of polyethylene oxide (PEO), resulting in a protein repellent surface. Excimer laser micromachining was used to pattern the PEO to control protein localisation. Subsequent removal of a sacrificial layer of polycarbonate resulted in the patterned polymer coating only within the channels of a simple fluidic device. Following a final diffusion bonding fabrication step the devices were filled with a buffer containing streptavidin conjugated with fluorescein, and visualized under a confocal fluorescence microscope. This confirmed that protein adhesion occurred only in laser-patterned areas. The ability to control protein adhesion in microfluidic channels leads to the possibility of generating arrays of proteins or cells within polymer microfluidics for cheap automated biosensors and synthesis systems.