Assay technologies capable of detecting low biomarker concentrations in complex biological samples are fundamental for biological research and for applications in medical diagnostics. In this paper we address the challenge to perform protein biomarker detection homogeneously in one single step, applying a minute amount of reagent directly into whole human blood plasma, avoiding any sample dilution, separation, amplification, or fluid manipulation steps. We describe a one-step homogeneous assay technology based on antibody-coated magnetic nanoparticles that are spiked in very small amount directly into blood plasma. Pulsed magnetic fields and a double-linker molecular architecture are used to generate high biomarker-induced binding and low nonspecific binding between the nanoparticles. We demonstrate dose-response curves for prostate specific antigen (PSA) measured in undiluted human blood plasma with a detection limit of 400-500 femtomol/L, in a total assay time of 14 min and an optically probed volume of only 1 nL. We explain the dose-response curves with a model based on discrete binding of biomarker molecules onto the nanoparticles, which allows us to extract reaction parameters for the binding of biomarker molecules onto the nanoparticles and for the biomarker-induced binding between nanoparticles. The demonstrated analytical performance and understanding of the nanoparticle assay technology render it of interest for a wide range of applications in quantitative biology and medical diagnostics.

Dengue is a tropical vector-borne disease without cure or vaccine that progressively spreads into regions with temperate climates. Diagnostic tools amenable to resource-limited settings would be highly valuable for epidemiologic control and containment during outbreaks. Here, we present a novel low-cost automated biosensing platform for detection of dengue fever biomarker NS1 and demonstrate it on NS1 spiked in human serum. Magnetic nanoparticles (MNPs) are coated with high-affinity monoclonal antibodies against NS1 via bio-orthogonal Cu-free ‘click’ chemistry on an anti-fouling surface molecular architecture. The presence of the target antigen NS1 triggers MNP agglutination and the formation of nanoclusters with rapid kinetics enhanced by external magnetic actuation. The amount and size of the nanoclusters correlate with the target concentration and can be quantified using an optomagnetic readout method. The resulting automated dengue fever assay takes just 8 minutes, requires 6 μL of serum sample and shows a limit of detection of 25 ng/mL with an upper detection range of 20000 ng/mL. The technology holds a great potential to be applied to NS1 detection in patient samples. As the assay is implemented on a low-cost microfluidic disc the platform is suited for further expansion to multiplexed detection of a wide panel of biomarkers.

We describe an optomagnetic bionanotechnology for rapid and sensitive solution-based affinity assays. Nanoactuators made from bioactive magnetic nanoparticles undergo rotational motion in the volume of a fluid under frequency-controlled magnetic actuation. The nanoactuators show a time-dependent scattering cross-section to an incoming light beam. We demonstrate that the temporal behavior of the scattered light intensity relates to the number, the magnetic properties and the size distribution of the nanoactuators, independently revealing the average value and variation in the magnetic properties of the nanoparticles as well as the concentration of nanoactuators. The method is applied to detect biomolecules in fluid by interparticle binding. In a total assay time of less than 3 min, we demonstrate a limit of detection lower than 400 fM in buffer and 5 pM in human plasma.