Versatile Amplification Method for Single-Molecule Detection in Liquid Biopsy

Robust detection of single molecules in complex biological fluids is the ultimate goal in the field of disease biomarker analysis. Conventionally, to enable the quantitative analysis of individual molecules in macroscopic volumes, analyte pre-concentration and sample partitioning into fL-nL compartments has been combined with the amplification of the specific recognition events. In these setups, the positive or negative detection of fluorescence signal is triggered by enzymatic reactions occurring in each compartment. Binary readout based on Poisson statistics quantifies ultra-low concentrations of analyte molecules. This approach has been adopted for nucleic acids analysis in current digital PCR, and is also available for proteins in a technique coined as digital ELISA.

The objective of VerSiLiB is to develop an enzyme-free amplification strategy for the analysis of both protein and nucleic acid analytes with the single digital platform that offers means to access additional information on target analytes not achievable with current technologies. Method is based on novel affinity-mediated-transport amplification, where affinity interaction of target analyte with a specific ligand attached to a magnetic nanoparticle transporter is accompanied with rapid shuttling of fluorescent tracers that serve as reporters. By applying external magnetic field, tracers are transported from the tracer storage side (where they are dark) to tracer active side (where they become bright) only if target analyte is present in the small reaction compartment. Tailored plasmonic nanostructures will be prepared at the storage and active sides of the compartment to render the tracer either dark or bright. The aim is to perform technology validation for the novel VerSiLiB proteogenomics amplification platform in cancer management using biobanked liquid biopsy samples.