Novel strategies for biosensing with DNA origami and solid state nanopores

Protein sensing is crucial in understanding biological processes and diagnosis of diseases as many relevant biomarkers are proteins. Sensitive and specific detection of such proteins is crucial for disease diagnosis and studying molecular interactions at nanoscale in complex biological systems. Traditional methods for such detection involves ensemble or bulk measurement which average out the output lacking sensitivity in detecting proteins that are in low abundance. Here, a sensitive biosensor based on nanopipette is developed which can detect the precise positioning of proteins on DNA origami.
DNA origami can be precisely functionalised with proteins at varying positions enabling spatial control and signal enhancement facilitating the detection of proteins which are otherwise difficult to detect. In this work, I show the specific attachment of protein binders called affimers at multiple location which are specific to the infection biomarker C-Reactive protein (CRP). For this, I have developed a DNA origami frame with a central pocket in which different combinations of affimers can be functionalised that capture CRP to observe that at least half of the origami structures are functionalised by CRP protein when captured with one or more affimers. The formation of a stable affimer-oligonucleotide conjugate is demonstrated of which the oligonucleotide is complimentary to the DNA strands in the central pocket of DNA origami.
The oligonucleotides or functional molecules like proteins are always added in excess during origami folding and functionalisation. There needs to be a strategy for removing these excess molecules to prevent their interference in downstream applications, at the same time provide high functionalisation efficiency, reduced structural damage and high yield. I have shown the use of SPRI beads, which are traditionally used in DNA sequencing, for the purification of DNA origami from excess biomolecules. I have discussed about the optimum ratio of beads to be utilised and demonstrated the successful purification of DNA origami while maintaining the structural integrity and high yield comparing with column purification.
Finally, I demonstrate the difference in the ion current signals in percentage based on the number of affimer-functionalised origami structures using nanopipette. I notice an increasing trend in the percentage of origami occupied with affimer proteins with increasing number of functionalisation. Thus, it enables the detection of precise positioning of proteins opening up possibilities of developing highly sensitive label-free sensors for biosensing.