The continued development of diagnostic and therapeutic techniques for biological samples requires robust single-molecule detection techniques. Protein assays that are currently commonly used, such as ELISA, do not fit the specificity and sensitivity requirements, and also involve significant sample processing. In short, they do not provide what is needed for biomarker qualification and quantification. One technique that has the potential to offer improved single-molecule detection is nanopore sensing. Nanopore sensing uses electrical voltage to drive molecules across a
small pore, one that is either biological or solid state. For this research, as solid state pore at
the end of a pipette, a nanopipette, is used. In this technique, the current is continuously monitored and as specific molecules, usually DNAs or proteins, cross through the pore (translocate) the current changes. As nanopore sensing is a single-molecule technique, it
easily fulfils the sensitivity requirements. Unfortunately, on its own, it is not able to select
for specific molecules, and this limitation, in addition to difficulties that are encountered with protein translocations, leads to the necessity of DNA carriers. DNA carriers are typically double-stranded DNAs that has been modified to bind specifically to certain proteins. The translocations of these DNA tethered protein molecules then look significantly different
compared to DNA on its own, such that it is possible to select for them specifically. The distinguishing figure for these events is typically another peak inside these events, or rather a subpeak. This research investigates two DNA carriers, a DNA dendrimer and a DNA plasmid carrier.
The DNA dendrimer has great potential due to its customisability and ability to perform multiplexed sensing. To form the dendrimer, Y-shaped DNAs, each made of three oligonucleotides, was combined in stoichiometric ratios. For the first generation (G1) dendrimer, four Y-shaped DNAs were combined. This G1 could have three protein binding sites or be combined with another six Y-shaped DNAs to form a second generation (G2) with six protein binding sites. However, unfortunately, at the low concentrations required for the nanopore, the binding of the oligonucleotides was not strong enough. Many of the dendrimer structures simply fell apart or could not stay in a reliable shape with the required salt conditions. Therefore, there were few translocation events recorded and it was established that the dendrimers were not feasible DNA carriers.
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The plasmid, like the dendrimer, is completely customisable and has the potential to perform multiplexed protein sensing. A 10 Kbp plasmid was modified such that a short
single-stranded section was replaced with a sequence including both the original sequence as well as a biotin. Theoretically, any aptamer could be used in place of the biotin, but the biotin-streptavidin bond was used for the proof of concept. The plasmid was also linearised. Successful modification of the plasmid so that it could bind to streptavidin was confirmed. Several protein binding curves with both monovalent streptavidin and quadrivalent streptavidin were performed, and events with subpeaks were regularly identified. Additionally, a binding curve for a biotinylated phosphatase bound to quadrivalent streptavidin was performed. This allowed for an investigation of a sandwichlike assay as it moved through the nanopore. While there was a significant difference in the binding curve for this sandwich-like assay, individual events did not have as many differences as was expected. This plasmid carrier has a lot of potential, as it overcomes the issue of introducing selectivity into the nanopore platform without diminishing its sensitivity. The long term goal for this plasmid carrier is for it to be used to identify specific proteins, ideally with multiplexed sensing capabilities, at extremely low concentrations.Open Acces
