Single molecule multiplexed nanopore protein screening in human serum using aptamer modified DNA carriers

Abstract

The capability to screen a range of proteins at the single-molecule level with enhanced selectivity in biological fluids has been in part a driving force in developing future diagnostic and therapeutic strategies. The combination of nanopore sensing and nucleic acid aptamer recognition comes close to this ideal due to the ease of multiplexing, without the need for expensive labelling methods or extensive sample pre-treatment. Here, we demonstrate a fully flexible, scalable and low-cost detection platform to sense multiple protein targets simultaneously by grafting specific sequences along the backbone of a double-stranded DNA carrier. Protein bound to the aptamer produces unique ionic current signatures which facilitates accurate target recognition. This powerful approach allows us to differentiate individual protein sizes via characteristic changes in the sub-peak current. Furthermore, we show that by using DNA carriers it is possible to perform single-molecule screening in human serum at ultra-low protein concentrations.

Analysis of 2D DNA Origami with Nanopipettes

“…The successful assembly of DNA origami folding from an M13mp18 ssDNA scaffold following previous reports [16,18] was confirmed by atomic force microscopy (AFM). Figure 2 shows representative examples of all four assembled DNA origami structures.…”

“…Moreover, the adaptation of the nanopipette with a DNA origami "gate" structure has been shown to enable the orchestration of controlled DNA translocation. [15] An alternative strategy for single-molecule sensing employed by Edel et al involved protein detection using nucleic aptamer-modified DNA carriers [16] and DNA aptamer-functionalized gold nanoparticles. [15] An alternative strategy for single-molecule sensing employed by Edel et al involved protein detection using nucleic aptamer-modified DNA carriers [16] and DNA aptamer-functionalized gold nanoparticles.…”

The applications of nanopores in studies of proteins

“…In the same year, Meervelt et al [117] carried out a similar experimental study by using ClyA nanopores to study the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). In 2017, Sze et al [118] demonstrated simultaneous nanopore-based detection of multiple protein targets each bound to a specific aptamer hybridizing to a specific segment of a dsDNA (Fig. 8).…”

The Utility of Nanopore Technology for Protein and Peptide Sensing

“…Sze et al. developed a rapid and simple aptamer assay that grafts a selective binding region to a long λ‐DNA carrier . This work is also noteworthy because it demonstrates the possibility of nanopore sensing in complex physiological fluids.…”