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New Study Uses DNA Origami, Nanopores for Multiplex Antibody Detection

NEW YORK (GenomeWeb) – A new study from University of Cambridge researchers demonstrates the use of quartz nanopores to recognize dumbbell-shaped DNA constructs, which can in turn be used to detect antibodies.

Nicholas Bell and Ulrich Keyser leveraged the principles of DNA origami to design structures that could carry a unique digital barcode made up of groups of the dumbbell-shaped DNA hairpins. Using a nanopore, they were able to read a three-bit barcode with 94 percent accuracy. When they incorporated antigens onto DNA strands bearing unique origami barcodes, they were able to detect up to four antibodies at a time at nanomolar concentration levels.

They published their results Monday in Nature Nanotechnology.

The new paper builds on Bell and Keyser's method of using DNA carriers to selectively drive proteins through a nanopore. Keyser has also led work on controlling the size of nanopores, which Oxford Nanopore has licensed. Here, they created a double-stranded DNA construct with one strand bearing dumbbell-shaped hairpin protrusions.

They first worked on creating a single digital bit that could be read using a nanopore. To register the bit against background noise, Bell and Keyser found they needed to incorporate multiple hairpins spaced at intervals on a double-stranded DNA backbone.

Varying the number of hairpins used to form a single bit presents a tradeoff between encoding more data and establishing a stronger signal; fewer hairpins per bit allows more bits per DNA strand, while more hairpins provides a stronger single and higher read accuracy. The researchers settled on 11 hairpins per bit.

They then created a three-bit digital system, where each grouping of 11 hairpins — the bit— was either present or absent, leading to eight unique barcodes. These barcodes were then incorporated into antigen-presenting DNA carrier molecules, to which antibodies could bind.

"The presence of the antibody does not significantly affect the dynamics of the DNA," the authors wrote, meaning that the barcodes could be used to read out the presence of an attached antibody.

To demonstrate this, Bell and Keyser created barcoded constructs presenting biotin, BrdU, puromycin, and digoxigenin, and successfully used them for multiplexed detection of the corresponding antibodies at 10 nanomolar antibody concentrations.

"Our basic design of a three-bit code could be significantly expanded," the authors wrote, perhaps by using a longer single-stranded DNA scaffold. "Advances in high-bandwidth amplifiers and high-sensitivity nanopores in thin membranes will also enable greater multiplexing via a reduction in the number of dumbbell hairpins required to signal one bit."

Moreover, they said a sandwich assay approach to improve molecular weight contrast could be taken, by adding an antibody to bind to the DNA-analyte complex.

These properties could enable the technique to be applied to both research and clinical diagnostics, they said.