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Nanopore Detection Aims Mainstream.

Traditional methods for detecting DNA segments include microarrays or PCR followed by quantification through electrophoresis or optics. These approaches are complex and costly. But there's a new avenue researchers are exploring: nanopore detection.

In the same way that a flow cytometer picks up cells, a solid-state nanopore can detect individual segments of DNA that travel through it to provide thousands of measurements within minutes (1). Moreover, research has demonstrated the use of nanopores and peptides linked to nucleic acids for detection of double-stranded DNA, requiring precise and <5 nm pore diameters (2, 3).

William Dunbar, cofounder and Chief Technology Officer of the nanopore detecting company, Two Pore Guys (TPG), says the field of nanopore research and commercialization is charging toward sequencing. Like their colleagues, Dunbar and his company began in that position. It wasn't until the arrival of the first full time hire at TPG, Trevor Morin, a biochemist, that they saw another path.

"Think of all the kids on the soccer field running to the same ball," says Dunbar. "We went another direction."

So here we discuss what TPG--which promises "a digital, handheld, testing platform that's as accurate as medical laboratory equipment, but is as inexpensive and easy to use as a blood glucose monitor"--brings to the field of nanopore detection (4).

What Is the Innovation?

"Trevor [Morin] was used to thinking about molecules to be Lego-like," says Dunbar. Using click chemistry principles to join substrates, Morin demonstrated that nanopores could be used to detect DNA segments.

"It is easier to fabricate a massive number of nanopore chips if you're willing to use bigger nanopores," explains Dunbar. Specifically, this means nanopores with diameters >25 nm. As had others, Dunbar combined nanopores with sequence-specific probes for DNA detection, but he went further to make it practical, and today it is on a commercialization path.

"While exploratory research studies often employ precise nanopore geometries, it is challenging to make two or more solid-state pores that have precisely the same geometry and electrical performance," he describes in a recent report (5).

He and his colleagues modified the backbones of peptide nucleic acid probes to include polymer scaffolds such as polyethylene glycol. These scaffolds can be further linked (via click chemistry) to larger molecules that increase the size and features of the probes, making them easier to detect by a larger nanopore. Further, with this design you can have an immunoassay, molecular assay, or both at one time.

"This is a radical departure from what is available today, with immunoassays using one detection paradigm and molecular assays using an entirely different paradigm," says Dunbar.

How Does It Work?

The analogy to flow cytometry fails in how the molecules pass through the detector.

There is no pressure-driving flow through the tiny pores. Rather, the sample is incubated with its target binding ligand and polymer scaffold (such as peptide nucleic acid-polyethylene glycol), and voltage is applied. Molecules diffuse in the chamber at room temperature, and stochastically a molecule that enters a volume within a few microns of the pore is captured by electrophoresis into and through the pore to the other side. As a charged macromolecule travels down the voltage gradient through the pore, a transient current shift is measured across the pore. The shift in conductance (quotient of current change and voltage) and time it takes for the molecule to pass through the pore are measured and plotted. The event signatures of the probe linked to the target are distinctive from DNA alone and provide the means for detection.

The engineered molecules are "not just about adding bulk, we engineer them to give a discriminatory signal," explains Dunbar. "One of the knobs you turn is size, another is surface charge." A well-engineered molecule can display unique interactions with the pore--for example, it may leverage the fact that negative DNA wants to avoid the sides of the pore.

The name of the game is signal boosting. A secondary antibody added to the macromolecule will not interfere with the nanopore, and can provide a "secondary boost-adding feature," says Dunbar.

How Can It Fit in the Laboratory?

Dunbar sees an application as a cancer companion to guide treatment on the basis of the presence and levels of target mutations.

"It is an easy thing to come to work for," he says.

TPG has developed a handheld device that fits a testing component (the "test strip"), which houses reagents and a nanopore chip (Fig. 1).

Christopher Price, an expert in point-of-care testing and Honorary Senior Fellow, Nuffield Department of Primary Care Health Sciences, University of Oxford, feels they have a ways to go before adoption in the point-of-care area.

"They do not appear to have tested the nanopore approach with a range of biological samples to demonstrate the analytical specificity of the approach they are investigating," he wrote in an e-mail to Clinical Chemistry.

Price would like to see data on "the variability of nanopore fabrication and the impact on test result, comparison with laboratory based methods, and 'time to result'."

Dunbar says some of this information is forthcoming--the team is finishing a report now that looks at HIV antibody detection and quantification, including the presence of serum and saliva background. It plans to submit the report soon, as well as publically post the raw data.

Recently, investor Vinod Khosla made a sizable investment in TPG (6). He said of the company's handheld nanopore diagnostics:

"What Silicon Valley is good at is taking an old idea and doing it so much better with new breakthrough science that it becomes an attractive platform just when everybody thought it's not interesting. [TPG's technology] dramatically changes the economics of testing for almost anything, and not just DNA: DNA, RNA, proteins, small molecules, it could test any of those things."

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

References

(1.) Venkatesan BM, Bashir R. Nanopore sensors for nucleic acid analysis. Nat Nanotechnol 2011;6:615-24.

(2.) Singer A, Wanunu M, Morrison W, Kuhn H, Frank-Kamenetskii M, Meller A. Nanopore based sequence specific detection of duplex DNA for genomic profiling. Nano Letters 2010;10:738-42.

(3.) Singer A, Rapireddy S, Ly DH, Meller A. Electronic barcoding of a viral gene at the single-molecule level. Nano Letters 201212:1722-8.

(4.) Two Pore Guys. Home. http://twoporeguys.com/index.html (Accessed July 2017).

(5.) Morin TJ, Shropshire T, Liu X, Briggs K, Huynh C, Tabard-Cossa V, et al. Nanopore-based target sequence detection. PLoS ONE 2016;11:e0154426.

(6.) Herper M. This is neat: billionaire Vinod Khosla is betting on a handheld diagnostic test. Forbes. April 25, 2017. https://www.forbes.com/sites/matthewherper/2017/ 04/25/this-is-neat-billionaire-vinod-khosla-is-betting-on-a-handheld-diagnostictest/#2da25f6f449a (Accessed July 2017).

DOI: 10.1373/clinchem.2016.266908

Vikram S. Kumar [1] * and Molly Webster [2]

[1] Dimagi, Inc., Boston, MA; [2] Science writer and producer, Brooklyn, NY.

* Address correspondence to this author at: 585 Massachusetts Avenue, Suite 4, Cambridge MA02139. Fax 61.7-899-8944; e-mailvkumar@dimagi.com.

Received July 21, 2017; accepted August 10, 2017.

Caption: Christopher Price

Caption: Bill Dunbar

Caption: Fig. 1. Prototype of the nanopore diagnostic device. "Testing strip" with its nanopore chip (A). Handheld unit that provides power and data management to the removable testing strip (B). Image reproduced with permission from William Dunbar.
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Title Annotation:the Clinical Chemist: Technology Corner
Author:Kumar, Vikram S.; Webster, Molly
Publication:Clinical Chemistry
Date:Nov 1, 2017
Words:1266
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