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Nanopore Platform Developers Aim to Bring Multiplex MDx Testing to Point of Care

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NEW YORK (GenomeWeb) – Researchers in the UK and Italy are collaborating to develop single-molecule DNA technology for use in a multiplex diagnostic platform and testing at the point of care.

The technology uses solid-state nanopore current sensing and a carrier with attached DNA probes that could give the diagnostic device, if commercialized, the capability to do multiplexing and high-throughput analysis of infectious analytes, according to a study published recently in Analytical Chemistry.

Such a diagnostic test would use a liquid medium, such as saliva or serum, and operate without requiring labeling of fluorescent tags often needed for detection in existing diagnostic systems, but which add to cost and testing complexity, Tim Albrecht, a professor of physical chemistry at the University of Birmingham and study author, said in an interview.

"The point of differentiation of the platform we are developing is its simplicity," he said.

The group designed molecular probes that bind to target DNA with a predefined sequence, enabling identification of the presence of the molecules by detecting the location and binding along a DNA carrier.

The nanopore sensor is made of a liquid cell split into two compartments by a nanopipet, which is an insulating membrane, and the nanopore is the only way that the two compartments can exchange liquids or analytes consisting of DNA.

In order to initiate movement through the pore, the system applies an electric field that leads to a current that's altered by the moving DNA. The nanopore sensor accordingly detects the specific binding event by a change in the magnitude of the ionic current. The sensing approach enables detecting the volume of molecules that pass the through the pore and analyzing changes in molecular weight.

"The nice thing about this is that you can increase the density of those probes and put specific probes in specific locations," Albrecht said. "There's a lot of flexibility in what we add, and the information density of an individual translocation event increases accordingly."

The device developed by Albrecht and his colleagues is a proof of concept and far from being fully developed as a commercial tool. Developing a system that can compete with existing diagnostic tests — ranging from antibody and molecular diagnostics to sequencing — is a tall order, and the current platform would need to be adapted to make it suitable for use as a high-throughput, solid-state platform, Albrecht said.

However, the potential for accurate, point-of-care multiplexing involving the detection of many different disease analytes is appealing to clinicians and researchers alike not the least because it could significantly cut the time to receive a test result and avoid delays in receiving results associated with sending samples to laboratories.

In using nanopipets to electrically detect DNA sequences associated with infectious disease pathogens — including an 88-mer target from the RV1910c gene in Mycobacterium tuberculosis, which is associated with antibiotic resistance in TB — the group has proved that the technology works in the way that they had envisioned it would, Albrecht said. The research team is currently developing the system for sepsis diagnosis, which needs to be particularly quick. He further noted that clinicians and patients would benefit from having a sepsis diagnostic tool available at the point of care.

"We have to establish the technology a bit more before we can talk about spinning it out and potentially forming a startup," Albrecht said.

Looking at diagnostic commercial timelines, the technology could be advanced from the present stage of development and become a commercial test within about 3 or 4 years, Albrecht said.

He added that use of nanopipets doesn't provide the most suitable means of implementing commercial-scale nanopore sensing, and a fully developed platform would need to include solid-state chips with nanopores mass produced using semiconductor processing.

He and his colleagues are also working on implementing sample processing along with sensing in a single workflow. "If we can show that sample processing and sensing go hand in hand, then we'll be able to exploit the true potential of the technique," he said.

Further, the group has developed a process to modify DNA enzymatically, which would be more amenable for production than the current method of building DNA probes through self-assembly. They are also evaluating adding antibody and RNA probes to the carrier.

Other research groups in the UK have been working on similar technologies. The Imperial College London, for example, has reported the development of a 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.

In that platform, protein bound to an aptamer produces unique ionic current signatures that facilitate target recognition, enabling the researchers to differentiate individual protein sizes by characteristic changes in current. Further, the group reported demonstrating that it is possible to perform single-molecule screening in human serum at ultra-low protein concentrations by using DNA carriers.

Ulrich Keyser and Nicholas Bell, researchers at the University of Cambridge, published a study last year in Nature Biotechnology in which they demonstrated the use of nanopipets and a carrier based on DNA self-assembly to include a site for antibody binding and barcodes identifying DNA.

Keyser said in an interview that Albrecht and his colleagues have also shown "that nanopores are ideally suited for barcoding applications with nucleic acids. The method presented in this paper [published by Albrecht and his coworkers] is especially relevant for DNA and RNA binding to targets, due to high specificity and high binding affinity, which reduces false positive events."

The authors have designed sequences for specific targets and thus created the necessary specificity, Keyser said, adding, "Interestingly, the method allows one to detect relatively short DNA sequences … and hence expands the range of detectable molecules towards the shorter end of nucleic acids." With smaller nanopores and higher resolution current detection, the method should be able to detect much smaller structures as well, and possible targets include short RNA molecules, he noted.

The main challenges associated with the approach is making available solid-state nanopores, "ideally in a massively parallel configuration with hundreds or thousands of nanopores working in parallel," Keyser said. "The authors also measure in relatively controlled conditions and do not yet show the applicability in clinically relevant samples," he said.

However, other groups, including Imperial College London, have shown that it is in principle possible to detect single proteins in more complex solutions like serum, he added.

Keyser also noted that the concentrations of the target molecules need to be in the nanomolar range because "the resolution of the presented technique pushes the limits of detection, and the authors need hundreds of events to confirm the correct detection."

However, Keyser said, "The study demonstrates the vast opportunities that a combination of DNA nanotechnology and nanopore sensing offers for highly specific and facile nanopore sensing."