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New Nanopore Sequencing-Based Method Enables Multiplexed Biomarker Detection


NEW YORK – Researchers from Imperial College London have developed a new nanopore sequencing-based workflow in collaboration with Oxford Nanopore Technologies that can simultaneously analyze multiple types of biomarkers in one sample.

Described in a Nature Nanotechnology study published last month, the method combines nanopore sequencing with DNA-barcoded molecular probes. By demonstrating its ability to quickly and cost-effectively detect dozens of analytes — including microRNA, proteins, and small molecules — the proof-of-concept study opens the door for potential diagnostics applications with further development.

"The motivation behind the study was to come up with a new sensor probe that can bind to certain biomarkers in [a biological sample]," said Caroline Koch, a PhD student in chemistry at Imperial College London and the co-first author of the study. "By combining the probes with nanopores in a multiplexed approach, we were hoping to come up with a strategy that is portable, gives you fast results, and is cheaper than what you can currently do."

Conventionally, to interrogate different types of biomarkers in a sample, researchers typically have to carry out separate assays that can be tedious and costly, Koch said. For instance, to detect microRNAs (miRNAs) — short non-coding RNA molecules that play a role in gene expression — researchers primarily rely on RT-qPCR, she noted. Meanwhile, enzyme-linked immunosorbent assays (ELISA) are commonly used to detect protein targets.

As multi-class biomarker analysis is playing an increasingly important role in patient diagnosis and treatment, especially for precision medicine, the study authors noted that there is "a great need" to develop an assay that can investigate various classes of analytes in one go.

To achieve this, Koch and her collaborators sought to develop a highly multiplexed detection workflow by combining nanopore sequencing and DNA-barcoded molecular probes, which are engineered to recognize a spectrum of analytes.

According to Koch, the probes consist of three key regions: an adapter on the 5' end, a unique DNA barcode, and a target-binding region on the 3' end. More specifically, she said the adapter, which is supplied by Oxford Nanopore, is universal across the probes and contains a motor protein that controls the translocation speed of the probe to ensure appropriate sequencing and base calling.

Meanwhile, the DNA barcode is 35 bases long and functions as a unique identifier for different analytes, making multiplexed detection possible. Lastly, the target-binding region is specific to the biomarker of interest, being either a complementary sequence that can bind to miRNAs or an aptamer that can target proteins and small molecules. During sequencing, the adapter part of the probe first flows through the pore, followed by the barcode, whose sequence can be used to identify the target analyte associated with the probe.

As the probe continues to pass through the nanopore, its translocation will be slowed if the target-binding region recognizes an analyte, which creates a structural obstacle for the passage, Koch said. This, in turn, can result in a delay in the electrical signal that is notably different from the signal generated by an unbound probe, indicating the presence of the target molecule.

In addition, by analyzing the proportion of reads with a delayed signal, the concentration of different biomarkers can also be informatically inferred.

"A lot of optimization has gone into the barcode design," said Benedict Reilly-O'Donnell, a postdoctoral researcher at Imperial College London who is the other first author of the study. Additionally, he said the team has come up with "very stringent criteria" for acceptable sensing events to ensure the assay's accuracy.

Koch said the team owns the IP for the probe design, which is currently licensed to Oxford Nanopore. She also noted that Oxford Nanopore provided studentship funding for her PhD and contributed flow cells in-kind to this study.

Oxford Nanopore did not provide more details regarding the company's plans with the probe technology.

To benchmark the method's performance, in their current study, the researchers designed and tested probes for 40 synthetic targets, including miRNAs, proteins, and small molecules such as neurotransmitters, associated with cardiovascular diseases. Furthermore, they used the assay on blood serum from eight healthy individuals to detect the presence of 40 target miRNAs.

Overall, the results demonstrated that the technology "offers a highly accurate and sensitive analyte sensing platform" to detect and quantify multiple types of analytes with a single-molecule resolution," the study authors noted. According to them, the assay only requires sample input of less than 30 μl, and its current turnaround time, from sample to results, can be within an hour. Moreover, it costs less than $100 per workflow.

In addition, the assay's compatibility with human blood serum indicates its future potential for biomarker detection in a clinical setting. "That was like a cherry on the top" to show that the technology can detect microRNAs in human samples, Reilly-O'Donnell noted.

"I think [the method] is a pretty powerful approach because it really extends the Oxford Nanopore platform to the detection of a range of different biomarkers," said Jeff Nivala, a professor at the University of Washington with nanopore research expertise who was not involved in the study.

Nivala said to his knowledge, this is the first published method that enables multiplex biomarker detections across different analyte types using nanopores. In addition, he praised the study authors for going "above and beyond" many other nanopore biomarker detection papers by demonstrating the approach's sensitivity is good enough to detect targets at relevant concentrations in real biological samples.

Besides biomarker detection, Nivala said the approach could also be potentially deployed for applications such as environmental sensing to detect small molecule contaminants or biohazards.

Meanwhile, since the method is affinity-based, Nivala noted that one of its limitations of is that it can only detect known analytes. In addition, researchers have to be able to design probes that can bind to the targets strongly enough to generate the stall signal when passing through the nanopore in order for the assay to work, he pointed out.

Reilly-O'Donnell said another limitation of the method is that currently, when performing blood serum assays, the proteins in the sample can block the pore, curtailing the detection events. That said, the authors noted that solving unwanted pore blockage is "an active research area" that the team is working on.

In the current study, all the experiments were conducted using the Oxford Nanopore R9 flow cells, Koch noted. As the company continues to roll out new chemistry, she said the team is also in the process of updating the workflow to leverage the newer R10 flow cells, which promise to have improved sensitivity.

To that end, Koch said another further direction for the team is to continue improving the performance of the assay. According to her, the current detection limit of the method for miRNA is around 50 pM.

With the transition to the new nanopores and other optimization strategies, the goal is to push the platform into the lower femtomolar detection range, which would be more clinically relevant, Koch said.