A blood test that covers a wide range of diseases at a low cost has been the holy grail in diagnostics. One major hurdle in achieving this is that most available blood tests detect a single biomarker associated with the target disease. As Imperial College London postdoctoral researcher Caroline Koch explained, this often isn’t enough. “The biomarkers we currently have are not specific enough for certain diseases,” said Koch. “We need better techniques that are able to analyze multiple biomarkers simultaneously.”

In a recent paper published in Nature Nanotechnology, Koch and her collaborators achieved exactly that (1). The team developed a diagnostic platform that could detect multiple RNA molecules, small molecules, and proteins at once. “With this technique, it could be possible to detect those biomarker species in a multiplex way, which is not something that any other technique can achieve at the moment,” said Koch.

The approach is based on nanopore sequencing, which involves passing DNA molecules through small protein pores embedded in a membrane (2). Researchers apply an external voltage to the membrane and measure the resulting current. Each DNA base causes a distinct change to the current as the molecule passes through the nanopore. Researchers can then convert the signal, or series of changes in the current, to a sequence of bases. 

Nanopore sequencing has recently gained a lot of traction in the realm of diagnostics, especially with the MinION, a cheap and portable laptop-powered device that contains thousands of nanopores and can detect many molecules at a time. However, attempts at nanopore-based blood testing have so far also involved detecting single biomarkers.

To detect multiple biomarkers in a sample, Koch’s team designed a system of molecular probes that each bind a different target. Each probe consists of three covalently linked parts: an adapter enzyme that anchors the probe to the nanopore, a barcode or DNA tag unique to each probe, and an aptamer or DNA polymer that binds to its target biomarker. Each probe can pass through the pore and produce a signal without its target. However, when bound to its target, the probe does not fit through the pore. In this case, only the barcode region will pass through the pore before the signal stalls; only once the aptamer region releases the target can the rest of the probe pass through and resume the signal. 

To identify biomarkers present in a sample, Koch’s team looked for signal stalling, sequenced the preceding barcode region, and mapped the barcode back to the corresponding biomarker. Because each barcode creates a signal, and each probe binds to a unique biomarker, Koch’s team can use several probes on the same sample and in the same MinION device without any interference. 

Koch’s team tested a system of 40 probes that each bound to a different RNA target. They incubated all the probes together and tested several combinations of RNA. Each probe only exhibited signal stalling when the target RNA was present, confirming that they had achieved their goal of multiplex detection. “I was really happy to see that because you always start with a hypothesis, but then sometimes you can't prove it,” said Koch. 

Nanopore detection of non-nucleic acid biomarkers is still an area of active development with many unsolved challenges (3). Yet, Koch and her team bypassed these hurdles by passing their probes through the nanopore and sequencing the DNA barcodes rather than the target biomarkers themselves. Using their platform, they successfully detected the small molecule serotonin, as well as proteins related to cardiac fibrosis: thrombin, B-type natriuretic peptide, and cardiac troponin. By mixing the corresponding probes, they simultaneously detected thrombin, RNA, and serotonin.

“The big breakthrough with this paper is that they used these aptamers for any classic target molecule,” said Jeff Nivala, a computer scientist at the University of Washington who specializes in nanopore technology development but who was not involved in the study. “If you can develop an aptamer for it, you can sense it now using their nanopore approach.” Nivala pointed out that the platform relies on aptamers that had been previously tested; this method would not be suitable to test how well a new aptamer bound a particular target. “Your assay is really only as good as your aptamer,” he said. 

Finally, the researchers used their platform to test blood serum from eight healthy people for 40 different miRNA molecules associated with cardiovascular disease. Koch explained that they first had to filter out proteins in the serum: “Nanopores are super small, and serum is quite sticky material. So, they block quite easily,” she said.

I am hoping that clinicians will reach out, and we can start collaborations to validate this technology on different types of datasets. 
- Caroline Koch, Imperial College London

Nivala also explained that sample preparation is an open question for nanopore diagnostics; Koch’s team will likely have to optimize different filtering protocols for different biomarker tests. In the end, they found signal stalling that corresponded to significant increases in miRNA levels, which they were able to validate with RT-qPCR. 

“For me, it was always important that my research has an impact on patients,” said Koch. Her team is in the process of writing a more translational paper, for which they are working with a cohort of patients with cardiac fibrosis and using the platform to detect a more clinically relevant selection of biomarkers. She anticipates that the paper will be published by the end of 2024.

Ultimately, Koch envisions using their MinION platform to detect various types of cancer, cardiac disease, and neurological disorders at the point of care. “I am hoping that clinicians will reach out, and we can start collaborations to validate this technology on different types of datasets,” she said.

References

1. Koch, C. et al. Nanopore sequencing of DNA-barcoded probes for highly multiplexed detection of microRNA, proteins and small biomarkers. Nat Nanotechnol  18, 1483-1491. (2023).
2. Wang, Y.H. et al. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol  39, 1348-1365. (2021).
3. Ying, Y.L. et al. Nanopore-based technologies beyond DNA sequencing. Nat Nanotechnol  17, 1136-1146 (2022).