As chronic age-related diseases rise, so does the urgency for diagnostic tools capable of detecting illness early, ideally before symptoms appear. One of the key challenges lies in identifying subtle changes in the shape and structure of proteins. These conformational shifts often mark the earliest biological signals of disease but remain largely invisible to conventional diagnostics.
Most assays measure biomarker concentrations or genetic sequences. They miss finer structural variations, such as misfolded proteins, which are central to diseases like Alzheimer’s, Parkinson’s, and prion disorders. Capturing these elusive changes at the molecular level could enable earlier, more accurate diagnoses.2
That’s where nanopore sensing comes in.
A Label-Free Window Into Molecular Behavior
At the centre stage of nanopore technology is a nanoscale aperture that allows individual biomolecules to pass through. As each molecule travels through this gap, it uniquely disrupts an ionic current, revealing data about its size, charge, and shape. Looking at molecular behaviour through this lens provides label-free, single-molecule analysis in real time with remarkable precision.4
Unlike traditional techniques that rely on chemical tags or extensive sample prep, nanopore platforms require minimal intervention. Their portability and potential for miniaturization make them particularly attractive for decentralized and point-of-care settings.3-5
Researchers have already demonstrated that nanopores can detect post-translational modifications, such as phosphorylation and glycosylation, and can distinguish between closely related peptide isoforms. These advances suggest a new class of biosensors, ones that assess the presence of biomarkers and their structural behavior.3
Signals From the Heart and Tumor Microenvironment
In cardiovascular research, nanopore systems have identified natriuretic peptides at concentrations matching guideline thresholds from the European Society of Cardiology. Though still experimental, this reflects a growing ability to parse clinically relevant proteins with molecular-level detail.5
Similar findings have emerged in oncology. Lab studies show that nanopore sensors can detect prostate-specific antigen and human epididymis protein 4, as well as different structural variants and degradation products. This could result in improved diagnostic resolution over traditional immunoassays.3-5
Notably, nanopore sequencing was an essential technology during the COVID-19 pandemic. While that application focused on DNA and RNA rather than proteins, it demonstrated the platform’s adaptability and field-readiness, which are valuable traits for future diagnostic tools.
Engineering the Right Pore
The design of nanopores is central to their performance. Early platforms used biological pores like α-hemolysin, but researchers have since developed synthetic variants made of materials such as silicon nitride and graphene. While these solid-state pores offer durability and tunability, they are more prone to electrical noise, especially 1/f fluctuations that affect signal clarity.5
Hybrid systems, which combine the mechanical strength of synthetic pores with the specificity of biological components, aim to overcome these limitations. But pore behavior depends on a host of variables, including pH, electrostatic potential, and surface chemistry, making design optimization a cumbersome process. Achieving consistent results across batches and under physiological conditions remains an issue.5
Real-Time Kinetics and Protein Interactions
One of nanopore sensing’s most powerful abilities is its monitoring of dynamic molecular events. When a ligand binds to a protein, or when proteins form complexes, the resulting conformational shifts produce measurable changes in ionic current, and these changes can be measured.3
For instance, researchers have used a ClyA nanopore to monitor ubiquitination, a key process in protein degradation. They were able to distinguish between unmodified enzymes and those bearing one or more ubiquitin units, observing this interaction in real time. Such data could be integral to understanding disrupted signaling pathways in diseases like cancer and neurodegeneration.3
Scaling this technique to complex biological samples will require enhanced data processing and pore control, but it represents a meaningful step toward functional proteomics.
Unlocking disease mechanisms with multiomic nanopore sequencing
Video Credit: Oxford Nanopore Technologies/Youtube.com
Detecting Chemical Modifications at Single-Residue Resolution
Post-translational modifications play critical roles in cellular regulation. More than 400 of these modifications are known, including acetylation, methylation, hydroxylation, and glycosylation. Among these, phosphorylation is especially relevant due to its involvement in signaling pathways.
Mutated biological nanopores, such as a modified α-hemolysin channel, have been used to detect phosphorylation, glycosylation, and glutathionylation on peptides. A more advanced system, using a double-mutated aerolysin nanopore, successfully slowed the passage of tau protein fragments to resolve individual phosphorylation sites, a key biomarker of Alzheimer’s disease.3
Even higher resolution was achieved using a helicase-assisted system, in which peptide-oligonucleotide hybrids were translocated through a pore using a DNA motor. This setup was able to detect phosphorylation on a single threonine residue within the cancer-related BCAR3 peptide.3
These systems remain confined to idealized conditions and short peptide sequences. Transitioning to full-length proteins in biological fluids is a future goal.5
Download this in PDF form to read offline!
Chirality at the Nanoscale
Another emerging capability is enantiomer detection. Enantiomers, molecules that are mirror images of each other, share the same mass and charge but often behave differently in biological systems due to their different conformations. Their presence in long-lived proteins, such as crystallins in the eye, has been linked to age-related diseases.
Nanopore sensors have distinguished amino acid enantiomers using α-hemolysin pores in combination with copper-complexed cyclodextrins. Other studies engineered nanopores like CytK and FraC to recognize enantiomeric peptide pairs, including variants of Leu-enkephalin, based on differences in their current blockades.3
The OmpF nanopore, with its uniquely asymmetric constriction, has detected chirality differences in amyloid-beta peptides, distinguishing L-Asp from D-Asp within the same sequence. Meanwhile, an aerolysin pore differentiated between L- and D-forms of vasopressin, a hormone relevant to diabetes insipidus.3
Still at the proof-of-concept stage, these findings suggest that nanopore sensing may become a valuable tool for identifying subtle biochemical variants in neurodegenerative and metabolic diseases.
Bridging Innovation and Application
Despite advances, several barriers stand in the way of widespread clinical adoption of nanopores. Signal drift, protein unfolding, and inconsistent translocation speeds remain technical challenges. Most critically, interpreting nanopore data, especially when dealing with minute structural differences, requires sophisticated analytics, including machine learning approaches that are still evolving.3-5
Researchers must also address throughput, multiplexing, and long-term reproducibility under physiological conditions to move from lab to clinic. But if these challenges can be overcome, nanopore sensing could shift the diagnostic paradigm, from bulk biomarker detection to single-molecule structural profiling.
Originally developed for DNA sequencing, nanopore technology is rapidly evolving into a broader platform for proteomics. Its ability to sense not just what a molecule is, but how it behaves, may define the next generation of early, personalized, and precise diagnostics.
References and Further Reading
- Bellantuono, I. (2018). Find drugs that delay many diseases of old age. Nature, 554(7692), 293-295. DOI: 10.1038/d41586-018-01668-0, https://www.nature.com/articles/d41586-018-01668-0
- Califf, R. M. (2018). Biomarker definitions and their applications. Experimental Biology and Medicine, 243(3), 213-221. DOI: 10.1177/1535370217750088, https://journals.sagepub.com/doi/abs/10.1177/1535370217750088
- Ratinho, L., Meyer, N., Greive, S., Cressiot, B., & Pelta, J. (2025). Nanopore sensing of protein and peptide conformation for point-of-care applications. Nature Communications, 16(1), 1-19. DOI: 10.1038/s41467-025-58509-8, https://www.nature.com/articles/s41467-025-58509-8
- Zeng, X. et al. (2021). Nanopore Technology for the Application of Protein Detection. Nanomaterials, 11(8), 1942. DOI: 10.3390/nano11081942, https://www.mdpi.com/2079-4991/11/8/1942
- ?oldanescu, I., Lobiuc, A., Adriana, O., Covasa, M., Mangul, S., & Dimian, M. (2025). The Potential of Nanopore Technologies in Peptide and Protein Sensing for Biomarker Detection. Biosensors, 15(8), 540. DOI: 10.3390/bios15080540, https://www.mdpi.com/2079-6374/15/8/540
Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.