The device is built on MspA, a mushroom-shaped porin drawn from the bacterium Mycobacterium smegmatis. MspA has long been a favorite scaffold in nanopore research because of its conical geometry and its narrow, well-defined constriction, which sits at the narrowest point of the pore and acts as the readout zone where passing molecules modulate an ionic current. What the team led by Shuo Huang of Nanjing University did was to graft a bespoke chemical adaptor into that constriction. The adaptor, called maleimido-C2-formylphenylboronic acid, or maleimido-C2-FPBA, combines two reactive functions in one compact molecule: a maleimide group that covalently anchors the adaptor to a single cysteine residue engineered into the pore wall, and an ortho-formylphenylboronic acid group that juts into the lumen of the pore where it can interact with passing analytes.

The chemistry of that boronic acid head group is what gives the pore its versatility. Boronic acids are famous for forming reversible covalent bonds, known as boronate esters, with cis-diols — the paired hydroxyl groups found abundantly on sugars — which explains why FPBA-equipped pores excel at recognizing saccharides. But the formyl group adjacent to the boron adds a second recognition mode: it can condense with the N-terminal amino groups of amino acids and peptides to form iminoboronate adducts, a reversible linkage that has become a powerful tool in chemical biology. This dual chemistry means that amino acids, sugars and even nucleotides, which carry phosphate and hydroxyl groups capable of engaging the boron center, each generate their own characteristic electrochemical fingerprints as they dock at and escape from the constriction.

When a voltage is applied across a lipid membrane containing a single MspA-FPBA pore, ions stream through the opening and produce a steady baseline current. Each time an analyte molecule enters the pore and binds transiently to the FPBA adaptor, the ionic current drops in a brief blockade whose depth, duration, noise profile and shape encode information about the molecule’s identity. The researchers systematically characterized these events for each of their target molecules. Notably, the earlier versions of related sensors had struggled: an MspA pore carrying only a phenylboronic acid adaptor failed to detect the amino acid phenylalanine, and a nickel-nitrilotriacetic acid modified pore registered no events for fructose or guanosine monophosphate. Only the combined formyl-boronate chemistry of FPBA proved capable of capturing this chemically diverse set of analytes with strong, well-resolved signals.

The catalog of molecules the engineered pore can discriminate is remarkable. On the amino acid side, it resolves all twenty standard proteinogenic building blocks plus selenocysteine, the twenty-first genetically encoded amino acid, and it further distinguishes three post-translationally modified residues: N6-acetyllysine, O-phosphotyrosine and asymmetric dimethylarginine, representing acetylation, phosphorylation and methylation, respectively. On the nucleotide side, it identifies the four canonical nucleoside monophosphates — AMP, UMP, CMP and GMP — as well as three epigenetically relevant variants: pseudouridine monophosphate, N6-methyladenosine monophosphate and inosine monophosphate. Four monosaccharides, including iduronic acid, L-arabinose, D-fructose and N-acetyl-D-glucosamine, and five short peptides round out the panel. Some analytes produce more than one characteristic event type — cysteine, histidine and lysine each yield two event classes, and arabinose produces three — yet the machine learning layer absorbs this complexity without difficulty.

That machine learning layer is central to the system’s performance. The researchers extracted nine numerical features from each nanopore event, including the mean current blockade, its standard deviation, a local noise ratio, the minimum and maximum current values, the event range, the dwell time, and the skewness and kurtosis of the blockade shape, forming a nine-dimensional fingerprint for every single-molecule encounter. They then benchmarked several classifiers using ten-fold cross-validation and found that an ensemble of bagged decision trees — an approach rooted in Leo Breiman’s classic random forests methodology — performed best, reaching the headline accuracy of 98.7 percent across the full panel of 24 amino acids, 7 nucleotides, 4 sugars and 5 peptides. The resulting confusion matrix shows cleanly separated classes even for chemically subtle distinctions, such as proline versus methionine, which the model resolves with validation accuracies of 98.8 and 99.4 percent respectively.

Perhaps the most consequential demonstration is the sensor’s ability to generalize. Because each class of analyte — amino acids, nucleotides, saccharides, peptides — produces predictable families of event signatures, the trained model can recognize the category of a molecule it has never seen in its training data, a capability the team calls few-shot learning. When the researchers filtered a yeast cell extract into the pore chamber, the classifier correctly assigned events to their proper analyte classes and further identified individual amino acids and nucleotides within those classes, despite the mixture being far messier than any of the purified standards used in training. This out-of-database classification matters enormously for real-world applications, where an unknown sample cannot be presumed to contain only molecules with pre-archived reference signatures.

To showcase practical analytical chemistry, the team applied MspA-FPBA to the compositional analysis of glycopeptides — peptides decorated with sugar chains, which are central to protein glycosylation, one of the most important and clinically consequential post-translational modifications in biology. They digested a glycosylated heptapeptide with leucine aminopeptidase, an enzyme that clips amino acids from the N-terminus until it stalls at the bulky glycosylation site. As the enzyme released glycine, methionine, glutamine and arginine one by one, the nanopore and its machine learning classifier identified each in real time, while the residual glycosylated tripeptide fragment generated its own distinctive events. In this way, a single measurement revealed both the amino acid composition preceding the sugar attachment site and the presence of the glycosylated remnant — information that would ordinarily require multiple orthogonal techniques.

The researchers are candid that this is a waypoint rather than a destination. In their abstract they note that future integration of a hydrolase enzyme with MspA-FPBA could turn the system into a self-consuming reader that degrades larger biomolecules into their building blocks and identifies them sequentially — an architecture that would echo the motor-protein ratcheting used in commercial nanopore DNA sequencing and that has already been explored conceptually for exopeptidase-driven protein sequencing. Challenges remain, including throughput, the concentration ranges required for reliable detection, and extending the panel beyond the current analyte set. Still, the convergence of a single multifunctional chemical adaptor, a rugged bacterial pore and modern machine learning delivers something the field has been missing: one sensor that speaks the chemical languages of proteomics, glycomics and transcriptomics at the single-molecule level. If the promised hydrolase coupling matures, the humble mycobacterial porin may find itself at the heart of a universal molecular reader, one blockade at a time.

Subject of Research: An engineered MspA nanopore with a formylphenylboronic acid adaptor for simultaneous single-molecule identification of amino acids, nucleotides, saccharides and peptides

Article Title: An engineered nanopore identifies saccharides, amino acids, peptides and ribonucleotides

Article References: Yao, L., Wang, Z., Chen, J., Sun, W., Wang, K., Xiao, Y., Zhang, H., Li, W., Wang, Y., Zhao, L., Dai, X., Qian, L., Zhang, P., & Huang, S. (2026). An engineered nanopore identifies saccharides, amino acids, peptides and ribonucleotides. Nature Biotechnology. https://doi.org/10.1038/s41587-026-03308-9

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03308-9

Keywords: nanopore sensing, MspA, formylphenylboronic acid, amino acid identification, nucleoside monophosphates, saccharide detection, peptide sensing, machine learning, single-molecule analysis, glycopeptide, post-translational modifications, epigenetic modifications

The heart of the device is MspA, a porin from the soil bacterium Mycobacterium smegmatis that has already earned a storied reputation in DNA sequencing. MspA forms a stable, conical pore roughly one nanometre wide at its constriction, a scale at which a single small molecule can obstruct the flow of ions and produce a characteristic electrical signature. When a voltage is applied across a membrane containing the pore, analytes that wander into the aperture transiently block or modulate the ionic current, and the resulting blips carry fingerprints of the molecule’s size, shape, charge and chemistry.

The problem has always been versatility. Nanopores are exquisitely selective, which is a virtue for detecting one target molecule but a liability when the target is unknown. Different chemical classes interact with the pore environment in different ways: saccharides are neutral and hard to trap, amino acids span a dramatic range of charge and hydrophobicity, and nucleotides carry dense negative charges that make them rush through too quickly to be read. Earlier engineered pores succeeded with individual analyte classes—discriminating all twenty proteinogenic amino acids in one design, or distinguishing monosaccharides in another—but no single pore had tackled all of them at once.

The new work solves this by grafting a chemical adapter into the pore. The team modified MspA with a maleimido-C2-FPBA moiety, a benzaboronic acid derivative attached through a short linker to a defined site inside the pore lumen. Boronic acids are famous in chemical biology for forming reversible covalent complexes with cis-diols, the pairing of hydroxyl groups found abundantly on sugars. But the adapter does more than catch carbohydrates. Inspired by iminoboronate chemistry, the reversible interaction between boronic acids and nitrogen-containing functional groups allows the adapter to transiently capture amines as well, slowing the passage of amino acids and peptides so that their signals can be recorded.

The result is a pore that no longer lets any of these small molecules simply tumble through. Instead, each analyte is repeatedly captured, held and released at the adapter site, producing long trains of current fluctuations rather than a single fleeting blip. Amino acids produce residence events whose depths and durations reflect their side chains; nucleoside monophosphates generate distinct blockade patterns shaped by their base and phosphate; saccharides, bound through their diols, yield slow, stuttering signals; and peptides produce composite signatures reflecting both their terminal amines and their residue composition. Crucially, all four classes can be present in the same solution and still be told apart, because their event statistics cluster in separate regions of feature space.

Distinguishing those clusters is where machine learning enters. The researchers extracted features from thousands of individual events—mean and variance of the blockade current, dwell times, recurrence rates and higher-order statistics of the fluctuation patterns—and trained classifiers on labelled mixtures. Using a few-shot learning strategy, which requires only a small number of labelled examples per analyte, the system assigned unseen events to the correct analyte with 98.7% accuracy across the four chemical classes. The approach is notable for its data efficiency: instead of demanding enormous training sets, the engineered pore’s physically distinct binding chemistry produces such characteristic signals that a handful of reference measurements per molecule suffices.

The most striking demonstration involves glycopeptides, molecules that marry a peptide backbone to one or more covalently attached glycans. Glycopeptides are central to biology—most secreted and membrane proteins carry them—and their analysis is a major bottleneck in proteomics, typically requiring enzymatic release of the sugars and elaborate liquid chromatography tandem mass spectrometry workflows. Because the FPBA adapter engages the cis-diols of the glycan while the pore constriction senses the peptide, the sensor reads both parts of the molecule in one event. The team showed that the device could determine the compositional makeup of native, unlabelled glycopeptides, distinguishing variants that differ in their sugar content without any prior chemical derivatization.

Experts in bioanalytical chemistry will recognize how much engineering subtlety underlies this apparent simplicity. The site-specific attachment of the adapter required a genetically introduced handle in the MspA protein, and the linker length and chemistry had to be tuned so that analytes of wildly different sizes—from a single amino acid of around one hundred daltons to glycopeptides several times larger—could all access and interact with the boronic acid. The ionic current readout itself is straightforward, but converting raw fluctuation trains into reliable chemical identities demands rigorous control of pH, salt concentration and voltage, conditions the authors establish and characterize in the study.

The broader significance lies in what a practical four-class nanopore sensor could enable. A benchtop or even portable device that identifies metabolites, nucleotides, sugars and peptide fragments from a crude mixture would be transformative for point-of-care diagnostics, metabolic flux studies, quality control in biopharmaceutical manufacturing and the emerging field of glycomics. It also complements the rapid progress in nanopore peptide sequencing, where related engineered pores have recently been used to read stepwise-shortened peptides, and with nanopore single-molecule chemistry more generally, which now extends far beyond its nucleic-acid origins. A universal small-molecule reader, in other words, no longer seems fanciful.

Challenges remain before the technology migrates from research lab to routine use. Real biological samples contain hundreds of analytes, some present at vanishingly low concentrations, and the classifier’s performance on such dense, unbalanced mixtures will need to be demonstrated. Sensor lifetime, throughput and the standardization of pore fabrication will also matter for adoption. But the conceptual hurdle has been cleared: a single chemically versatile protein pore, read by a modest machine-learning model, can simultaneously name molecules from four different chemical worlds. As nanopore sensing matures, the humble ionic-current trace is quietly becoming one of the most information-dense signals in all of analytical science.

Subject of Research: An engineered MspA nanopore sensor that simultaneously identifies amino acids, nucleoside monophosphates, saccharides and peptides, including native glycopeptides, using machine-learning classification.

Article Title: A versatile nanopore identifies four classes of analytes simultaneously

Article References: A versatile nanopore identifies four classes of analytes simultaneously. (2026). Nature Biotechnology. https://doi.org/10.1038/s41587-026-03322-x

Image Credits: AI Generated

DOI: 10.1038/s41587-026-03322-x

Keywords: nanopore sensing, MspA, biosensor, machine learning, amino acids, saccharides, peptides, nucleoside monophosphates, glycopeptides, single-molecule detection, bioanalytical chemistry, chemical biology

The study, led by Mingqian Zhang, Ziyi Li, Lei Liu and Hai-Chen Wu of the Institute of Chemistry at the Chinese Academy of Sciences together with colleagues, reconstitutes the complete catecholamine biosynthetic pathway in a test tube using purified enzymes and their required cofactors. The researchers then coupled this miniature chemical factory to an engineered protein nanopore — a nanoscale pore embedded in a membrane through which an ionic current flows. Whenever a metabolite from the pathway interacts with the pore, it briefly perturbs that current, producing an electrical fingerprint specific to that molecule. By reading these fingerprints as they accumulate, the team could follow the transformation of each intermediate into the next, second by second.

The technical heart of the work lies in how the researchers achieved specificity. Small molecules are notoriously difficult for nanopores to distinguish because they zip through the pore too quickly and too subtly to leave clean, separable signals. The team solved this by integrating two orthogonal molecular recognition modalities within a single nanopore platform. In essence, different chemical strategies were used to make different members of the pathway register at the pore: some metabolites, such as the catecholamines themselves, generate characteristic blockade patterns as they translocate, while the amino acids phenylalanine and tyrosine were detected through a tailored probe chemistry involving a copper-coordinated molecular adaptor and a cucurbituril host that captures the amino acid and presents it to the pore as a distinct complex.

With this dual recognition scheme in place, the researchers demonstrated highly specific detection of all six key players in the pathway: phenylalanine, tyrosine, levodopa, dopamine, noradrenaline and adrenaline. Extended data traces show characteristic current blockades and dwell-time distributions for each analyte at 100 micromolar concentration, recorded in a high-salt buffer at a transmembrane potential of +100 millivolts. Gaussian fits to the blockade histograms and single-exponential fits to the dwell times confirm that each metabolite produces a statistically distinct signature, which is the prerequisite for unambiguous identification in a mixed sample.

Having validated each molecule individually, the team turned to the full cascade. They initiated the enzymatic sequence and sampled the reaction mixture every five minutes, feeding each sample to the nanopore. The resulting single-channel recordings amount to a time-lapse film of neurotransmitter synthesis: phenylalanine signals fade as tyrosine rises, tyrosine gives way to levodopa, and levodopa is converted onward to dopamine, noradrenaline and finally adrenaline. Because the readout is label-free and single-molecule, nothing was added to the reaction to make it visible, and no amplification step could distort the quantitative picture of pathway dynamics.

This time-resolved view matters because the catecholamine pathway is not a simple conveyor belt. It is a branched network whose flux is governed by enzyme kinetics, cofactor availability and regulatory feedback, and disruption at any node is implicated in conditions ranging from Parkinson’s disease and attention-deficit hyperactivity disorder to orthostatic hypotension and stress-related endocrine disorders. Conventional analytical methods — liquid chromatography, mass spectrometry, electrochemistry, ELISAs — can measure metabolite concentrations at chosen endpoints, but they are generally snapshot techniques that require aliquots, labels or lengthy separation steps, making continuous, coupled monitoring of a multi-enzyme cascade cumbersome.

The platform also opened a window on pathology. A striking application involves iodotyrosine dehalogenase 1, or DEHAL1, an enzyme best known for recycling iodide in the thyroid, where it removes iodine from iodinated tyrosine residues. Defects in the DEHAL1 gene cause hypothyroidism, and 3-iodo-L-tyrosine, the substrate of this enzyme, has long been known to inhibit tyrosine hydroxylase, the rate-limiting enzyme that converts tyrosine to levodopa. The researchers used their nanopore assay to examine what happens to the catecholamine cascade when dehalogenase activity is compromised. They found that 6-methylisothiocyanate, a compound that disrupts the dehalogenase, perturbs catecholamine biosynthesis in a way that offers a mechanistic explanation for how DEHAL1 deficiency may compromise neurotransmitter production — potentially linking an inherited thyroid disorder to impaired catecholamine output.

Figure 5 of the study, titled ‘Effect of thyroiditis-like disorders on catecholamine metabolism,’ presents this perturbation experiment, with extended recordings showing how the cascade changes when monoiodotyrosine is present. The single-molecule records reveal shifts in the relative abundance of the pathway intermediates, illustrating the value of the method: rather than inferring a block from diminished end-product levels, researchers can see exactly where flux stalls and which intermediates accumulate. That level of mechanistic detail is precisely what drug developers and enzymologists need when trying to dissect regulatory nodes in metabolic networks.

Broader implications extend well beyond catecholamines. The authors describe their integrated enzymology-and-nanopore-sensing platform as a general framework for studying complex biochemical pathways with single-molecule resolution. Because protein nanopores can be engineered with different recognition elements, the same design philosophy could in principle be applied to other branched metabolic cascades, to drug-metabolism studies, or to screening enzyme inhibitors in real time. The approach builds on two decades of stochastic sensing, in which engineered pores have been taught to identify analytes as diverse as metal ions, nucleotides, amino acids, peptides, proteins and microRNAs, and it extends that lineage from static identification to dynamic pathway monitoring.

The team has deposited its source data publicly on Zenodo, and the paper — received in December 2025, accepted in August 2026 and published on 1 September 2026 — carries no reported competing interests. What the work ultimately delivers is a new kind of laboratory instrument: a single pore through which the chemistry of a neurotransmitter can be watched being born. If the framework generalizes as its authors hope, metabolic biochemists may soon spend far less time taking snapshots of pathways and far more time watching them move.

Subject of Research: Time-resolved single-molecule monitoring of catecholamine biosynthesis from phenylalanine using an engineered nanopore sensor

Article Title: Nanopore-enabled time-resolved monitoring of catecholamine-related phenylalanine metabolism

Article References: Zhang, M., Li, Z., Hao, W., Yi, Y., Zhou, K., Liu, L., & Wu, H.-C. (2026). Nanopore-enabled time-resolved monitoring of catecholamine-related phenylalanine metabolism. Nature Nanotechnology. https://doi.org/10.1038/s41565-026-02273-3

Image Credits: AI Generated

DOI: 10.1038/s41565-026-02273-3

Keywords: nanopore sensing, catecholamines, dopamine, adrenaline, phenylalanine metabolism, single-molecule detection, neurotransmitter biosynthesis, DEHAL1, iodotyrosine dehalogenase, label-free biosensing, Nature Nanotechnology, enzymology