Xiaoyu Du

Formaldehyde (HCHO) is a reactive electrophile that covalently modifies diverse amino acid residues in proteins. Although ensemble studies have characterized reactions between HCHO and amino acids, the reaction dynamics at individual residue sites have yet to be resolved at the single-molecule level. Here, we engineer a Mycobacterium smegmatis porin A (MspA) nanopore by positioning a single lysine residue at its constriction, enabling real-time single-molecule monitoring of HCHO–lysine chemistry. Distinct current blockade states and reversible transitions between them are observed, consistent with multistep HCHO–lysine reactions involving monoadducts, bisadducts, and Schiff bases under nanoconfinement. The reactions exhibited concentration- and pH-dependent kinetics and generated characteristic electrical signatures that differentiate HCHO from potential interferents, supporting label-free detection of airborne HCHO via aqueous trapping, as well as identification of HCHO in food matrices. This work establishes a residue-confined nanopore platform for real-time probing of HCHO–amino acid chemistry and selective detection of HCHO.

Many cancers are characterized by the coordinated dysregulation of multiple small noncoding RNAs (sncRNAs), yet fluorescence-based assays such as quantitative reverse transcription PCR (qRT-PCR) fundamentally limit multiplexing. Here, we report an amplification-encoded nanopore sensing strategy in which sncRNA identity is written during RT-PCR amplification and decoded by single-molecule nanopore sensing without chemical labeling or optical detection. Target sncRNAs are converted into stem-loop amplicons whose electrical signatures are modulated by the nucleotides at the 3' terminus of sncRNA, allowing for the encoding of many distinct sncRNA identities and enabling single-nucleotide discrimination within the highly homologous Let-7 family. As a demonstration, we concurrently analyzed gastric cancer-associated microRNAs and tRNA-derived small RNAs, achieving classification accuracies exceeding 97% with machine-learning-assisted decoding. This method also enables absolute quantification of multiple sncRNAs simultaneously through capture-efficiency correction and an internal reference sequence. This amplification-encoded nanopore strategy provides a scalable framework for multiplex single-molecule analysis of diverse sncRNAs and highlights its potential for liquid biopsy applications.

Biogenic amines (BAs) are nitrogenous biomolecules with essential physiological functions. However, excessive levels of BAs in food matrices can lead to adverse health effects. Conventional analytical techniques such as chromatography and mass spectrometry, are often limited by solvent dependency and derivatization requirements. Nanopore, an emerging single-molecule detection platform, was used to overcome these limitations. In this work, a single nickel ion-modified nanopore enabled the detection of aliphatic, aromatic, and heterocyclic BAs. Six BAs, including putrescine, cadaverine, β-phenylethylamine, tyramine, tryptamine and histamine were distinctly identified, with mechanistic insights elucidated via detailed event characterization. Furthermore, a machine learning classifier was developed for automated biogenic amine identification, achieving an accuracy of 99.0%. The strategy was also applied to detect BAs in real food samples, where five types of biogenic amines were successfully identified in shrimp, highlighting its significant potential for applications in food freshness monitoring and safety assurance.

As global pollution continues to escalate, timely and accurate monitoring is essential for guiding pollution governance and safeguarding public health. The increasing diversity of pollutants across environmental matrices poses a significant challenge for instrumental analysis methods, which often require labor-intensive and time-consuming sample pretreatment. Nanopore technology, an emerging single-molecule technique, presents a promising solution by enabling the rapid identification of multiple targets within complex mixtures with minimal sample preparation. A wide range of pollutants have been characterized using natural biological nanopores or artificial solid-state nanopores, and their distinct advantages include simple sample preparation, high sensitivity, and rapid onsite analysis. In particular, long-read nanopore sequencing has led to dramatic improvements in the analyses of environmental microbial communities, allows species-level taxonomic assignment using amplicon sequencing, and simplifies the assembly of metagenomes. In this Perspective, we review the latest advancements in analyzing chemical and biological pollutants through nanopore sensing and sequencing techniques. We also explore the challenges that remain in this rapidly evolving field and provide an outlook on the potential for nanopore environmental analysis to transform pollution monitoring, risk assessment, and public health protection.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Isomers of some chemical compounds may be dynamically interconvertible. Due to a lack of sensing methods with a sufficient resolution, however, direct monitoring of such processes can be difficult. Engineered Mycobacterium smegmatis porin A (MspA) nanopores can be applied as nanoreactors so that chemical reactions can be directly monitored. Here, an MspA modified with a phenylboronic acid (PBA) adapter was prepared and was used to observe dynamic interconversion between chiral configurations of boronate esters, which appears as telegraphic switching on top of nanopore events. The mechanism of this behavior was further confirmed by trials with different halogenated catechols, dopamine, adenosine, 1,2-propanediol, and (2R,3R)-2,3-butanediol, and its generality has been demonstrated. These results suggest that an engineered MspA possesses an exceptional resolution in its monitoring of chemical reaction processes and may inspire the future design of nanopore small-molecule sensors.

Ribonucleotides, which widely exist in all living organisms and are essential to both physiological and pathological processes, can naturally appear as ribonucleoside mono-, di-, and triphosphates. Natural ribonucleotides can also dynamically switch between different phosphorylated forms, posing a great challenge for sensing. A specially engineered nanopore sensor is promising for full discrimination of all canonical ribonucleoside mono-, di-, and triphosphates. However, such a demonstration has never been reported, due to the lack of a suitable nanopore sensor that has a sufficient resolution. In this work, we utilized a phenylboronic acid (PBA) modified Mycobacterium smegmatis porin A (MspA) hetero-octamer for ribonucleotide sensing. Twelve types of ribonucleotides, including mono-, di-, and triphosphates of cytidine (CMP, CDP, CTP), uridine (UMP, UDP, UTP), adenosine (AMP, ADP, ATP), and guanosine (GMP, GDP, GTP) were simultaneously discriminated. A machine-learning algorithm was also developed, which achieved a general accuracy of 99.9% for ribonucleotide sensing. This strategy was also further applied to identify ribonucleotide components in ATP tablets and injections. This sensing strategy provides a direct, accurate, easy, and rapid solution to characterize ribonucleotide components in different phosphorylated forms.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and β-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Blockers of pore-forming toxins (PFTs) limit bacterial virulence by blocking relevant channel proteins. However, screening of desired blockers from a large pool of candidate molecules is not a trivial task. Acknowledging its advantages of low cost, high throughput, and multiplicity, DiffusiOptoPhysiology (DOP), an emerging nanopore technique that visually monitors the states of individual channel proteins without using any electrodes, has shown its potential use in the screening of channel blockers. By taking different α-hemolysin (α-HL) mutants as model PFTs and different cyclodextrins as model blockers, we report direct screening of pore blockers solely by using fluorescence microscopy. Different combinations of pores and blockers were simultaneously evaluated on the same DOP chip and a single-molecule resolution is directly achieved. The entire chip is composed of low-cost and biocompatible materials, which is fully disposable after each use. Though only demonstrated with cyclodextrin derivatives and α-HL mutants, this proof of concept has also suggested its generality to investigate other pore-forming proteins.