Single Molecule Reactions - Nanopore Technology

New research suggests that nanopore-based techniques could provide unprecedented insights into chemical bond formation and breakage at the single-molecule level. By transforming protein nanopores into tiny reactors, scientists can observe transient intermediates in real time—details often obscured in traditional bulk measurements. This approach holds encouraging potential for advancing fields like biomarker detection and drug development, though further refinements are needed to broaden its applications.

The Challenge of Observing Chemical Bonds Up Close

Fundamental chemistry revolves around the making and breaking of bonds, which dictate a molecule's properties and behavior. However, capturing these processes in individual molecules has long been difficult. Ensemble methods, which study large groups of molecules, average out signals and miss fleeting intermediates. As Yi-Lun Ying and Yi-Tao Long, lead authors of the review, explain, "Real-time observation of chemical bonding at the single-molecule level offers insights into transient intermediates that are normally inaccessible via ensemble measurements."

Traditional tools like optical or force spectroscopy often require labels, which can introduce ambiguities. Biological nanopores—tiny protein channels inspired by nature—offer a label-free alternative. These pores, with diameters on the scale of a few nanometers (comparable to the width of a DNA strand), confine one molecule at a time, allowing precise monitoring through changes in ionic current.

Nanopores as Miniature Reactors: The Core Methodology

Nanopore techniques work by embedding a protein channel, such as alpha-hemolysin (α-HL) or aerolysin (AeL), into a lipid bilayer membrane. An electric field drives a single reactant molecule into the pore, where it interacts with engineered reactive sites on the inner wall. This interaction alters the flow of ions, producing measurable current changes that reveal bond dynamics.

Think of the nanopore as a narrow tunnel: the molecule enters under electrophoretic force (like a charged particle pulled by an electric field) or electroosmotic flow (solvent movement driven by the pore's charged surface). As the molecule collides with the reactive site, bonds form or break, generating distinct current signatures. "The successive entry of each reactant ensures the continuous recording of individual single-molecule reactions," note Ying and colleagues.

Kinetics are analyzed using models that account for confinement effects. For instance, the rate of bond formation (k_on) and breakage (k_off) can be derived from event durations and frequencies, providing a pseudo-first-order description. Results indicate that nanopore kinetics often align with bulk systems but can differ due to spatial constraints, which orient molecules and influence collision efficiency.

Engineering Nanopores for Precision and Efficiency

Designing effective nanopore reactors involves strategic modifications. Reactive sites are introduced via site-directed mutagenesis, using natural amino acids like cysteine (with its thiol group) or unnatural ones incorporated through genetic code expansion. For example, selenocysteine can be added to α-HL to monitor hydroperoxide reduction by glutathione.

To enhance molecule capture, engineers tweak the pore's charge distribution. Mutating residues in AeL to increase positive charges doubles electroosmotic flow, aiding the entry of neutral or variably charged peptides. Wider pores like cytolysin A (ClyA) accommodate larger enzymes, such as dihydrofolate reductase, revealing conformational changes during catalysis that are hidden in averages.

Strengthening collisions extends residence times, boosting reaction probabilities. Replacing a positively charged lysine with cysteine in AeL creates an electrostatic barrier, prolonging interactions and accelerating disulfide bond formation—rates up to eight orders of magnitude faster than in bulk solution. "By adopting an advanced biological engineering approach, desired reactive groups or ligands can be introduced into the nanopore," the authors highlight, emphasizing chemo- and site-selectivity.

Revealing Hidden Intermediates and Pathways

Nanopores excel at identifying short-lived states, with temporal resolution down to microseconds. Early studies monitored cysteine modifications in diphtheria toxin channels; more recent work tracks organoarsenic-thiol interactions or polymerization steps through stepwise current blockages.

In one example, α-HL nanopores detected no long-lived intermediates in tetrazine cycloadditions, aligning with computational predictions of femtosecond lifetimes. Another application trapped light-induced nitronate intermediates, quantifying metastable states. These capabilities extend to health-relevant reactions, such as probing protein interactions or detecting biomarkers at ultrasensitive levels.

The review summarizes progress in sequencing biopolymers, proteomics, and pollutant detection, underscoring nanopores' dual role as reactors and sensors.

Future Applications and Cautions

Encouraging results point to nanopores' role in elucidating complex kinetics, with implications for personalized diagnostics—such as early disease detection through single-molecule biomarkers—or designing targeted therapies; this could mean faster, more precise tools for monitoring molecular changes.

Yet, as the authors caution, challenges remain: optimizing capture for diverse molecules, improving signal-to-noise ratios, and scaling for practical use.

"This Review summarizes the state of the art... which offer great potential but also present many challenges that remain to be solved."