Biological nanopores are nanoscale sensors employed for high-throughput, low-cost, and long read-length DNA sequencing applications. The analysis and sequencing of proteins, however, is complicated by their folded structure and non-uniform charge. Here we show that an electro-osmotic flow through Fragaceatoxin C (FraC) nanopores can be engineered to allow the entry of polypeptides at a fixed potential regardless of the charge composition of the polypeptide. We further use the nanopore currents to discriminate peptide and protein biomarkers from 25 kDa down to 1.2 kDa including polypeptides differing by one amino acid. On the road to nanopore proteomics, our findings represent a rationale for amino-acid analysis of folded and unfolded polypeptides with nanopores.

Solid-state nanopores promise a scalable platform for single-molecule DNA analysis. Direct, real-time identification of nucleobases in DNA strands is still limited by the sensitivity and the spatial resolution of established ionic sensing strategies. Here, we study a different but promising strategy based on optical spectroscopy. We use an optically engineered elongated nanopore structure, a plasmonic nanoslit, to locally enable single-molecule surface enhanced Raman spectroscopy (SERS). Combining SERS with nanopore fluidics facilitates both the electrokinetic capture of DNA analytes and their local identification through direct Raman spectroscopic fingerprinting of four nucleobases. By studying the stochastic fluctuation process of DNA analytes that are temporarily adsorbed inside the pores, we have observed asynchronous spectroscopic behavior of different nucleobases, both individual and incorporated in DNA strands. These results provide evidences for the single-molecule sensitivity and the sub-nanometer spatial resolution of plasmonic nanoslit SERS.

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76–81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

The ability to confine and to study single molecules has enabled important advances in natural and applied sciences. Recently, we have shown that unlabeled proteins can be confined inside the biological nanopore Cytolysin A (ClyA) and conformational changes monitored by ionic current recordings. However, trapping small proteins remains a challenge. Here, we describe a system where steric, electrostatic, electrophoretic, and electro-osmotic forces are exploited to immobilize a small protein, dihydrofolate reductase (DHFR), inside ClyA. Assisted by electrostatic simulations, we show that the dwell time of DHFR inside ClyA can be increased by orders of magnitude (from milliseconds to seconds) by manipulation of the DHFR charge distribution. Further, we describe a physical model that includes a double energy barrier and the main electrophoretic components for trapping DHFR inside the nanopore. Simultaneous fits to the voltage dependence of the dwell times allowed direct estimates of the cis and trans translocation probabilities, the mean dwell time, and the force exerted by the electro-osmotic flow on the protein (≅9 pN at −50 mV) to be retrieved. The observed binding of NADPH to the trapped DHFR molecules suggested that the engineered proteins remained folded and functional inside ClyA. Contact-free confinement of single proteins inside nanopores can be employed for the manipulation and localized delivery of individual proteins and will have further applications in single-molecule analyte sensing and enzymology studies.