The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm per read.

Gold nanoparticles (AuNPs) have attracted increasing attention as catalysts for pollutant degradation because of their unique reactivity. Direct use of gold nanoparticles in water treatment faces prohibitive challenges from nanoparticle aggregation and post-treatment separation. To prevent nanoparticles from aggregating and eliminate the need for separation, we affixed AuNPs on hierarchical carbon nanotube membrane (HCNM) that was approximately 50 μm thin with 10 μm × 10 μm openings as pores for water passage. HCNM was fabricated by growing vertically aligned carbon nanotube (CNT) arrays on stainless steel mesh. Using p-nitrophenol (PNP) as model pollutant, we showed that in batch experiments HCNM-supported AuNPs retained 78% of their catalytic capability compared to suspended AuNPs. The slight reduction in reactivity was attributed to the blockage of part of the gold surface at the AuNP−CNT juncture. When the membrane was used in continuous flow-through operation, HCNM-supported AuNPs achieved 71% of the maximum catalytic ability measured in batch. The rapid kinetics obtained with HCNM-supported AuNPs was in great contrast to the slow kinetics that one would expect for a rigid membrane of similar configuration. For a rigid membrane, water passing through microscopic pores was confined as laminar flow and thus would not mix well with catalysts affixed on pore walls. For HCNM, CNTs aligning pore walls were flexible so that they could move vigorously to create a chaotic mixing condition and promote AuNP-catalyzed PNP reduction.

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Recent advances in top-down mass spectrometry enabled identification of intact proteins, but this technology still faces challenges. For example, top-down mass spectrometry suffers from a lack of sensitivity since the ion counts for a single fragmentation event are often low. In contrast, nanopore technology is exquisitely sensitive to single intact molecules, but it has only been successfully applied to DNA sequencing, so far. Here, we explore the potential of sub-nanopores for single-molecule protein identification (SMPI) and describe an algorithm for identification of the electrical current blockade signal (nanospectrum) resulting from the translocation of a denaturated, linearly charged protein through a sub-nanopore. The analysis of identification p-values suggests that the current technology is already sufficient for matching nanospectra against small protein databases, e.g., protein identification in bacterial proteomes.

The size of an ion affects everything from the structure of water to life itself. In this report, to gauge their size, ions dissolved in water are forced electrically through a sub-nanometer-diameter pore spanning a thin membrane and the current is measured. The measurements reveal an ion-selective conductance that vanishes in pores <0.24 nm in diameter—the size of a water molecule—indicating that permeating ions have a grossly distorted hydration shell. Analysis of the current noise power spectral density exposes a threshold, below which the noise is independent of current, and beyond which it increases quadratically. This dependence proves that the spectral density, which is uncorrelated below threshold, becomes correlated above it. The onset of correlations for Li + , Mg 2+ , Na + and K + -ions extrapolates to pore diameters of 0.13 ± 0.11 nm, 0.16 ± 0.11 nm, 0.22 ± 0.11 nm and 0.25 ± 0.11 nm, respectively—consonant with diameters at which the conductance vanishes and consistent with ions moving through the sub-nanopore with distorted hydration shells in a correlated way.

The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore informs with extreme sensitivity on the sequence of amino acids that constitute the protein. The current blockade signals measured during the translocation are called a nanospectrum of the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS identifies proteins by database search but often fails to provide high protein sequence coverage. It is also not very sensitive requiring about a femtomole for protein identification. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting nanospectra. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting theoretical nanospectra from protein sequences, which are essential for protein identification.

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.