Polysaccharides have key biological functions and can be harnessed for therapeutic roles, such as the anticoagulant heparin. Their complexity—e.g., >100 monosaccharides with variety in linkage and branching structure—significantly complicates analysis compared to other biopolymers such as DNA and proteins. More, and improved, analysis tools have been called for, and here we demonstrate that solid-state silicon nitride nanopore sensors and tuned sensing conditions can be used to reliably detect native polysaccharides and enzymatic digestion products, differentiate between different polysaccharides in straightforward assays, provide new experimental insights into nanopore electrokinetics, and uncover polysaccharide properties. We show that nanopore sensing allows us to easily differentiate between a clinical heparin sample and one spiked with the contaminant that caused deaths in 2008 when its presence went undetected by conventional assays. The work reported here lays a foundation to further explore polysaccharide characterization and develop assays using thin-film solid-state nanopore sensors.

In this study, we investigated the voltage and pH responsiveness of human serum transferrin (hSTf) protein using silicon nitride (Si x N y ) nanopores. The Fe(III)-rich holo form of hSTf was dominant when pH > pI, while the Fe(III)-free apo form was dominant when pH < pI. The translocations of hSTf were purely in an electrophoretic sense, thus depended on its pI and the solution pH. With increasing voltage, voltage driven protein unfolding became prominent which was indicated by the trends associated with change in conductance, due to hSTf translocation, and in the excluded electrolyte volume. Additionally, analysis of the translocation events of the pure apo form of hSTf showed a clear difference in the event population compared to that of the holo form. The results obtained demonstrate the successful application of nanopore devices to distinguish between the holo and apo forms of hSTf in a mixture and to analyze its folding and unfolding phenomenon over a range of pH and applied voltages.

Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling—most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope—nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters (3— ∼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (∼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive—sodium hypochlorite—that resulted in dramatically different nanopore surface chemistry including ∼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.

Nanopores are a prominent enabling tool for single-molecule applications such as DNA sequencing, protein profiling, and glycomics, and the construction of ionic circuit elements. Silicon nitride (SiN x ) is a leading scaffold for these <100 nm-diameter nanofluidic ion-conducting channels, but frequently challenging surface chemistry remains an obstacle to their use. We functionalized more than 100 SiN x nanopores with different surface terminationsacidic (Si−R−OH, Si−R−CO 2 H), basic (Si−R−NH 2 ), and nonionizable (Si−R−C 6 H 3 (CF 3 ) 2 )to chemically tune the nanopore size, surface charge polarity, and subsequent chemical reactivity and to change their conductance by changes of solution pH. The initial one-reaction-step covalent chemical film formation was by hydrosilylation and could be followed by straightforward condensation and click reactions. The hydrosilylation reaction step used neat reagents with no special precautions such as guarding against water content. A key feature of the approach was to combine controlled dielectric breakdown (CDB) with hydrosilylation to create and functionalize SiN x nanopores. CDB thus replaced the detrimental but conventionally necessary surface pretreatment with hydrofluoric acid. Proof-of-principle detection of the canonical test molecule, λ-DNA, yielded signals that showed that the functionalized pores were not obstructed by chemical treatments but could translocate the biopolymer. The characteristics were tuned by the surface coating character. This robust and flexible surface coating method, freed by CDB from HF etching, portends the development of nanopores with surface chemistry tuned to match the application, extending even to the creation of biomimetic nanopores.

Silicon nitride is a ubiquitous and well-established nanofabrication material with a host of favourable properties for creating nanofluidic devices with a range of compelling designs that offer extraordinary discovery potential. Nanochannels formed between two thin silicon nitride windows can open up vistas for exploration by freeing transmission electron microscopy to interrogate static structures and structural dynamics in liquid-based samples. Nanopores present a strikingly different architecture—nanofluidic channels through a silicon nitride membrane—and are one of the most promising tools to emerge in biophysics and bioanalysis, offering outstanding capabilities for single molecule sensing. The constrained environments in such nanofluidic devices make surface chemistry a vital design and performance consideration. Silicon nitride has a rich and complex surface chemistry that, while too often formidable, can be tamed with new, robust surface functionalization approaches. We will explore how a simple structural element—a ∼100 nm-thick silicon nitride window—can be used to fabricate devices to wrest unprecedented insights from the nanoscale world. We will detail the intricacies of native silicon nitride surface chemistry, present surface chemical modification routes that leverage the richness of available surface moieties, and examine the effect of engineered chemical surface functionality on nanofluidic device character and performance.

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A method to directly electrolessly plate silicon-rich silicon nitride with thin gold films was developed and characterized. Films with thicknesses <100 nm were grown at 3 and 10 °C between 0.5 and 3 h, with mean grain sizes between ∼20 and 30 nm. The method is compatible with plating free-standing ultrathin silicon nitride membranes, and we successfully plated the interior walls of micropore arrays in 200 nm thick silicon nitride membranes. The method is thus amenable to coating planar, curved, and line-of-sight-obscured silicon nitride surfaces.

This paper describes a method to gauge the stiffness of nanosized liposomes – a nanoscale vesicle – using a custom‐made recapture platform coupled to a solid‐state nanopore sensor. The recapture platform electrically profiles a given liposome vesicle multiple times through automated reversal of the voltage polarity immediately following a translocation instance to re‐translocate the same analyte through the nanopore – provides better statistical insight at the molecular level by analyzing the same particle multiple times compared to conventional nanopore platforms. The capture frequency depends on the applied voltage with lower voltages (i.e., 100 mV) permitting higher recapture instances than at higher voltages (>200 mV) since the probability of particles exiting the nanopore capture radius increases with voltage. The shape deformation was inferred by comparing the normalized relative current blockade (ΔI/I0̂false) at the two voltage polarities to that of a rigid particle, i.e., polystyrene beads. We found that liposomes deform to adopt a prolate shape at higher voltages. This platform can be further applied to investigate the stiffness of other types of soft matters, e.g., virus, exosomes, endosomes, and accelerate the potential studies in pharmaceutics for increasing the drug packing and unpacking mechanism by controlling the stiffness of the drug vesicles.