Researchers report on the development of a solid-state nanopore sensing platform they claim demonstrates far greater sensitivity than existing nanopore technologies for the label-free detection of single proteins. The new technology represents a generic platform that allows high-affinity receptors to just about any target protein to be anchored within the pore. It also minimizes nonspecific binding of proteins to the pore surface and enables receptor-ligand binding affinity to be fine-tuned to aid throughput.
Described in Nature Nanotechnology, the nanopore sensing platform has been developed by a team at the Walter Schottky Institute & Institute for Advanced Study at the Technische Universität München (TUM) in collaboration with biochemists at Goethe-University Frankfurt. The TUM researchers have pioneered the development of single-molecule nanopore sensors over many years. In their published paper, the team describes use of the new platform to detect His-tagged proteins and also to discriminate between subclasses of hamster and rat IgG antibodies. Previously, the authors state, achieving the level of selectivity displayed by their platform would have come at the expense of single-molecule sensitivity. The paper is titled “Stochastic sensing of proteins with receptor-modified solid-state nanopores.”
Nanopore sensors effectively detect molecules passing one at a time through nanoscale pores in a membrane by measuring changes in conductivity. Stochastic nanopore sensors in which specific receptors are immobilized inside a nanopore can probe the chemical nature of analytes by recording for how long the individual molecules bind to the receptor. However, while sensors based on the naturally occurring α-haemolysin nanopore have been shown to discriminate metal ions, small molecules and oligonucleotides, biological nanopores are too small to study natively folded proteins and other large molecules. Moreover, the pores must be genetically engineered to incorporate the receptors.
In contrast, solid-state nanopores can be produced with pore diameters big enough to allow the passage of proteins and other large biomolecules. Biochemical selectivity can be built in by anchoring a molecular receptor in the pore. However, the researchers note, with these platforms it's difficult to maintain single-molecule sensitivity because inability to accurately position a single receptor in the nanopore means reversible binding of ligand to receptor can’t be controlled.
The TUM’s platform aims to address these issues. The sensor is based on a silicon nitride membrane in which conical tapered pores are generated using electron beam lithography and reactive ion etching. The pore-containing membrane is then coated in a thin layer of gold, which effectively reduces the pore size down to about 25 nm. Then the nanopores are chemically modified by covering the gold layer within each pore with a monolayer of alkane-thiols (SAM), further reducing the pore size.
Critically, by mixing the desired receptor head groups into the SAM matrix, the pore adopts the chemical properties of the receptors, the researchers explain. The receptors thus act as the selective binders for target molecules, while the SAM layer prevents unspecific binding of protein to the pore itself. In fact just about any functionality can be built in to the pore, depending on the receptor chosen.
The team first confirmed the utility of their basic concept by generating a sensor in which the pores contained anchored nitrilotriacetic acid (NTA) receptors to act as binding sites for His-tagged proteins. Tests confirmed that the sensor selectively detected His6-tagged A/G/L protein, which is a recombinant fusion protein consisting of IgG binding domains. In essence, the proteins are detected as a transient reduction in the trans-pore ion current as they pass through the pore one by one and bind with the receptor. The duration of this dip in current (the ‘off’ time) is a measure of how long each protein remains bound to a receptor. As would be expected, adding a competitive binder to interfere with NTA-His6 interaction within the pore led to a decrease in off time.
The team then combined data on binding kinetics with results from assays to define receptor multivalence and evaluate the strength of receptor-ligand binding to device a generic scheme for constructing highly selective and sensitive protein-protein interaction nanopores. In this optimized system the SAM monolayer is embedded with trisNTA headgroups, to which the receptor proteins (or primary proteins) are attached.
To demonstrate the utility of the platform the researchers modified the gold-layered nanopores with SAM layer containing trisNTA headgroups linked to His-tagged primary proteins to act as the receptors for specific subclasses of IgG antibodies. Studies confirmed that this platform demonstrated the sensitivity and selectivity to distinguish between different subclasses of IgG in both rat and hamster antibody samples.
The researchers claim that while most recent nanopore research has focused on DNA detection and sequencing, their platform will represent a valuable new tool for protein research, and even pave the way to the development of protein nanopore sensors for diagnosing protein biomarkers in clinical samples.
One conceptual problem with the general approach relates to how long each individual ligand might remain bound to its receptor, they admit. Because the molecules pass through the pores one at a time, high-affinity interactions could mean the ligands stay bound to their receptors for perhaps tens of seconds, in which case it could take hours to process adequate numbers of molecules in a single sample. However, the team stresses, even this can be overcome by adjusting the pH, salinity, or concentration of competitive binders, to weaken the ligand-receptor interaction. In fact, for the IgG fingerprinting assays reported, the solution pH was reduced to 6.2 to facilitate unbinding of the IgG molecule from the receptor.
And importantly, the investigators add, the fabrication and chemical modification methods applied to produce the sensors are well-established and easily available. In addition, the use of trisNTA anchors means that the platform can easily be adapted to examine a wide range of protein-protein or protein-nucleic acid interactions. “A single-molecule sandwich assay can readily be implemented with any of the thousands of available recombinant His-tagged proteins, without the need for adapting the coupling method,” they write. “With this approach, proteins can be distinguished based on biochemical selectivity, and their abundance in a mixed analyte can be quantified from a single-molecule dwell time analysis.”