Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

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Label-free biosensors enable the monitoring of biomolecular interactions in real-time, which is key to the analysis of the binding characteristics of biomolecules. While refractometric optical biosensors such as SPR [Surface Plasmon Resonance] are sensitive and well-established, they are susceptible to any change of the refractive index in the sensing volume caused by minute variations in composition of the sample buffer, temperature drifts and most importantly nonspecific binding to the sensor surface. Refractometric biosensors require reference channels as well as temperature stabilisation and their applicability in complex fluids such as blood is limited by nonspecific bindings. Focal molography does not measure the refractive index of the entire sensing volume but detects the diffracted light from a coherent assembly of analyte molecules. Thus, it does not suffer from the limitations of refractometric sensors since they stem from non-coherent processes and therefore do not add to the coherent molographic signal. The coherent assembly is generated by selective binding of the analyte molecules to a synthetic binding pattern -the mologram. Focal Molography has been introduced theoretically [C. Fattinger, Phys. Rev. X 4, 031024 (2014)] and verified experimentally Nat. Nanotechnol. 12, 1089 (2017)] in previous papers. However, further understanding of the underlying physics and a diffraction-limited readout is needed to unveil its full potential. This paper introduces refined theoretical models which can accurately quantify the amount of biological matter bound to the mologram from the diffracted intensity. In addition, it presents measurements of diffraction-limited molographic foci i.e. Airy discs. These improvements enabled us to demonstrate a resolution in real-time binding experiments comparable to the best SPR sensors, without the need of temperature stabilisation or drift correction and to detect low molecular weight compounds labelfree in an endpoint format. The presented experiments exemplify the robustness and sensitivity of the diffractometric sensor principle.

Focal molography is a next-generation biosensor that visualizes specific biomolecular interactions in real time. It transduces affinity modulation on the sensor surface into refractive index modulation caused by target molecules that are bound to a precisely assembled nanopattern of molecular recognition sites, termed the 'mologram'. The mologram is designed so that laser light is scattered at specifically bound molecules, generating a strong signal in the focus of the mologram via constructive interference, while scattering at nonspecifically bound molecules does not contribute to the effect. We present the realization of molograms on a chip by submicrometre near-field reactive immersion lithography on a light-sensitive monolithic graft copolymer layer. We demonstrate the selective and sensitive detection of biomolecules, which bind to the recognition sites of the mologram in various complex biological samples. This allows the label-free analysis of non-covalent interactions in complex biological samples, without a need for extensive sample preparation, and enables novel time- and cost-saving ways of performing and developing immunoassays for diagnostic tests.