Principles for Sensitive and Robust Biomolecular Interaction Analysis: The Limits of Detection and Resolution of Diffraction-Limited Focal Molography

Abstract

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.

Critical assessment of relevant methods in the field of biosensors with direct optical detection based on fibers and waveguides using plasmonic, resonance, and interference effects

“…It is claimed that the resolution in real-time binding experiments is comparable with that of the best SPR sensors without the need of temperature stabilization or drift correction. The method even allows the label-free detection of lowmolecular weight compounds in an endpoint format [271].…”

Ultra Stable Molecular Sensors by Submicron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part II. Experimental Demonstration

“…where N is the effective refractive index of the guided mode, n c is the refractive index of the cover medium, θ is the outcoupling angle with respect to the surface normal, m is the diffraction order, λ is the wavelength and Λ is the grating period. Sensitive diffractometric biosensors that are based on outcoupling gratings are relatively new and offer exciting new applications [26,27]. In particular, they enable real-time label-free measurements in complex fluids as well as the label-free observation of signaling cascades within living cells [33,34].…”

“…These sensors detect binding events by a change in the diffraction efficiency of the outcoupling grating due to binding of the molecules in a coherent, i.e., ordered, fashion. Compared to previous implementations of diffractometric biosensors [44,45], the arrangement described in [26,27] has substantially improved stability and performance. This is due to multiple reasons [46].…”

“…This is due to multiple reasons [46]. First, the spatial modulation frequency is considerably higher than previously because the grating period is submicron [26,27,46]. This achieves an effective separation of signal and environmental noise by a high-frequency spatial affinity lock-in [28].…”

“…However, even when stabilized, they experience significant drift, which over the timescale of an experiment limits the resolution to 1-5 µRIU (1-5 pg/mm 2 ) [25]. On the other hand, diffractometric biosensors were demonstrated to be largely unaffected by these extrinsic influences, even in the complete absence of stabilization [26,27]. The reason for this stability lies in the intrinsic architecture of the diffractometric biosensors and in the arrangement of the binding sites in particular.…”