Ultra-Stable Molecular Sensors by Sub-Micron Referencing and Why They Should Be Interrogated by Optical Diffraction—Part I. The Concept of a Spatial Affinity Lock-in Amplifier

Abstract

Label-free optical biosensors, such as surface plasmon resonance, are sensitive and well-established for the characterization of molecular interactions. Yet, these sensors require stabilization and constant conditions even with the use of reference channels. In this paper, we use tools from signal processing to show why these sensors are so cross-sensitive and how to overcome their drawbacks. In particular, we conceptualize the spatial affinity lock-in as a universal design principle for sensitive molecular sensors even in the complete absence of stabilization. The spatial affinity lock-in is analogous to the well-established time-domain lock-in. Instead of a time-domain signal, it modulates the binding signal at a high spatial frequency to separate it from the low spatial frequency environmental noise in Fourier space. In addition, direct sampling of the locked-in sensor’s response in Fourier space enabled by diffraction has advantages over sampling in real space as done by surface plasmon resonance sensors using the distributed reference principle. This paper and part II hint at the potential of spatially locked-in diffractometric biosensors to surpass state-of-the-art temperature-stabilized refractometric biosensors. Even simple, miniaturized and non-stabilized sensors might achieve the performance of bulky lab instruments. This may enable new applications in label-free analysis of molecular binding and point-of-care diagnostics.

Nonspecific Binding—Fundamental Concepts and Consequences for Biosensing Applications

“…(c) Introducing a single reference channel with nonbinding probes does not improve the signal-to-noise situation significantly, but (d) coherently arranged many reference channels or areas can move the signal to the low-noise regions in Fourier space (to 1/Λ where Λ represents the periodicity of the coherent pattern). Figure reproduced with permission from ref Copyright 2021 MDPI.…”

“…However, the smaller feature size comes at the price of a more sophisticated fabrication, surface functionalization, and depending on the implementation, also the readout. This spatial lock-in amplifying concept is a unifying theory that is applicable to all sensors that employ referencing in space …”

Investigating Complex Samples with Molograms of Low-Affinity Binders

“…However, macroscopic references fail to capture micron-scale differences, such as surface composition or temperature, in sensing and referencing channels. Diffractometric biosensors, − in particular focal molography, , provide the ability to address this challenge by employing a molecular diffraction grating, which may serve the double purpose of readout and nanoscale referencing. , However, diffractometric biosensors can also be affected by nonspecific binding if they are designed carelessly as has been pointed out previously . To estimate the fraction of occupied binding sites due to coherently, nonspecifically bound molecules, let us start with the general case of affinity biosensors.…”

Quantitative Diffractometric Biosensing

“…It is important to state already here that a diffractometric biosensor is fundamentally different and more stable than a double referenced refractometric biosensor. This was explained in detail in two previously published papers [9,10].…”