Nanoscale biosensors provide the possibility to miniaturize optic, acoustic and electric sensors to the dimensions of biomolecules. This enables approaching single-molecule detection and new sensing modalities that probe molecular conformation. Nanoscale sensors are predominantly surface-based and label-free to exploit inherent advantages of physical phenomena allowing high sensitivity without distortive labeling. There are three main criteria to be optimized in the design of surface-based and label-free biosensors: (i) the biomolecules of interest must bind with high affinity and selectively to the sensitive area; (ii) the biomolecules must be efficiently transported from the bulk solution to the sensor; and (iii) the transducer concept must be sufficiently sensitive to detect low coverage of captured biomolecules within reasonable time scales. The majority of literature on nanoscale biosensors deals with the third criterion while implicitly assuming that solutions developed for macroscale biosensors to the first two, equally important, criteria are applicable also to nanoscale sensors. We focus on providing an introduction to and perspectives on the advanced concepts for surface functionalization of biosensors with nanosized sensor elements that have been developed over the past decades (criterion (iii)). We review in detail how patterning of molecular films designed to control interactions of biomolecules with nanoscale biosensor surfaces creates new possibilities as well as new challenges.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.