No abstract

Putative riboflavin receptors are considered as biomarkers due to their overexpression in breast and prostate cancers. Hence, these receptors can be potentially exploited for use in targeted drug delivery systems where dendrimer nanoparticles with multivalent ligand attachments can lead to greater specificity in cellular interactions. In this study, the single molecule force spectroscopy technique was used to assess the physical strength of multivalent interactions by employing a riboflavin (RF)-conjugated generation 5 PAMAM dendrimer G5(RF)n nanoparticle. By varying the average RF ligand valency (n = 0, 3, 5), the rupture force was measured between G5(RF)n and the riboflavin binding protein (RFBP). The rupture force increased when the valency of RF increased. We observed at the higher valency (n = 5) three binding events that increased in rupture force with increasing loading rate. Assuming a single energy barrier, the Bell-Evans model was used to determine the kinetic off-rate and barrier width for all binding interactions. The analysis of our results appears to indicate that multivalent interactions are resulting in changes to rupture force and kinetic off-rates.

be capable of sensing the interactions. Field-effect transistors (FET) have been widely used as biosensors, but are generally used with a large number of molecules to obtain a sufficient signal. The measurements taken with the FET-based biosensors are mostly ''on'' or ''off'' measurements, which are determined by the presence or absence of a reaction. A specific type of FET, the p-type metal oxide semiconductor FET (pMOSFET) appears to be a promising biosensor device. The pMOSFET contains holes in the channel, also known as the inversion layer, opposite in carrier type to the substrate. An atomic force microscope (AFM) has shown the ability to measure molecular interactions down to the single molecular level and to control the distance between a ligand and a receptor protein up to subatomic resolution. The integration of AFM and FET technologies has the potential to provide not only valuable information about these biomolecular interactions that has not been accomplished by other methods, but also a much more rapid drug screening technique. The ability to control single molecules will allow for the comprehensive study of biomolecular interactions at the singlemolecular level. This presentation will show how the AFM and FET were integrated into one functioning biosensor. The efficiency at detecting single molecular binding and unbinding events will be demonstrated by probing the interactions between avidin-biotin complexes. There are strong indications that mechanical forces are particularly relevant in immune recognition. For the immune system, the enormous variety of antigenic ligands imposes a fundamental challenge to the discriminative power; the mechanism for discriminating between activating and non-activating ligands has remained enigmatic. In a recent theoretical study we showed how forces alters the potency for receptor ligand discrimination by orders of magnitudes(1). For the T cell receptor, which specifically binds to peptides presented by MHC on an antigenpresenting cell, discrimination can be realized with kinetic proofreading, which fails when ligands have only marginal differences in their off-rates. We showed, however, that the specificity of antigen-recognition can be massively improved by putting the TCR-pMHC bond under load: while under no force the bond rupture probability decays exponentially with time, force-induced bond rupture leads to much narrower distributions. Here, we present cellular traction force microscopydata to measure forces involved during T cell activation. Hydrogels were prepared with variable stiffness. Fluorescent beads carrying CD3 antibodies were immobilized onto the top layer of the hydrogel. Forces are read out by measuring the fluorescent bead movement throughout T cell attachment and activation. The bead movement was directly correlated to forces applied to the antibodies immobilized on the beads. Moreover, discrimination between lateral and transversal applied forces was possible by tracking the beads' positions in 3D.