Modified Pluronic F127 Surface for Bioconjugation and Blocking Nonspecific Adsorption of Microspheres and Biomacromolecules.

Many experiments and applications require the chemical coupling of target molecules to surfaces, during which the elimination of nonspecific interactions presents a difficult challenge. We report on a technologically accessible surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer (F127-NHS). To quantify interactions between the F127-NHS surface and magnetic microspheres, we developed a simple assay: the microsphere adhesion by gravity, inversion, then counting, or "MAGIC" assay. To improve blocking of microspheres while maintaining the ability to chemically couple additional molecules, we implemented a pH-dependent two-step chemical modification process for amine microspheres. This process achieves an extremely high level of blocking nonspecific interactions (less than 2% nonspecific adhesion) for a variety of microsphere surface charges and chemical functionalities. We also demonstrate the ability to specifically tether magnetic microspheres to an F127-NHS surface, using single DNA molecules. Using the DNA microspheres, we establish the applicability of the surface for force spectroscopy (stable with an applied load >30 pN) via the massively parallel technique of centrifuge force microscopy. Finally, we demonstrate that the surface can be used in fluorescence studies with a fluorogenic peptide cleavage assay, with high levels of blocking achieved for both the fluorogenic peptide and trypsin. These results suggest applications including, but not limited to, single-molecule force spectroscopy and fluorescence, biosensors, medical implants, and anti-biofouling, which could make use of the F127-NHS surface.

Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization.

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.