High Density Single-Molecule-Bead Arrays for Parallel Single Molecule Force Spectroscopy

Abstract

The assembly of a highly-parallel force spectroscopy tool requires careful placement of single-molecule targets on the substrate and the deliberate manipulation of a multitude of force probes. Since the probe must approach the target biomolecule for covalent attachment, while avoiding irreversible adhesion to the substrate, the use of the polymer microsphere as force probes to create the tethered bead array poses a problem. Therefore, the interactions between the force probe and the surface must be repulsive at very short distances (< 5 nm) and attractive at long distances. To achieve this balance, the chemistry of the substrate, force probe, and solution must be tailored to control the probe-surface interactions. In addition to an appropriately designed chemistry, it is necessary to control the surface density of the target molecule in order to ensure that only one molecule is interrogated by a single force probe. We used gold-thiol chemistry to control both the substrate’s surface chemistry and the spacing of the studied molecules, through a competitive binding of the thiol-terminated DNA and an inert thiol forming a blocking layer. For our single molecule array, we modeled the forces between the probe and the substrate using DLVO theory and measured their magnitude and direction with colloidal probe microscopy. The practicality of each system was tested using a probe binding assay to evaluate the proportion of the beads remaining adhered to the surface after application of force. We have translated the results specific for our system to general guiding principles for preparation of tethered bead arrays and demonstrated the ability of this system to produce a high yield of active force spectroscopy probes in a microwell substrate. This study outlines the characteristics of the chemistry needed to create such a force spectroscopy array.

Unraveling protein–protein interactions in clathrin assemblies via atomic force spectroscopy

“…While other SMFS measurement systems exist (36-41) , AFM-based force spectroscopy is arguably the most widely adopted. AFM uses a flexible cantilever with a sharp probe (2-20 nm tip radius) that is scanned over a sample surface, in its imaging mode, sensing inter-atomic forces between the probe tip and the sample.…”

Progress toward the application of molecular force spectroscopy to DNA sequencing

“…DNA is attached to the substrate using gold‐thiol chemistry described elsewhere . A mixture of 3′ thiol‐modified DNA was combined in a 200:1 ratio with (11‐Mercaptoundecyl)tetra(ethylene glycol) (MutEG) in pH 7.4 phosphate buffer with 1 M NaCl.…”

“…This approach queries the composition of the DNA strand by mechanically stretching individual molecules to determine the success or failure of the addition of a complement (as in sequencing by synthesis) through differentiation of the physical characteristics of dsDNA and ssDNA. To ensure that such analysis can be practiced on systems of many molecules, we are developing a highly parallel single molecule force spectroscopy platform using magnetic or dielectrophoretic (DEP) tweezers . In this article, we review the principles of the proposed mechanical sequencing of DNA, report on initial experiments indicating its feasibility, and discuss challenges to demonstrate this technology.…”

“…To achieve manipulation of many molecules, one can attach a microscopic force probe (superparamagnetic or dielectric bead) to each DNA strand and apply a force field, via either magnetic or electric field gradient , to pull on these probes, thus generating force versus extension curves for multiple DNA molecules in parallel . A uniform magnetic field gradient can be set up over relatively large areas (0.01–1 mm 2 ).…”

A combined magnetophoresis/dielectrophoresis based microbead array as high-throughput biomolecular tweezers

“…A number of biophysical methods have been developed more recently to interrogate molecular interactions at the micro/nanoscale, including: atomic force spectroscopy 1 , optical 2 and magnetic 3,4 tweezers, micropipette based methods 5,6 , and dielectrophoresis based tweezers [7][8][9] . Th ese methods, while powerful in specifi c applications, have a number of limitations.…”