The Princess and the Pea: A Story of Cell Mechanics

1785-Plat Surface-Free Single-Molecule Force Spectroscopy Yan Jiang1,2, Wesley Wong1,2. PCMM, Boston Children’s Hospital, Boston, MA, USA, Bcmp, Harvard Medical School, Boston, MA, USA. Mechanical forces play key roles throughout biology. Most existing methods for probing forces in/between molecules and cells, such as the AFM, optical tweezers, magnetic tweezers, traction force microscopy, etc., require molecules to be tethered to the surfaces of cover glasses or beads. However, surface tethering has a number of drawbacks. First, the conjugation chemistry for tethering can be challenging. Second, the spatial confinement and the nonspecific attraction from the surface can induce artifacts to the dynamics of the molecule. For semi-rigid objects, such as actin filaments, surface attachment could also induce bending and twisting of the filament, complicating the applied force profile. To overcome these issues, we present a surface-free force spectroscopy method that uses a high-speed cross-slot hydrodynamic trap, capable of stretching molecules and cells with hydrodynamic drag. The trap is based on a glass microfluidic cross-slot flow chamber. Buffer flows in from two opposite directions and exits via the two orthogonal outlets to create an elongational flow field with a stagnation point in the center. As a result, objects near the stagnation point are stretched by the viscous drag from the flow. In addition, the pressure in one of the outlet reservoirs is electronically controlled with a high-speed feedback algorithm to stabilize the object at the stagnation point. Thanks to the high-speed feedback, we can apply much higher flow rate and therefore much higher stretching force on the trapped object than with the cross slot alone. We demonstrate tension-dependent actin severing, extension of vonWillebrand Factor (a key protein in haemostasis that changes conformation in response to hydrodynamic stress in blood stream), overstretching of double strand DNA, and stretching deformation of red blood cells. In summary, the high-speed cross-slot hydrodynamic trap can be a powerful, surface-free alternative to more commonly used force spectroscopy methods.