Until 1993, 2D kinetics measurements have not been available experimentally (50). From that on, many experimental assays have been developed by coordinating the biological experiments and mechanical measurements. These include micropipette aspiration (24-29), optical tweezer (30, 31), biological force probe (32-34), atomic force microscopy (AFM) (35-40, 46, 47), flow chamber (42-45, 50), micro-cantilever needle (51), centrifugation (52), rosetting (53-55), cone-plate viscometer (56-58), surface force apparatus (59, 60), and fluorescence recovery after photobleaching (FRAP) (61, 62). Here two assays of micropipette aspiration and atomic force microscopy are reported to demonstrate how the assays work.
In a micropipette aspiration assay, two cells (generally a human red blood cell (RBC) and a nucleated cell respectively expressing or coated with a receptor and the counterpart ligand) are respectively aspirated by two micropipettes with diameters of ~1.5-3 μ m via a suction pressure of 1-4 mmH2O (24-29) (Figure 1a). Adhesion between the RBC and the nucleated cell is staged by placing them onto controlled contact via micromanipulation (Figure 1b). The presence of adhesion and the adhesion force at the end of a given contact period are detected mechanically by observing microscopically the deflection of the flexible RBC membrane upon retracting it away from the nucleated cell (Figures 1c and 1d). This contact-retraction cycle is repeated one hundred times to estimate the adhesion probability, Pa, at that contact duration, t. ~100 pairs of cells are used to obtain several Pa vs t curves that correspond to different receptor and ligand densities, mr and ml. Eq. 3 is used to estimate the zero-force reverse rate, r 0, and effective binding affinity, AcmlKa 0 (if mr was known) or AcKa 0 (if both mr and ml were known).
In an atomic force microscopy assay, a receptor or ligand is coated directly or captured via capturing monoclonal antibody (mAb) onto AFM cantilever tip. Purified counterpart ligands or receptors are incorporated in lipid vesicles and then reconstituted by vesicle fusion in a polyethylenimine (PEI) polymer-supported lipid bilayer onto mica or glass surface before use (Figure 2b) (38, 41, 46, 47). The ligand-or receptor-reconstituted lipid bilayer is placed on the AFM stage, which is repeatedly driven to approach the receptor- or ligand-coated cantilever tip, to make contact to allow reversible bond formation and dissociation, and to retract away to allow observation of the adhesion event and measurement of lifetime or rupture force, if any (Figure 2c). The adhesion and lifetime or force signals for each approach-contact-retract cycle are collected from a quad photodetector (Figure 2a). Different locations on each lipid bilayer are tested for 150-400 cycles at each location to collect a set of adhesion events and lifetimes or rupture forces, and all experiments are repeated using a set of different lipid bilayers. Measured Pa vs t data is fitted to the model (Eq. 3) to obtain the kinetic parameters (kr 0 and AcmrmlKa 0) (41). Bond lifetime data, vs f, are measured to test the forced-dissociation hypotheses (Eq. 4) (46, 47), and bond rupture forces are measured at given force loading rates (38, 41).
It should be pointed out that slight differences might exist in determining quantitatively kinetic parameters, bond rupture force, and bond lifetime when different assays are used for same molecular system. This should not be surprising since experimental conditions are hardly to be kept identical from one assay to another. Nevertheless, these assays provide the new insight into quantifying the binding kinetics and force dependence of dissociation of receptor-ligand interactions.