The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.

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This paper presents a theoretical and computational investigation of convection, diffusion, and adsorption in surface-based biosensors. In particular, we study the transport dynamics in a model geometry of a surface plasmon resonance (SPR) sensor. The work, however, is equally relevant for other microfluidic surface-based biosensors, operating under flow conditions. A widely adopted approximate quasi-steady theory to capture convective and diffusive mass transport is reviewed, and an analytical solution is presented. An expression of the Damkoḧler number is derived in terms of the nondimensional adsorption coefficient (Biot number), the nondimensional flow rate (Pećlet number), and the model geometry. Transient dynamics is investigated, and we quantify the error of using the quasi-steady-state assumption for experimental data fitting in both kinetically limited and convection-diffusionlimited regimes for irreversible adsorption, in specific. The results clarify the conditions under which the quasi-steady theory is reliable or not. In extension to the well-known fact that the range of validity is altered under convection-diffusion-limited conditions, we show how also the ratio of the inlet concentration to the maximum surface capacity is critical for reliable use of the quasi-steady theory. Finally, our results provide users of surface-based biosensors with a tool for correcting experimentally obtained adsorption rate constants.

Phospholipid hydrolysis to free fatty acid and 1-lyso-phospholipid by water-soluble phospholipase A2 (PLA2) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis. In the present study we apply for the first time cryo-transmission electron microscopy (cryo-TEM) and high-performance liquid chromatography (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis. Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become perforated and degrade into open vesicles, bilayer fragments, and micelles. This structural instability extends throughout the system in the activity burst regime. In agreement with earlier reported correlations between initial phospholipase activity and substrate morphology, our results suggest that the lag-burst phenomenon reflects a cascade process. The PLA2-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA2 activity and the morphology of the lipid suspension.