Label-Free, Real-Time Phospholipase-A Isoform Assay

Abstract

Phospholipase-A (PLA) enzymes catalyze the hydrolysis of ester bonds in select glycerophospholipids. Sensors for rapidly measuring the PLA activity in biological samples have relevance in the study of venom compositions and in medical diagnostics for the diagnosis of diseases such as acute pancreatitis. Current PLA sensor technologies are often restricted by the time it takes to prepare an assay, the necessity of using fluorescent labels, or the fact they might require strict pH control of the buffer vehicles used. Here we present a tethered bilayer lipid membrane (tBLM) impedance sensor array for the rapid and real-time detection of PLA, which includes the ability to selectively detect phospholipase-A 2 (PLA 2 ) from phospholipase-A 1 (PLA 1 ) isoforms. Comparing the activity of PLA 1 and PLA 2 in an array of tBLMs composed of ether phospholipids, ester phospholipids or ether-ester phospholipids allows for the rapid and reliable distinction between the isoforms, as measured using swept-frequency electrical impedance spectroscopy. After testing the assay using pure enzymes, we demonstrate the capacity of the sensor to identify specific PLA 2 -type, calcium-dependent activity from the venom of the South American bullet ant, Paraponera clavata, at a concentration of 1 μg/mL. The specificity of the phospholipase activity was corroborated using matrixassisted laser-desorption/ionization time-of-flight mass spectrometry. As further validation, we tested the activities of a PLA 1 isoform in the presence of different buffers commonly used in biology and biochemistry experiments. Sensitivity testing shows that PLA 1 can be detected at an activity as low as 0.06 U/mL. The rapid and reliable detection of phospholipases presented in this study has potential applications in the study of animal venoms as well as in lipase bioreactors and point-of-care devices.

Triglyceride-Tethered Membrane Lipase Sensor

“…This is to be contrasted with previous studies using phospholipid tBLMs exposed to phospholipases. 17 When the membrane seal is eliminated, the Bode diagrams would show no minimum in the phase plot, and the apparent conduction (G m ) values dramatically increase beyond the relevance of the model from which these measures are derived. These data suggest that the oleic acids formed from the triglyceride hydrolysis are not sequestering themselves out of the membrane and any alterations in membrane conduction are solely due to a rearrangement of the packing of the lipid molecules.…”

“…Following this, each membrane is rapidly solvent exchanged by washing out the mobile phase lipids with 3 × 400 μL tris buffer to enable the lipids to self-assemble into a membrane around the tether moieties. , Three different mobile phase lipids were used to create the tethered lipid substrates (Figure B), namely: the triglyceride triolein ((1,2,3-tri-(9Z-octadecenoyl)-glycerol), the ester phospholipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and a diether phospholipid with a structure, akin to DOPC, designated diether-PC (1,2-di-O-(9Z-octadecenyl)-sn-glycero-3-phosphocholine). In these experiments, because it lacks any hydrolizable ester groups, diether-PC is used to create negative control tethered membrane substrates . Experiments looking into mixtures of triolein with single-chained lipids included the use of 18:1 Lyso-PC (1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine; Avanti Polar Lipids, USA) and Oleic acid ((9Z)-Octadec-9-enoic acid; Sigma-Aldrich, Australia).…”

“…In these experiments, because it lacks any hydrolizable ester groups, diether-PC is used to create negative control tethered membrane substrates. 17 Experiments looking into mixtures of triolein with single-chained lipids included the use of 18:1 Lyso-PC (1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine; Avanti Polar Lipids, USA) and Oleic acid ((9Z)-Octadec-9-enoic acid; Sigma-Aldrich, Australia).…”

“…The sensor uses the same tethering molecules used to form sparsely tethered bilayer lipid membranes (tBLMs). 17 When used with electrical impedance spectroscopy (EIS), this sparsely tethered lipid membrane enables a direct and real-time measurement of lipase enzymatic activity.…”

“…This involves a wash step that converts the lipids from being dissolved in an ethanolic mobile phase to an aqueous solution where the presence of the hydrophobic moieties of the lipids dictates they must form into anisotropic layer(s) around the tethering molecules. The sensor uses the same tethering molecules used to form sparsely tethered bilayer lipid membranes (tBLMs) . When used with electrical impedance spectroscopy (EIS), this sparsely tethered lipid membrane enables a direct and real-time measurement of lipase enzymatic activity.…”