1. Label-Free, Real-Time Phospholipase-A Isoform Assay
- Author
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Samira R. Aili, Matthew P. Padula, Alvaro Garcia, Axel Touchard, Christopher Murphy, Evelyne Deplazes, Graham M. Nicholson, Bruce Cornell, Upeksha Mirissa Lankage, Charles G. Cranfield, Ecologie des forêts de Guyane (UMR ECOFOG), and Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-AgroParisTech-Université de Guyane (UG)-Centre National de la Recherche Scientifique (CNRS)-Université des Antilles (UA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
- Subjects
Tethered bilayer lipid membranes (tBLMs) ,Gene isoform ,[SDV]Life Sciences [q-bio] ,0206 medical engineering ,Biomedical Engineering ,electrical impedance spectroscopy (EIS) ,02 engineering and technology ,Phospholipase ,Glycerophospholipids ,Biomaterials ,0903 Biomedical Engineering ,Diether lipids ,Animals ,Protein Isoforms ,Lipase ,Lipid bilayer ,Phospholipids ,chemistry.chemical_classification ,Phospholipase A ,biology ,Bilayer ,021001 nanoscience & nanotechnology ,Venom ,020601 biomedical engineering ,Phospholipases A2 ,Enzyme ,Pancreatitis ,chemistry ,Biochemistry ,Acute Disease ,Ester lipids ,biology.protein ,lipids (amino acids, peptides, and proteins) ,0210 nano-technology - Abstract
International audience; 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 mu g/mL. The specificity of the phospholipase activity was corroborated using matrix-assisted 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.
- Published
- 2020