A Resistive Biosensor for the Detection of LC3 Protein in Autophagy

Abstract

Resistive sensing techniques have recently emerged as a powerful tool to sensitively detect proteins of interest. However, detecting intracellular proteins present in cell-derived lysates remains largely unexplored. Here, a resistive sensing technique was utilized to develop a biosensor for the detection of the autophagy-related LC3 protein using three different resistive transducers. Microfiber-, paper- and carbon nanofiber-based transducers were compared to determine optimal sensitivity. The microfiber- and paper-based transducers utilized a conductive poly-pyrrole, co-polymerized with carboxylic acid functional 3-thiopheneacetic acid, allowing for effective cross-linking between the conductive coated substrates and LC3 antibodies using glutaraldehyde as the cross-linking agent. Anti-LC3 antibodies were immobilized on the highly conductive carbon nanofiber using adsorbed 1-pyrenebutyric acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as the cross-linking agent. A resistive sensor capable of mediating the protein-antibody binding and accurately measuring a change in conductivity of the transducers was designed and manufactured. The portable sensor was integrated with a computer to generate and display measured results via a user-friendly interface. The various transducing elements were added to the sensor where LC3 detection took place. All three transducers proved effective at detecting LC3 protein in the solution buffer and were capable of distinguishing between different concentrations of LC3 protein in a highly sensitive manner. The results highlight how the unique combination of transducer- and system design, in combination with resistive sensing enables the rapid, robust and accurate detection of the LC3 protein, reporting on a critical biological process, here autophagy.