Conjugation Strategies for Low Solubility Proteins to Single-Walled Carbon Nanotubes as a Sensitive Fluorescent Assay to Protease Activity

Abstract

Single-walled carbon nanotube (SWCNT)-based optical biosensors are shown to detect hydrolase activity directly on target substrates. This study presents a metastable protein conjugation approach to immobilize hydrophobic proteins and enhance  sensitivity of protease detection. The method combines covalent conjugation of substrate proteins via carbodiimide chemistry with non-covalent polymer wrapping of SWCNT, carboxymethyl cellulose (CMC). The formation of protein-SWCNT complexes as a result of multi-site conjugation between the proteins and carboxyl groups, enabled iterative pelleting, washing, and resuspension steps to be applied to the probes which allowed for the removal of unbound proteins and residual materials, enhancing the sensor's sensitivity by approximately threefold, reaching a limit of detection (LOD) of 6.4 ng ml⁻¹ in a 5 minute reaction. This immobilization approach is applied to extracellular matrix (ECM) proteins such as gelatin and collagen to detect ECM degrading enzyme activity. ECM degrading enzymes caused a fluorescent intensity decrease of the SWCNT probes, enabling quantification of enzyme concentration between the range 160 ng ml⁻¹ to 100 µg ml⁻¹ within 5 minutes of reaction. This hybrid approach provides a rapid and sensitive platform for detecting extracellular degrading enzymes with potential applications in cancer diagnosis and prognosis, wound healing, high-throughput screening for enzyme inhibitors, and drug discovery.