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

Recently, single-walled carbon nanotube-based optical biosensors have been shown to detect hydrolase activity directly on target substrates such as proteins and peptides. This study presents a metastable protein conjugation approach to immobilize more hydrophobic proteins and enhance the sensitivity of protease detection. The method combines covalent conjugation of substrate proteins via carbodiimide chemistry (EDC/NHS) with non-covalent polymer wrapping of SWCNT, in this case, carboxymethyl cellulose. 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 removal of unbound proteins and residual materials, enhancing the sensor's sensitivity by approximately threefold, reaching an LOD of 6.4 ng/ml in a 5 minute reaction. This immobilization approach is applied to the ECM proteins such as gelatin and collagen and used to detect ECM degrading enzymes' activity. ECM degrading enzymes caused a fluorescent intensity decrease of the SWCNT probes, enabling quantification of enzyme concentration between the range 160 ng/ml to 100 µg/ml 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.