A Bio-Sensor for Human Neutrophil Elastase Employs Peptide-p-Nitroanilide Cellulose Conjugates

摘要

High levels of human neutrophil elastase (HNE) in chronic wounds have been associated with degradation of cytokine growth factors necessary for normal wound healing. Thus, accurate clinical detection and quantification of HNE will be important to the therapeutic management of chronic wounds. Colorimetric detection of HNE using immobilized HNE substrate-cellulose analogs has been assessed. The chromogenic peptide substrate Succinyl-Ala-Ala-Pro-Ala-pNA and its analog Succinyl-Ala-Ala-Pro-Val-pNA were attached to derivatized cellulose. Cellulose was treated with 3-aminopropyltriethoxysilane to form the amino-propyloxy ether of cellulose. The amino-propyloxy-ether of cellulose (Cell-AP) was reacted with the HNE chromogenic para-nitroanilide peptide substrates to form a covalently linked conjugate of cellulose (Cell-AP-suc-Ala-Ala-Pro-Ala-pNA or Cell-AP-suc-Ala-Ala-Pro-Val-pNA) through amide bond between the Cell-AP amine and the succinyl carboxylate of the substrate. The colorimetric response of the cellulose-bound chromophore was assessed in HNE buffered solutions by monitoring release of para-nitroaniline from the derivatized cellulose probe to determine HNE levels from 5.0 × 10{sup}(-3) to 6.0 units per ml. A comparison of the analogs rate of hydrolysis demonstrated that Cell AP-suc-Ala-Ala-Pro-Ala-pNA was faster and it gave slightly stronger absorption than the Cell-AP-suc-Ala-Ala-Pro-Val-pNA. Visual differentiation and detection of elastase activity units resulting from substrate hydrolysis was optimal at 2-4 minutes with Cell-AP-suc-Ala-Ala-Pro-Ala-pNA, and at 15-60 minutes with Cell-AP-suc-Ala-Ala-Pro-Val-pNA.