用于天冬氨酸组织蛋白酶的表面等离子体共振成像生物传感器：传感器的开发及其在生物材料中的应用

SPR imaging biosensor for aspartyl cathepsins: sensor development and application for biological material.

作者信息

Gorodkiewicz Ewa, Regulska Elzbieta

机构信息

Department of Electrochemistry, Institute of Chemistry, University of Bialystok, Al.J.Pilsudskiego11/4, 15-443 Bialystok, Poland.

出版信息

Protein Pept Lett. 2010 Sep;17(9):1148-54. doi: 10.2174/092986610791760450.

DOI:10.2174/092986610791760450

PMID:20394580

Abstract

A Surface Plasmon Resonance Imaging (SPRI) sensor has been developed for highly selective determination of cathepsin D (Cat D) or/and E (Cat E). The sensor contains immobilised pepstatin A, which binds aspartyl proteases from solution. Pepstatin A activated with N-Hydroxysuccinimide (NHS) and N-Ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) was immobilized on an amine-modified gold surface. Cysteamine was used for modification of the gold surface. Pepstatin A concentration and pH of interaction were optimised. A concentration of pepstatin equal to 0.5 microg mL(-1) and a pH of 3.75 were selected as optimal. The sensor's dynamic response range is between 0.25 and 1.0 ng mL(-1), and the detection limit is 0.12 ng mL(-1). However, the sensor cannot distinguish between Cat D and Cat E. In order to demonstrate the sensor's potential, Cat E was determined in human red blood cells, Cat D in human saliva, as well as total concentration of Cat D and Cat E in human nasal polyps.

摘要

已开发出一种表面等离子体共振成像（SPRI）传感器，用于高选择性地测定组织蛋白酶D（Cat D）或/和组织蛋白酶E（Cat E）。该传感器含有固定化的胃蛋白酶抑制剂A，其可从溶液中结合天冬氨酸蛋白酶。用N-羟基琥珀酰亚胺（NHS）和N-乙基-N'-(3-二甲基氨基丙基)碳二亚胺（EDC）活化的胃蛋白酶抑制剂A被固定在胺修饰的金表面上。半胱胺用于金表面的修饰。对胃蛋白酶抑制剂A的浓度和相互作用的pH值进行了优化。选择0.5μg mL(-1)的胃蛋白酶抑制剂浓度和3.75的pH值作为最佳值。该传感器的动态响应范围在0.25至1.0 ng mL(-1)之间，检测限为0.12 ng mL(-1)。然而，该传感器无法区分Cat D和Cat E。为了证明该传感器的潜力，测定了人红细胞中的Cat E、人唾液中的Cat D以及人鼻息肉中Cat D和Cat E的总浓度。