Surface Plasmon Resonance Imaging (SPRI) Sensor for Cystatin Determination Based on Immobilized Papain

E.      Gorodkiewicz; J.      Luszczyn

doi:10.2174/092986611794328663

Abstract

A Surface Plasmon Resonance Imaging (SPRI) sensor has been developed for specific determination of cystatin. The sensor contains immobilised papain, which binds cystatin from solution. Papain activated with N- Hydroxysuccinimide (NHS) and N-Ethyl-N-(3-dimethyl aminopropyl)carbodiimide (EDC) was immobilized on an amine-modified gold surface. Cysteamine was used for modification of the gold surface. Papain concentration and the pH of interaction were optimised. A concentration of papain of 1.5 μg mL-1 and a pH of 6.5 were selected as optimal. The specificity of interaction was verified by the lack of interaction with human albumin. The sensors dynamic response range is between 0 and 0.6 μg mL-1, and the detection limit is 0.09 μg mL-1. The results were validated by comparison with the PETIA (particle enhanced immunoturbidimetric assay) method showing good agreement. A calibration curve of chicken egg white cystatin or Cystatin C was used. In order to demonstrate the sensors potential, cystatin C was determined in blood plasma, urine and saliva, showing good agreement with data reported in the literature. The results for cystatin concentration in the blood plasma of people suffering from leukaemia were found to be below the normal level of cystatin.

Keywords: Biosensor, blood plasma, cystatin, papain, saliva, Surface Plasmon Resonance Imaging, urine, antigen presentation, Cystatin C, PENIA, PETIA, TOF-MS, antibody IgG, A Surface Plasmon Resonance (SPR), kDa-molecular protein, papain-cystatin interaction, egg cystatin, Papain Immobilization, amine-modified surface, HBS-ES buffer, cysteamine, Langmuir isotherm type, papain-cystatin complex, –, Cystatin Interaction, leukaemia, cathepsin concentration, CLL-Chronic lymphocytic leukaemia, 4, CMLChronic Myelogenous Leukaemia, MCL-Myeloid cell leukaemia, SPRI biosensor, immobilised papainBiosensor, blood plasma, cystatin, papain, saliva, Surface Plasmon Resonance Imaging, urine, antigen presentation, Cystatin C, PENIA, PETIA, TOF-MS, antibody IgG, A Surface Plasmon Resonance (SPR), kDa-molecular protein, papain-cystatin interaction, egg cystatin, Papain Immobilization, amine-modified surface, HBS-ES buffer, cysteamine, Langmuir isotherm type, papain-cystatin complex, –, Cystatin Interaction, leukaemia, cathepsin concentration, CLL-Chronic lymphocytic leukaemia, 4, CMLChronic Myelogenous Leukaemia, MCL-Myeloid cell leukaemia, SPRI biosensor, immobilised papain