Surface Plasmon Resonance Imaging (SPRI) Sensor for Cystatin Determination Based on Immobilized Papain
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 sensor's 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 sensor's 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.
The sensor's 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 sensor's 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: A Surface Plasmon Resonance (SPR); Biosensor; CLL-Chronic lymphocytic leukaemia-4; CMLChronic Myelogenous Leukaemia; Cystatin C; HBS-ES buffer; Langmuir isotherm type; MCL-Myeloid cell leukaemia; PENIA; PETIA; Papain - Cystatin Interaction; Papain Immobilization; SPRI biosensor; Surface Plasmon Resonance Imaging; TOF-MS; amine-modified surface; antibody IgG; antigen presentation; blood plasma; cathepsin concentration; cystatin; cysteamine; egg cystatin; immobilised papain; kDa-molecular protein; leukaemia; papain; papain-cystatin complex; papain-cystatin interaction; saliva; urine
Document Type: Research Article
Publication date: 01 January 2011
- Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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