Gorodkiewicz E, Luszczyn J
Department of Electrochemistry, Institute of Chemistry, University of Bialystok, Al.J. Pilsudskiego11/4, 15-443 Bialystok, Poland.
Protein Pept Lett. 2011 Jan;18(1):23-9. doi: 10.2174/092986611794328663.
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 mg μL(-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.
已开发出一种用于特异性测定胱抑素的表面等离子体共振成像（SPRI）传感器。该传感器含有固定化的木瓜蛋白酶，其可从溶液中结合胱抑素。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）活化的木瓜蛋白酶被固定在胺修饰的金表面上。半胱胺用于金表面的修饰。优化了木瓜蛋白酶浓度和相互作用的pH值。选择1.5μg mL(-1)的木瓜蛋白酶浓度和6.5的pH值作为最佳条件。通过与人类白蛋白无相互作用验证了相互作用的特异性。该传感器的动态响应范围为0至0.6mg μL(-1)，检测限为0.09μg mL(-1)。通过与颗粒增强免疫比浊法（PETIA）比较验证结果，显示出良好的一致性。使用了鸡卵清白蛋白胱抑素或胱抑素C的校准曲线。为了证明该传感器的潜力，测定了血浆、尿液和唾液中的胱抑素C，结果与文献报道的数据显示出良好的一致性。发现白血病患者血浆中胱抑素浓度的结果低于胱抑素的正常水平。
