Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey.
Anal Chim Acta. 2013 Sep 10;794:90-8. doi: 10.1016/j.aca.2013.07.041. Epub 2013 Jul 25.
Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB(2-)) anion as a result of reaction with biothiols was measured at 410nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57μM and acceptable linearity ranging from 0.4 to 29.0μM (r=0.998).
开发用于生物硫醇的灵敏和选择性测定方法很重要，因为它们在生物系统中具有重要作用。我们提出了一种新的光学传感器，该传感器使用胶体溶液中吸附的 Ellman 试剂 (DTNB)-金纳米粒子 (Au-NPs) (DTNB-Au-NP) 设计，用于从生物样品和药物中选择性地测定重要的生物硫醇 (生物硫醇)。5,5'-二硫代双(2-硝基苯甲酸) (DTNB) 是一种用于溶液中游离巯基定量的多功能水溶性化合物，通过非共价相互作用吸附在 Au-NPs 上，与生物硫醇反应后形成黄色 5-硫代-2-硝基苯甲酸 (TNB(2-)) 阴离子，测量其在 410nm 处的吸光度变化。该传感器在包含半胱氨酸、谷胱甘肽、同型半胱氨酸、半胱胺、二氢硫辛酸和 1,4-二硫赤藓糖醇的标准生物硫醇的宽浓度范围内给出了线性响应。构建了各生物硫醇的校准曲线，并确定了它们的摩尔吸光率和线性浓度范围。使用 DTNB-Au-NP 测定法测定各种生物硫醇的半胱氨酸当量巯基含量 (CETC) 值与传统 DTNB 测定法相当，表明固定化 DTNB 试剂对巯基仍具有反应性。常见的生物样品成分，如氨基酸、类黄酮、维生素和血浆抗氧化剂，不会干扰所提出的传感方法。该测定法通过线性、加和性、精密度和回收率进行了验证，表明该测定法可靠且稳健。与传统的 DTNB 试剂相比，吸附在 DTNB 上的 Au-NPs 探针在生物硫醇测定中提供了更高的灵敏度（即更低的检测限）。在优化条件下，通过所提出的测定法定量半胱氨酸 (Cys)，检测限 (LOD) 为 0.57μM，线性范围可接受，为 0.4 至 29.0μM（r=0.998）。
