Ho Ja-an Annie, Hung Chi-Hsiang
BioAnalytical Laboratory, Department of Chemistry, National Tsing Hua University, Hsin-chu, 30013 Taiwan.
Anal Chem. 2008 Aug 15;80(16):6405-9. doi: 10.1021/ac800850w. Epub 2008 Jul 23.
Although biotin is an important vitamin for cellular function and growth, there are no rapid, simple, and reliable analytical tools available for its quantitation in bodily fluids or foodstuffs. In this study, we developed an immunoaffinity chromatographic biosensing system for the direct determination of biotin. A stationary phase having affinity for biotin was synthesized by covalently bonding antibiotin monoclonal antibodies onto 90-microm, NHS-activated sepharose beads. The beads were then packed into 1.9-cm-diameter plastic tubes to form a column having a volume of 3.0 mL. The function of the proposed immunoaffinity chromatographic assay was based on competition between biotin and carboxyfluorescein (CF)-encapsulated, biotin-tagged liposomes (liposomal biolabels) for the limited number of antibiotin antibody binding sites. Buffers containing biotin standards at concentrations ranging from 10(-12) to 10(-3) M were passed through the column to trap and concentrate the biotin on the solid support. The unbound binding sites of the antibody were then occupied through subsequent addition of the liposomal biolabels. The addition of 35% methanol released the CF molecules from the lyzed bound liposomes; the fluorescence intensity of the released markers was then measured using a fluorometer. The calibration curve for biotin was linear over 8 orders of magnitude, from 10(-12) to 10(-4) M. The limit of detection of this immunoaffinity chromatographic biosensing system reached as low as 5.0 pg of biotin (equivalent to 500 microL of 4.10 x 10(-11) M biotin).
尽管生物素是细胞功能和生长所需的一种重要维生素，但目前尚无快速、简单且可靠的分析工具可用于定量测定体液或食品中的生物素。在本研究中，我们开发了一种用于直接测定生物素的免疫亲和色谱生物传感系统。通过将抗生物素单克隆抗体共价结合到90微米的NHS活化琼脂糖珠上，合成了对生物素有亲和力的固定相。然后将这些珠子填充到直径为1.9厘米的塑料管中，形成一个体积为3.0毫升的柱子。所提出的免疫亲和色谱分析方法的功能基于生物素与羧基荧光素（CF）包裹、生物素标记的脂质体（脂质体生物标记物）之间对有限数量抗生物素抗体结合位点的竞争。将含有浓度范围为10^(-12)至10^(-3) M生物素标准品的缓冲液通过柱子，以捕获并浓缩固定相上的生物素。随后加入脂质体生物标记物占据抗体未结合的位点。加入35%甲醇可从裂解的结合脂质体中释放出CF分子；然后使用荧光计测量释放标记物的荧光强度。生物素的校准曲线在10^(-12)至10^(-4) M范围内呈8个数量级的线性关系。这种免疫亲和色谱生物传感系统的检测限低至5.0皮克生物素（相当于500微升4.10×10^(-11) M生物素）。
