Using liposomal fluorescent biolabels to develop an immunoaffinity chromatographic biosensing system for biotin.
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).
Ho
Ja-an Annie
JA
BioAnalytical Laboratory, Department of Chemistry, National Tsing Hua University, Hsin-chu, 30013 Taiwan. jaho@mx.nthu.edu.tw
Hung
Chi-Hsiang
CH
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000032:analysis*; D002214:Capsules / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007118:Immunoassay / Q000379:methods*; D008081:Liposomes / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D002214:Capsules; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; C024098:6-carboxyfluorescein; D001710:Biotin