As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluor® 488 dye-labeled avidin with a quencher dye, 2-(4′-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET. HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor >0.9.

Introduction

The affinity of the proteins streptavidin and avidin for the small molecule biotin (K_d approximately 10^-15 M) is one of the strongest noncovalent interactions known (1). The extremely high affinity makes this interaction a useful tool; proteins and nucleic acids labeled with biotin are the basis of a large number of biological detection systems (1). A key parameter that affects the performance of many of these systems is the number of attached biotins per macromolecule (degree of labeling or DOL). However, biotin is difficult to detect and quantitate directly because its intrinsic absorption is difficult to distinguish from that of proteins and nucleic acids.

Many assays have been developed to work around this problem. One of the first assays developed was the absorbance-based 2-(4′-hydroxyazobenzene) benzoic acid (HABA) assay developed by Green (2). HABA binds with moderate affinity to the biotin binding site of avidin. In the presence of biotin, HABA is displaced, and its absorption maximum shifts from 500 to 350 nm. The amount of biotin is determined by measuring the decrease of absorbance at 500 nm. This assay has a detection limit of 200 pmol biotin. Another assay was developed by Huang et al. in the 1990s (3). Based on the competition of free biotin for binding sites of soluble avidin-β-galactosidase, the assay has a sensitivity of approximately 100 pmol but requires washing steps for maximal sensitivity. Another assay for biotin compound quantitation was described by Dorris et al. (4). This assay is more sensitive than the HABA assay—60 pmol—but requires the use of high-performance liquid chromatography (HPLC). Another assay that is more amenable to high-throughput analysis is a colorimetric competition assay developed by Gan and Marquardt (5) that can detect as little as 1.6 fmol biotin. However, like the Huang assay, it requires a wash step. More recently, Wu et al. developed a kinetic assay based on the fluorescence enhancement of fluorescein biotin when it is displaced from avidin (6). While this assay has an excellent limit of sensitivity (approximately 150 fmol), low concentrations of biotin require more than 1 h to achieve equilibrium. While useful, these assays all lack at least one of the following four operationally desired characteristics: no-wash, high-throughput, rapid readout, and sensitivity.

Here we describe a fluorogenic assay for biotin that addresses all four of these issues. This assay is based on fluorescence resonance energy transfer (FRET) quenching and dequenching (Figure 1) of the Biotective™ Green reagent. The Biotective Green reagent consists of avidin covalently labeled with the fluorescent dye Alexa Fluor® 488 and complexed with a quencher dye, HABA, occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence of the Biotective Green reagent via FRET. When biotin or one of its compounds (biotin covalently linked to another small molecule) binds to the Biotective Green reagent, HABA is displaced from the avidin, eliminating the quenching of the fluorescent Alexa Fluor 488 dye. The FRET mechanism results in an increase in fluorescence corresponding to the amount of biotin or biotin compound present in the sample. After nuclease or protease pretreatment of samples, the assay is complete within 5 min without washing, can detect as little as 4 pmol biotin in 0.1 mL volume, and is amenable to high-throughput assay formats.

Figure 1. Response mechanism of the Biotective Green reagent-based assay. (Click to enlarge)

Materials and Methods

5-(biotinylaminohexylacrylamido)-dUTP (biotin-aha-dUTP), 5-(biotinylaminohexylacrylamido)-dUMP (biotin-aha-dUMP), and 5-(biotinylaminohexylacrylamido)-uridine (biotin-aha-uridine) were obtained from Berry & Associates (Dexter, MI, USA). Biotective Green is commercially available from Molecular Probes Detection Technologies, Invitrogen (Eugene, OR, USA) as a component of the FluoReporter® Biotin Quantitation Assay kit. Biotin, biocytin (∊-biotinoyl-L-lysine), 6-((6-((biotinoyl)amino) hexanoyl)amino)hexanoic acid (biotin-XX), phosphate-buffered saline (PBS), and goat anti-mouse immunoglobulin G (IgG) labeled with an average of 5.5 biotin-XX moieties were all obtained from Molecular Probes Detection Technologies, Invitrogen. HPLC-purified hexabiotinylated oligonucleotide was purchased from Qiagen (Valencia, CA, USA), sequence C[biotin∼dT]CATAGC[biotin∼dT]CACGC[biotin∼dT]GTAGG [biotin∼dT]ATC[biotin∼dT]CAG[biotin∼dT]TCGG, where [biotin∼dT] is biotinylated deoxythymidine. Sodium borate, sodium hydroxide, calcium chloride, and Pronase E were obtained from Sigma-Aldrich (St. Louis, MO, USA). Micrococcal nuclease was from Worthington Biochemical (Lakewood, NJ, USA). The plates used were clear round-bottomed 96-well plates from Corning Costar (Lowell, MA, USA).