Biotin detection

To reduce the hydrazone bonds to more stable linkages, cool the solution to 4°C and add an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 min. Note If the presence of a reducing agent is detrimental to protein activity, eliminate this step. In most cases, the hydrazone linkage is stable enough for avidin-biotin detection experiments. 

NPH HCl) with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC HCl) as a coupling agent [593,594], The advantage is that with hydrazines it is possible to carry out the derivatization in milder conditions. An efficient postcolumn derivatization is obtained using avidin or streptavidin. In fact, these two proteins react in a very specific way with biotin, therefore they are bound with a fluorescent marker, such as fluorescein 5-isothiocyanate (FITC) to obtain fluorescent derivatives. Some authors [595] report the use of MS or MS/MS for biotin detection, but this method seems to be less sensitive than FLD. 

The result is high-quality arrays that can be used with a fluorescence detection system in a manner similar to the glass slide microarrays, although only single color detection is used. cDNA is synthesized from a test RNA sample by reverse transcription followed by second strand synthesis. This is then used as a template for an in vitro transcription reaction to produce biotin-labeled cRNA. As well as introducing the label for detection, this step permits amplification of the RNA and thus enables less starting material to be used. The cRNA is hybridized to the GeneChip and the biotin detected with an avidin-... 

High-performance liquid chromatography (HPLC) methods have been comprehensively applied to the determination of biotin in pharmaceutical multivitamin preparations. Some of them have also been used to analyse more complicated matrices of samples, e.g. food material (Livaniou et al. 2000). Biotin detection by HPLC can be achieved with (either pre- or post-column) or without sample derivatization (Livaniou et al. 2000). 

Two optical sensor designs for biotin detection are described above. In this section, we present a liposome-amplified signalling strategy for electrochemical immunosensors, which converts the bio-specific affinity binding reactions into an electrochemical signal (Ho et al. 2009a). 

---