Abstract: The assay described was based on the enhancement of the emission intensity of the fluorescein-labelled conjugate of streptavidin by the biotin moiety. A merging-zone FIA system was used in which 70 µL of streptavidin-fluorescein isothiocyanate reagent (1.0 mg/l) was injected into one buffer stream and 70 µL of sample was injected into a second buffer stream. Both streams consisted of 50 mM phosphate buffer of pH 8 (1 ml/min). The streams were merged and passed through a 2-m knitted open-tubular reactor coil into the detection cell were the emission signal at 518 nm was recorded (excitation at 495 nm). The calibration graphs for biocytin, biotin, biotin-thyroxine and biotinylated BSA were linear at low concentration. (0.4 µM) but the gradients varied considerably. The detection limit was 2 nM-biotin.

Abstract: Conventional biosensors, such as those based on surface plasmon resonance, lack spectral resoln. and employ a permanent sensing layer that needs to be activated and also regenerated after use. In contrast, scanning of the UV/VIS spectrum of agarose beads, trapped in a specially designed flow cell, allows real time monitoring of labeled and unlabeled biomolecules, with spectral resoln., on a surface that can be automatically renewed, by microfluidic manipulation. Agarose beads are identical with column materials used in affinity chromatography and therefore are readily available, derivatized with a wide choice of bioligands. In this way, flow injection absorptiometry on renewable surfaces provides a basis for a new class of biosensors with 'open architecture' that allows the development of novel types of immunoassays employing both unlabeled and labeled molecules. The method also has interesting implications for affinity chromatography, because it uses identical materials and investigates the same type of bioligand interactions. An improved configuration of the jet ring cell is introduced and it is shown that both large (IgG) and small molecules (biotin) can be detected down to the 25 ng level reproducibly and rapidly.

Abstract: We present imaging ellipsometry technique for kinetic measurement of bimolecular interactions with high sensitivity. When combined with surface plasmon resonance (SPR) effects, the ellipsometry becomes powerful technique for analyzing adsorption and desorption of biomolecules on gold layer based sensor chip surfaces. Because ellipsometric measurement gives ellipsometric parameters, namely Δ, that is very sensitive to surface layer changes. The SPR combined ellipsometry is realized by Kretschmann configuration SPR cell comprising with about 30-nm-thick gold film deposited on top of glass slides, SF10 glass prism, and flow injection system. We used nulling type of imagining ellipsometer to acquire two dimensional ellipsometric parameters with spatial resolution down to one micrometer. We present results of kinetic measurements of biotin-streptavidin interactions for custom-built sensor chip.