Sensitive detection of histamine using fluorescently labeled oxido-reductases.

ABSTRACT

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM-225 µM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 µM with a coefficient of variation of 11% for 10 measurements of 100nM histamine in a 100 µL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum.