Development of immunosensors based on optical waveguide lightmode spectroscopy (OWLS) technique for determining active substance in herbs

For the quick and reliable quantification of special active substances derived from herbs, a new type of immunosensor based on optical waveguide lightmode spectroscopy (OWLS) detection was investigated. Artemisinin, an antimalarial drug derived from the sweet wormwood plant Artemisia annua is a sesquiterpene lactone endoperoxide, and it is distilled from the dried leaves or flower clusters of A. annua. Numerous derivatives of artemisinin, including artesunate and artemether, are also being used in the treatment of malaria, and these compounds have recently gained utility also as anticancer agents. To quantitatively determine the presence of these biologically effective substances in various herbs, a novel OWLS-based immunosensor was investigated. To create regenerable sensitized surfaces in the OWLS technique so that the sensor can be applied several times, the antigen or the antibody were immobilized by covalent attachment to the silanized surfaces of the OWLS chips. When measuring with the antibody capture mode antibodies raised against the appropriate artemisinin derivative (artemisinin, artesunate or artemether) were immobilized on the sensor surface and the linear measuring range was determined. During the antigen capture measurement the protein conjugate of the analyte was immobilized on the waveguide surface. Standard solutions containing different amounts of the appropriate standard were mixed with antibodies at optimized concentration, the mixture was incubated and injected into the flow-injection analysis system of OWLS. Binding of the antibodies in the sample to the coated surface is competed for with free antigen in the sample, and only antibodies that remained in free form in the mixture bound to immobilized antigen-conjugates. The amount of antibodies bound to the surface of the chip was inversely proportional to the active substance content in the samples. The dynamic measuring range and the relative substrate specificity of the serum on artemisinin and its derivatives were studied. Herbs of different origin and herbal supplement products were analyzed using the newly developed method and a correlation was studied with HPLC/DAD/MS reference method.