Determination of Pharmaceuticals Based on Indole Alkaloids with Amperometric DNA-Sensors and Enzyme Immunoassay Test-System

Abstract

Bioaffine methods were developed for determining indole-containing alkaloids ajmaline and vincristine. These methods are based on the proposed amperometric DNA-sensors and on immunoenzyme test-system with a spectrophotometric indication of the analytical signal. The complexing between ajmaline and immobilized native DNA (n-IDNA) allows effective preliminary concentration from test solutions on the biosensor. The time of analysis is 25–30 min, limit of detection (LOD) for ajmaline is 1.0 × 10⁻¹⁰ M. The test-system utilized the immunological reaction of ajmaline with its antibodies and the enzyme marker, horseradish peroxidase, LOD is 1.5 × 10⁻⁹ M. Anti-cancer vincristine interaction with immobilized renatured DNA (r-IDNA) was studied and determination was carried out using an amperometric DNA-sensor. Bioaffine membrane drug concentration and reactivation of the sensor was performed. The affinity binding constant K_bind for vincristine-r-IDNA complex calculated by Scatchard's method was found to be high enough [(5.0 ± 0.4) × 10⁵ l/mol] confirming high specificity of the complexing with r-IDNA. The duration of the assay is 40 min. The developed method is characterized with an LOD of 1.1 × 10⁻⁹ M, with the lack of the need for long sample processing. The pharmaceuticals were determined by those methods in model solutions of blood serum and in tablets and solutions for injections.

Keywords:

• Ajmaline
• Amperometric DNA-sensor
• Drug analysis
• Immunoenzyme test system
• Vincristine

Acknowledgments

The study was financially supported by the Russian Foundation for Basic Research, project no.08-03-00749.