Development of an ultrasensitive immunochromatography test to detect nicotine metabolites in oral fluids

Passive exposure to tobacco smoke causes a variety of illnesses ranging from allergic responses to cancer. Assessment of exposure to second-hand tobacco smoke (SHS), particularly among vulnerable populations enables intervention and prevention of future disease. A minimally invasive oral fluids-based onsite test to detect such exposure would create a valuable tool for researchers and clinicians. Here we describe the development of a test that uses an inexpensive reader that utilizes a CMOS image sensor to reliably quantify a reporter signal and determine nicotine exposure. The rapid lateral flow test consists of a nitrocellulose strip with a control line containing goat anti-rabbit IgG, used as an internal standard, and a test line containing BSA-cotinine conjugate. To run the test, diluted sample containing antibodies against cotinine, the major metabolite of nicotine, is mixed with protein A-gold nanoparticles and placed on the sample pad. As the sample runs up to the nitrocellulose pad, antibodies in the running buffer bind to available cotinine. If cotinine is absent, the antibodies will bind to the BSA-cotinine derivative immobilized on the test line, resulting in an intense purple-red band. The concentration of cotinine equivalents in the sample can be estimated from interpretation of the test line. In this article we describe the effect of different cotinine derivatives, oral fluid pretreatment, and application and running buffers on assay sensitivity. The test can reliably detect as little as 2 ng mL(-1) cotinine equivalents. The assay is sensitive, simple, rapid, inexpensive, and easily implementable in point-of-care facilities to detect second-hand smoke exposure.