Brian A. Cadle¹, Kristin C. Rasmus¹, Leah S. Leverich¹, Casey E. O’Neill², Ryan K. Bachtell² and Donald C. Cooper<sup>1,2

1</sup>Institute for Behavioral Genetics, ²Department of Psychology and Neuroscience, University of Colorado, Boulder. 1480 30th St. Boulder, CO 80303.

Abstract

Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new diagnostic and forensics tool with health applications.  Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (~$1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service.  A central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros (http://www.Neurocloud.org).  To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine.  Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE) antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml.  This data was then used to create standard curves to allow quantification of COC/BE in biological samples.  Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography.  The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid in a sample.  To determine the effectiveness of the QRPDA method for quantifying cocaine in biological samples, mice were injected with a sub-locomotor activating dose of cocaine (5 mg/kg; i.p.) and were found to have detectable levels of COC/BE in their urine (160.6 ng/ml) and blood plasma (8.1 ng/ml) after 15-30 minutes. By comparison rats self-administering cocaine in a 4 hour session obtained a final BE blood plasma level of 910 ng/ml with an average of 62.5 infusions.  It is concluded that automated QRPDA is a low-cost, rapid and highly sensitive method for the detection of COC/BE with health, forensics, and bioinformatics application and the potential to be used with other rapid immunotest strips directed at several other targets.