On-Site Microfluidic Assay for the Detection of Multiple Drugs in Urine

Immunoassays are widely used for preliminary drug screening. While lateral flow assays (LFAs) offer rapid and portable testing, they often suffer from limited sensitivity and subjective interpretation. In contrast, laboratory-based immunoassay systems provide high precision but are unsuitable for field use due to their size and cost. To address these limitations, we developed a portable fluorescence-based microfluidic assay capable of detecting methamphetamine (MET), benzoylecgonine (BE), 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), and benzodiazepines (BZOs) in urine specimens within 5 min. They were selected based on both global and domestic prevalence, as well as the detection capability of the developed microfluidic chip, which was designed to simultaneously target all four. The assay comprises a portable reader and a chip with two channels, each aligned with drug-specific antibodies, enabling simultaneous detection. The assay was tested against standard concentrations of the target drugs, delivering accurate test/reference ratios and signal displacements at threshold levels. We evaluated urine specimens (<i>N</i> = 141) provided by the National Forensic Service in Korea, including drug-free controls (<i>N</i> = 30) and cases confirmed for MET (<i>N</i> = 30), THC-COOH (<i>N</i> = 30), and BZOs (<i>N</i> = 13), along with samples from Paraguay for BE (<i>N</i> = 38), using validated diagnostic techniques. Analysis of 141 authentic urine specimens, encompassing verified instances of both single and poly drug use, showed high sensitivity and specificity with minimal cross reactivity. This study confirms that the microfluidic assay is effective in detecting multiple drugs simultaneously, offering high accuracy, cost-effectiveness, and portability, with excellent sensitivity for each drug tested.