HIGH SENSITIVITY MULTIPLEXED IMMUNOCHROMATOGRAPHIC ASSAY ON THREADS G. Zhou 1,2* , D. Juncker 1,2 1 Biomedical Engineering, McGill University, Montreal, CANADA and 2 Genome Quebec Innovation Centre, Montreal, CANADA ABSTRACT Lateral-flow immunochromatographic assays allow detecting an analyte in a matter of minutes following application of a sample, but are only available in strip format. Yarn has recently been proposed as a support for liquid handling and carrying out low-cost assays, but its use for quantitative immunoassays has not yet been demonstrated. Here we show cotton thread as support for immunochromatographic assays, introduce a setup with multiple threads and knotted fiber bundles to carry out assays, and a method to quantify the assay results. Our results suggest that threads and yarn in general may be used for sim- ple-to-use, sensitive point-of-care diagnostics. KEYWORDS: Threads, Yarns, Immunochromatography, Low-cost Diagnostics INTRODUCTION We have introduced cotton threads as an economic material to make microfluidic circuits [1], characterized the flow properties, developed knot-based mixers and circuits [2], and developed a multi-step sandwich assay to human C-reactive protein (CRP) [3]. Here, we present a high-sensitivity, one-step multiplexed immunoassay in serum. We pre-immobilize reagents on common cotton threads and porous fibrous bundles, and use it to detect CRP (an inflammatory protein associated with coronary heart disease) and Interleukin-6 (IL-6, an inflammatory cyctokine) in human serum. We obtained a limit of detection in the picomolar range. To demonstrate the multiplexing capability of thread-based immunochromatography, we assay against three human proteins simultaneously, CRP, Leptin (LEP), and Osteopontin (OPN) in serum. THEORY Lateral-flow immunochromatographic assays have been widely used to detect biomolecules for diagnostic and for environmental applications. It is one of the most commonly used rapid test format besides flow-through and “dipstick” assay. Compared to conventional microtiter plate-based immunoassay, it is a one-step assay where sample containing the analyte is applied to the test strip, and requires no additional washing and incubation steps, and test results become available within a few minutes to less than an hour. This type of one-step immunodiagnostics is attributed to the pre-drying of the reagents and dissolution of those reagents during assay. It has been explored as a point-of-care diagnostics to be deployed in resource poor countries for global health, for its low-cost, portability, fast-turn around time, and no requirement for expensive infrastructure (i.e. detection system) and trained personnel to operate. Cotton threads are made of cellulose. They are low-cost, available with different material characteristics, i.e. size, porosity. In a previous work, we have introduced yarns as an economic material to make microfluidic circuits, because its porous structure gives them high surface-to-volume ratio, and is a natural fluid conduit [2]. Li et al. and Roche et al. have also reported that cotton threads could be used for small molecule detection in artificial urine, but the assay format is a rather simple single-step reaction [4, 5]. EXPERIMENTAL Device Assembly. Assay devices were made by weaving multiple threads on a customized support (Fig 1A). The thread- based assay device was assembled using two types of threads: 100% mercerized cotton threads and a porous fiber bundle. One-step Assay. Reagents were pre-immobilized, and thread was coated with 0.6 µl of capture antibodies, whereas the fiber bundle was loaded with 3 µl of detection antibodies conjugated to gold nanoparticles containing sucrose, as reported previously [3]. Upon the application of a sample, the detection antibodies were re-dissolved by the sample and carried along the thread. The analyte in the sample were first bound by the detection antibodies and then captured by the capture antibodies, leading to the accumulation of gold nanoparticles, which results in a red color (Fig 1B and 1C). The system was optimized using IgGs, and was then optimized for use with human C-reactive protein, Interluken-6, Leptin, and Osteopontin. Image Analysis. For quantitative analysis, images of the binding zones were acquired with a desktop scanner and proc- essed with ImageJ. Binding curves were established using SigmaPlot. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 1867 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA