A Kinesin Driven Microfluidic Concentrator Device for Ultrasensitive Detection of Analyte

Abstract

The discovery of a vast array of biomarkers has spurred the demand for diagnostic assays with lower detection limits for early disease detection. Microfluidics makes it possible to work with small sample volumes and has played a significant role in creating more sensitive diagnostic tools. Our goal is to adapt our previous biomolecular motor (kinesin) based concentrator (NanoLett.8:1041) and integrate antibody-functionalized microtubules into the device. Transforming this device into an immunoassay platform allows a variety of proteins or biomarkers to be actively captured and concentrated for detection. We believe this concentrator can improve typical ELISA assays by integrating two key features. First, concentrating the analyte-carrying microtubules into a small 625μm2 concentrator region increases the signal to noise ratio allowing for more sensitive fluorescence measurements. Second, by ensuring that the binding capacity of these functionalized microtubules is high, we allow for a large number of antibodies and antigen to be concentrated. To achieve these goals we have developed a protocol to covalently link a high density of monoclonal, polyclonal or f(Ab)'2 antibodies onto microtubules without significantly affecting the motility of the complex. Motility is critical for the device since the microtubules with captured and fluorescently-labeled analyte are rapidly transported by kinesin into the concentrator region. The intensity of the resulting fluorescent signal in the concentrator region directly corresponds to the concentration of analyte in the initial sample. Our results show that the fluorescence intensity of individual anti-BSA coated microtubules allows the detection of sub-picomolar concentrations of TMR-BSA by integrating the fluorescence signals along microtubules. In conclusion, our data suggest that integrating functionalized microtubules and raising the signal to noise ratio by concentration in this device improves the detection limits of a typical ELISA assay while significantly reducing the assay time.