Abstract
Smart fluid manipulation with automatically controlled paper valves will enable automated and multi-step immunoassays on paper-based microfluidic devices. In this work, we present an integrated paper-based microfluidic platform with shape-memory polymer (SMP)-actuated fluid valves capable of automated colorimetric enzyme-linked immunosorbent assays (ELISAs). A single-layer microfluidic paper-based analytical device (μPAD) was designed to store all the reagents on the chip, and sequentially transfer reagents to a paper test zone following a specific ELISA protocol through automatic fluidic flow control by the multiple SMP-actuated valves. The actuation of a paper valve was based on the thermally responsive, duel-state shape transformation of a SMP sheet attached to the root of a paper cantilever beam for driving a hydrophilic paper bridge to connect and disconnect two paper channels. A portable colorimetric reader was developed to control the on-chip valve operations, quantify the colorimetric signal output, display the assay result, and wirelessly transmit the data to a smart phone for the application of telemedicine. Reliable operations of the paper valve and the entire μPAD were demonstrated with success rates of 97% and 93%, respectively. A detection mechanism for valve malfunction was designed and confirmed effective to identify any mal-operation of individual valves, thus rendering our platform reliable in real assays. For device calibration, we conducted direct ELISAs of rabbit IgG in phosphate-buffered saline (PBS), and achieved a low limit of detection (LOD) of 27 pM (comparable to that of standard and paper-based ELISAs). In order to demonstrate the clinical application of our multi-step immunoassay platform, we also conducted sandwich ELISAs to quantify the protein level of an inflammatory cytokine, namely tumor necrosis factor (TNF)-α, in surgically injured laryngeal tissues of rats. The protein levels of TNF-α were shown similar between the conventional and μPAD ELISAs.