Simultaneous detection of C-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks.

We show a proof-of-concept in which we combine our previously published concepts of micromosaic immunoassays (microMIAs) with self-regulating microfluidic networks (microFNs) to detect C-reactive protein (CRP) and other cardiac markers such as myoglobin (Mb) and cardiac Troponin I (cTnI). The microFNs are microfabricated in Si, have a well-defined surface chemistry, and are affixed to a bibulous material so as to self-regulate the displacement of an aliquot of liquid through the microFNs using capillary forces. An open section of the channels of the microFNs is covered with a hydrophobic poly(dimethylsiloxane) (PDMS) slab that acts as the substrate for a solid-phase immunoassay. Here, individual assays are conducted using independent channels. These assays are "sequential": series of samples, reagents, and buffers are displaced one after the other over the PDMS surface, and, as these assays are conducted under "microfluidic" conditions, they are fast to perform, very economical in their use of reagents, extremely integrated, and yield high-quality signals. The combinatorial character of microMIAs is exploited to optimize the assay parameters for detecting CRP. In particular, we found it optimal to deposit the capture antibody for CRP on PDMS at a concentration between 20 and 500 microg ml(-1) in PBS in 1 min and to detect captured CRP in 2 min using a detection antibody having a concentration in PBS of 120 microg ml(-1). With this method, CRP is quantitatively detected within 10 min in one microliter of human plasma down to concentrations of 30 ng ml(-1), which suggests the possibility to detect CRP at clinically relevant concentrations for the management of coronary heart disease (CHD) and systemic inflammation.