Quantification of low-picomolar concentrations of TNF-alpha in serum using the dual-network microfluidic ELISA...

For both research and diagnostic purposes, the ability to detect low levels of proteins in a cost- and time-effective manner is essential. In this study, the cytokine TNF-alpha (tumor necrosis factor-alpha), a widely used protein indicator of inflammatory response, was chosen to demonstrate the ability of the dual-network microfluidic ELISA (enzyme-linked immunosorbent assay) platform developed by the authors to rapidly quantify low concentrations of this biomarker in serum. Through the optimization of several experimental parameters, the system was shown to meet the requirements for fundamental and applied studies, while also being relevant for challenging clinical applications such as the diagnosis of septic patients. A sensitivity of 45 pg/mL (2.6 pM) in both culture medium and serum, with inter- and intravariations of less than 15%, was attained for the quantification of human TNF-alpha to a concentration of up to 500 pg/mL. The overall time for completion of the assay in eight parallel reactions was less than 1 h.