Calorimetric sandwich-type immunosensor for quantification of TNF-α.

We report a lab-on-a-chip immunosesnor for quantification of the inflammatory cytokine TNF-α with picomolar sensitivity. The feasibility of the technology was demonstrated via accurate measurement of the concentration of TNF-α in astrocytes cell culture media. The immunoassay was performed in a microfluidic device with an integrated antimony/bismuth thermopile sensor and had a limit of detection of 14 pg mL. The device was fabricated using rapid prototyping xurography technique and consisted of two inlets and single outlet. Anti-TNF-α monoclonal antibody was used to capture the analyte while the detection was performed using glucose oxidase-conjugated secondary antibody. Glucose (55 mM) was injected through a sample loop into the fluid flowing within the microfluidic device. A nanovolt meter connected to the thermoelectric sensor recorded the voltage change caused by the enzymatic reaction. Computer simulations using COMSOL Multiphysics were performed to analyze the effect of fluid velocity on the concentration of glucose within the reaction zone. A standard calibration curve was created using serial dilutions of synthetic TNF-α (0-2000 pg mL) by plotting the area under the curve of the signal versus the concentration of the analyte. The efficacy of the device was evaluated by quantifying TNF-α in the cell culture medium of lipopolysaccharide stimulated and non-stimulated astrocytes. The results demonstrated high accuracy of the calorimetric immunoassay when compared with gold standard commercial ELISA microplate reader. The immunosensor offers excellent reproducibility, accuracy, and versatility in the choice of the detection enzyme.