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In this contribution, we propose a label-free immunosensor, based on a novel type of electrolyte-gated field-effect transistor (EGOFET), for ultrasensitive detection of the C-reactive protein (CRP). The recognition layer of the biosensor is fabricated by physical adsorption of the anti-CRP monoclonal antibody onto a poly-3-hexyl thiophene (P3HT) organic semiconductor surface. A supplementary nonionic hydrophilic polymer is used as a blocking agent preventing nonspecific interactions and allowing a better orientation of the antibodies immobilized onto the P3HT surface. The whole biomolecule immobilization procedure does not require any pretreatment of the organic semiconductor surface, and the whole functionalization process is completed in less than 30 min. Surface plasmon resonance (SPR) measurements were performed to assess the amount of biomolecules physisorbed onto the P3HT and to evaluate the CRP binding proprieties of the deposited anti-CRP layer. A partial surface coverage of about 23 % of adsorbed antibody molecules was found to most efficiently sense the CRP. The electrical performance of the EGOFET immunosensor was comparable to that of a bare P3HT EGOFET device, and the obtained CRP calibration curve was linear over six orders of magnitude (from 4 pM to 2 μM). The relative standard deviation of the individual calibration points, measured on immunosensors fabricated on different chips, ranged between 1 and 14 %, and a detection limit of 2 pM (220 ng/L) was established. The novel electronic immunosensor is compatible with low-cost fabrication procedures and was successfully employed for the detection of the CRP biomarker in the clinically relevant matrix serum. Graphical abstract Schematic of the EGOFET immunosensor for CRP detection. The anti-CRP monoclonal antibody layer is physisorbed on the P3HT organic semiconductor and the CRP is directly measured by a label-free electronic EGOFET transducer.

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The covalent coupling of antibody fragments to linkers embedded in a monolayer matrix of phosphatidylcholine and cholesterol was examined at the air-water interface by the means of a quartz crystal microbalance, QCM. Two linkers that bind the free thiols of the Fab′ fragment were investigated. The nonspecific binding of bovine serum albumin and the specific binding of antigen were also monitored with the QCM. Standardized radioimmunoassay was used to confirm the immunoreaction and determine binding parameters. The monolayer formation of the linker lipids in the ternary system of phosphatidylcholine and cholesterol was, moreover, demonstrated by film balance studies. The results demonstrate that the covalent coupling of Fab′ fragments to linking groups embedded in a phospholipid monolayer matrix is a promising approach to achieve a defined immobilization of antibodies at the sensor surface with high antigen binding effeciency.

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