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Recognizing and quantifying specific biomolecules in aqueous samples are constantly needed in research and diagnostic laboratories. As the typical detection procedures are rather lengthy and involve the use of labeled secondary antibodies or other agents to provide a signal, efforts have been made over the last 10 y to develop alternative label-free methods that enable direct detection. We propose and demonstrate an extremely simple, lowcost, label-free biodetector based on measuring the intensity of light reflected by the interface between a fluid sample and an amorphous fluoropolymer substrate having a refractive index very close to that of water and hosting various antibodies immobilized in spots. Under these index-matching conditions, the amount of light reflected by the interface allows straightforward quantification of the amount of antigen binding to each spot. Using antibodies targeting heterologous immunoglobulins and antigens commonly used as markers for diagnoses of hepatitis B and HIV, we demonstrate the limit of detection of a few picograms per square millimeter of surface-bound molecules. We also show that direct and real-time access to the amount of binding molecules allows the precise extrapolation of adhesion rates, from which the concentrations of antigens in solution can be estimated down to fractions of nanograms per milliliter.immunoassay | optical biosensor | protein microarray | biomolecular detection | reflective phantom interface

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Amorphous fluorinated plastic can be produced with a refractive index similar to that of water, a condition that makes it essentially invisible when immersed in aqueous solutions. Because of this property, even a small amount of adsorbed molecules on the plastic-water interface provides a detectable optical signal. We investigated two distinct substrates made of this material, characterized by different interface area: a prism and a newly realized micro-porous membrane. We demonstrate that both substrates enable the label-free detection of molecular compounds in water even without any surface functionalization. The adsorption of molecules on the planar surface of the prism provides an increase of optical reflectivity, whereas the adsorption on the internal surface of the micro-porous membrane yields an increase of scattered light. Despite the different mechanisms, we found a similar optical response upon adsorption. We confirmed this result by a theoretical model accounting for both reflection and scattering. We investigated the spontaneous adsorption process for different kinds of molecules: surfactants with different charges, a protein (lysozyme) and a constituent of gasoline (hexane). The measured equilibrium and kinetic constants for adsorption differed by orders of magnitudes among the different classes of molecules. By suitable analytical models, accounting for the effects of mass limitation and transport, we found a simple and general scaling of the adsorption parameters with the molecular size.