[Adopted from Materials Today, 21 June 2013]

New sensing strategies

Apart from science fiction, invisibility to the human eye is a property hard to achieve for macroscopic objects. The simplest situation in which invisibility could occur is when a homogeneous material is incorporated in another material with the same refractive index. Because the refractive index of water is exceptionally low, this condition cannot be achieved with solid objects in air and it is also hard to obtain in water; lower than the vast majority of dense materials.

Amorphous plastics of perfluorinated polymers are among the very few homogeneous transparent materials that can match the refractive index of water, hence becoming almost invisible when immersed in a aqueous solution. A recent work appeared on PNAS [1] demonstrates that this peculiar optical condition makes perfluorinated plastic a remarkable sensing substrate of a novel optical label-free biosensor technology, named Reflective Phantom Interface (RPI).

In that work we exploited the index matching between water (refractive index n = 1.333) and a specific plastic made of an amorphous copolymer of tetrafluoroethylene (n = 1.327). The interface between the two materials reflects light only by a few parts per million. In this condition, even a thin layer of carbon-based molecules adhering on such interface induces an increment of reflectivity that can be easily measured because of the low background.

This property can be exploited in biosensing by providing the plastic surface with bio-recognition functionality. This is done by immobilizing antibodies in 10-100 µm sized spots. The notoriously difficult conjugation of the perfluorinated surface with the carbon-based compounds was achieved through an acrylate copolymer with multifunctional groups in the chain. As the functionalized surface is placed in contact with blood serum containing biomarkers, they are captured on the spots carrying the specific antibodies that recognize them. This results in the increment of the reflectivity of the spots involved in the specific interactions. The surface reflectivity is detected through a simple imaging system based on LED illumination and CCD camera (see Figure).

Despite the simplicity of the approach, the RPI method enables a direct (label-free), multi-spot and real-time detection of biomarkers. The local amount of molecules on the surface is readily quantified through basic thin film reflection formulas. In the PNAS paper it was shown that diagnostically relevant concentrations (few ng/ml) of Hepatitis B and HIV markers can be simultaneously detected in few minutes without the need of adding fluorescent or colorimetric labeling agents.

The limit of detection of RPI is of about 2-3 proteins per µm2, comparable to the performance of the widely used biosensors based on Surface Plasmon Resonance, albeit the RPI is based on a markedly simpler concept. Indeed, the elementary design of RPI, together with the possibility of easily shaping the sensing plastic substrate and of employing low-cost components, provides an unprecedented flexibility in the design of biosensors for the analysis of macromolecular interactions.

1. Giavazzi, F. et al. Multispot, label-free biodetection at a phantom plastic-water interface. Proceedings of the National Academy of Sciences of the United States of America (2013) 110(23):9350-9355.

Optical set-up of RPI and acquired image of the antibody-spotted surface. The surface of a prism of perfluorinated material isorefractive with water was coated with a functional copolymer enabling the immobilization of spot (100 ?m diameter) of different antibodies toward p24 capsid protein and hepatitis B surface antigen. The light reflected by the surface is imaged on a CCD camera. The amount of molecules bound on each spot is directly obtained from the local brightness.

This story is reprinted from material from M. Buscaglia and T. Bellini, Department of Medical Biotechnology and Translational Medicine, University of Milano, Italy. Editorial changes made by Materials Today.