Immunosensors are widely used in medicine, microbiology and environmental monitoring. These versatile biochemical tests measure the concentration of substances in biological liquids such as blood or urine, by reacting antibodies with their corresponding antigens.

Immunosensors are often built onto tiny two dimensional substrates to create ‘on-chip’ sensors. This helps their production, improves sensitivity, and facilitates sensing in array formats, but the overall antibody activity can be reduced due to the spatial constraints on the surface. Now, Michael Himmelhaus and Sivashankar Krishnamoorthy at Fujirebio Inc. in Tokyo1 demonstrate that the antibody-antigen interactions can be greatly enhanced by confining the antibodies within artificial ‘nanopatterns’ on the chip surface.

The nanopatterns were fabricated by coating a silicon plate with a gold film, followed by a single molecular layer of an alkanethiol to immobilize antibodies. Then, 500 nm diameter polystyrene beads were deposited onto this surface.

Fig. 1: Atomic force microscope image of the new design for an on-chip immunosensor, with nano-sized ‘fouling patches’ (white dots) that have an affinity for antibodies. When antibody molecules are confined to these patches they tend to stand up straight – the preferred alignment for interacting with antigens.Copyright 2008 WILEY-VCH Verlag GmbH & Co.

A reactive ion process was used to remove the alkanethiol from everywhere except for areas that were covered by the polystyrene beads (Fig. 1). These areas remained as nanopatterned ‘fouling patches’ to trap antibodies.

The functionality of the nanopatterned surfaces was tested by applying matching pairs of Immunoglobin antibodies and antigens taken from mice. The researchers found that the antibody-antigen binding was 120% better than on non-patterned surfaces.

Atomic force microscopy showed a high density of antibodies to collect on the fouling patches. This close crowding on the fouling patches caused the antibody molecules to ‘stand up’ at right angles to the surface – the best orientation for binding to antigens. The molecules also have more space in between the patches to flip themselves the right way up, before they move to the patches by diffusion.

In the future, the researchers want to optimize the distribution of fouling patches for their immunosensor, to improve binding efficiency. They will also test their design with other antibody-antigen pairs to see if the technique can be generalized. In the meantime, their study stands as proof that the performance of an immunosensor can be vastly improved by tailoring its surface with patterns on a similar scale to the antibodies.