An improved approach to use protein A for signal enhancement of miniaturized immunoarrays

Abstract

Miniaturization of microarrays is a crucial step towards development of portable biosensors for simultaneous detection of multiple (biological) targets. The main challenge in the process of miniaturization of the active site (spot) is the loss of signal (roughly proportional to the area), resulting in low sensitivity. One way to improve the performance of IgG-based sensors is oriented immobilization of capture antibodies. Here we suggest an improved protocol for antibody orientation, mixing the Fc receptor protein A with the IgG molecule and immobilizing the resulting complex. This protocol is compared with both randomly-oriented (covalent) immobilization of IgG onto reactive slide surfaces, and to the widely used protein A coated slides. We performed this comparison both in a standard microarray format (˜100 μm spot diameter) and on miniaturized arrays (2 μm–15 μm diameter) and found that our method yielded higher signal, increased dynamic range and higher binding density compared with randomly-oriented (covalent) binding and the commonly used protein A approach. This approach results in up to a 2 fold increase in signal compared to randomly-oriented immobilization, is suitable for miniaturized arrays as well as standard microarray format and presents clear advantages in simplicity and cost. Moreover, it is the only method that is suitable for spots with diameters < 2 μm.