Samples and Substrates
Although protein microarray technology borrows extensively from the science behind DNA microarrays, there are fundamental differences between proteins and DNA, primarily due to the markedly increased heterogeneity of proteins by comparison. Whereas DNA microarrays can be printed onto a range of surface chemistries with a range of buffers, protein microarrays require printing buffers, which are in some cases specific for each individual protein.
In DNA microarrays, capture/hybridization is based on primary structure only, i.e. base sequence. In contrast, a given protein’s complex 3-D structure must be maintained for capture or detection to be possible. This has led to the development of substrate chemistries (Table 1) and printing buffers designed with the intention of preserving protein structure and/or activity.
The buffers themselves, as well as the proteins in them, can affect viscosity tremendously. It is common, for example, to find proteins dissolved in a buffer containing high percentages of glycerol, as this has been shown to improve protein stability. Alternatively, protein samples have been kept at low temperatures during printing for the same purpose. In either case, the end result is a set of samples of differing viscosities, which need to be printed onto what is most likely a fragile surface.
Arrayjet technology is suited to printing high-quality protein microarrays: a modern piezoelectric inkjet print head is used for printing the arrays. The print head, a Xaar XJ126, has been adapted by Arrayjet from an altogether unrelated industry—printing ink onto paper and textiles. This adaptation is the subject of two key patents filed by Arrayjet in the area of microarray production, one of which has already been granted. As such the print head, which was developed for industrial-scale printing and is therefore highly robust, is working almost at idle in microarray applications.
The print head contains 126 nozzles in a linear arrangement. The nozzles are employed in subsets for printing microarrays of a variety of sample types. Importantly, the print head is suitable for printing viscous samples and can easily print samples of up to 20 centipoise (cP) in fluid viscosity (1 cP is equivalent to the viscosity of water), including high concentrations of expressed proteins and highly complex protein mixtures, such as cell lysates, irrespective of lysis method.
Concerning stability, protein printing with a series of glycerol concentrations has already been tested successfully, as have other buffers used in protein microarray production, such as phosphate-buffered saline. Additionally, Arrayjet printing technology has been tested successfully down to temperatures of 6°C.