Protein microarrays are many things to many people. Like the more mature DNA “chips”, they are often patterns of tiny spots arrayed on a microscope-type slide, used to determine the makeup and amount of protein in a sample in a multiplex fashion. But not always.
Just as there are a wide variety of uniplex protein assays, there are a variety of multiplex protein assays as well. Chips, yes—but they’re also found arrayed on the bottom of microwell plates and as beads in a tube. These, and others, were hot topics at BIT Life Sciences’ recent “PepCon” protein and peptide conference.
Protein chips are typically laid out by using a contact pin spotter or a noncontact microarrayer to immobilize ligands on a substrate. These can be antibodies, aptamers, peptides, or other molecules whose binding affinity is used to query samples. In the case of peptides, arrays can be created from random libraries for biomarker discovery, for example, or from series of overlapping protein sequences for epitope mapping.
For PEPperPRINT’s PEPperCHIP® microarrays, peptides are synthesized directly on a functionalized glass slide with a 20-color laser printer. The activated amino acids, which are embedded in the toner, are released from the solid matrix material upon melting. “The toner matrix liquefies and the activated amino acids can approach amino groups on the surface of the chip,” explained CEO Volker Stadler, Ph.D., “and that way they can couple covalently to form the peptides on the chip.” Up to 20 mers are produced by sequential rounds of heating, washing, and deprotection.
Toner is heated to just above the melting temperature of the amino acids so there is little or no evaporation. “And because the toner matrix has an oily consistency, it doesn’t spread on the slide surface. This is a key feature that allows us to achieve much higher spot densities compared to other technologies.”
Another major advantage of this approach, Dr. Stadler added, is that the combinatorial laser-printing process allows for one-off microarrays to be easily produced. “It’s the same effort to synthesize hundreds of different or hundreds of the same peptide microarrays.” Thus the Heidelberg-based biotech can offer custom microscope slide-sized arrays with about 9,000 peptides on a slide for around c900, or about c0.10 per feature. These high-density arrays allow for low per-feature reagent consumption as well, typically requiring only about 5–10 microliters of sample per chip.
Standard PEPperCHIPs can be processed and read with standard lab equipment. PEPperPRINT also offers larger slide formats with up to 275,000 features.
The company, which has been developing its technology for the past decade, began offering screening services in early 2010. In March of this year it started marketing chips for its customers to use in-house. It is currently focused on serology and antibody analysis—“it’s the most straightforward way to work with peptide chips,” Dr. Stadler said.
The chips can also be used for kinase, acetylase, deacetylase, and other enzymatic analyses, “but we don’t have enough experience in the field to really know that they work as well as for standard applications,” Dr. Stadler confessed. “So we have collaborators testing the chips now” to see how they perform and to elicit feedback on how to improve them for enzymatic assays.