April 15, 2003 | The advent of DNA chips in the mid-1990s coincided nicely with rapid progress in the pace of DNA sequencing of the human and other genomes. Not only were thousands of gene sequences now available, but the solid-state technology was there, borrowed from the semiconductor and high-tech industries — from silicon chips to inkjet printing to photolithography.

Most drug targets are proteins, and many researchers are interested in tools that can do for protein expression profiling what DNA chips have done for RNA expression. Analyzing protein expression can help researchers understand the molecular basis of disease, including disease susceptibility, diagnosis, progression, and potential points of therapeutic interference, says Peter Wagner, chief technology officer at Zyomyx, a biotech company that has recently unveiled such a "protein chip."

Arraying proteins to a solid surface in an active form is neither simple nor straightforward. Proteins possess great variation in structure and are far less stable than DNA. "The fundamental challenge in developing protein chips derives from the interplay between protein structure and the molecular complexities of surface chemistry," Wagner says. "Denaturation is a particular problem when proteins find themselves at a solid-liquid interface."

As with DNA chips, Zyomyx's chips start with a silicon wafer. But that's where the similarity stops. "With protein chips, there has been a paradigm shift from density to quality and accuracy of the individual data point," Wagner says, "and this creates a whole different set of analytical and process tools."

Although the details are proprietary, Zyomyx has published a prototype chip design (Ruiz-Taylor et al., Proceedings of the National Academy of Sciences 2001; 98: 852). The wafers are subjected to photolithography to create raised pillars that are then coated with an inorganic material to give the surface a negative charge. An organic polymer, poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG), forms an ordered monolayer on the negatively charged chip surface. PLL-g-PEG contains a PEG moiety derivatized with biotin that points upward into the aqueous solution on the chip. PEG compounds resist nonspecific adsorption of proteins from the biological fluid being assayed, while the biotin molecule provides an attachment point for specific capture molecules — usually antibodies or antibody fragments. The capture molecules remain in solution — permitting analysis of much smaller sample volumes than conventional technologies — without drying out.


The Secreted Life of Chemokines 
The first product exploiting this technology is the Human Cytokine Biochip, which can monitor 30 biologically relevant cytokines, enabling the comprehensive analysis of these important mediators of inflammatory and autoimmune disease. Part of the logic behind creating a chemokine chip was to exploit the advantages that a protein chip format provides over a DNA chip for certain applications. Chemokines are secreted proteins that localize to very specific regions of the immune system — often quite distant from the cell type in which their messenger RNA was produced. DNA chips are simply not suited to such soluble analytes.

Zyomyx's biochips can be used to evaluate a variety of sample agents, ranging from synovial fluid and cell lysates to serum and plasma. These chips are only one component of its Protein Profiling Biochip System, which also includes the Assay 1200 automated workstation, the Scanner 100 biochip reader, and data analysis software. The system can simultaneously process 12 chips, representing 55 patient samples, in less than two hours.

The Protein Profiling Biochip System is already in use at pharmaceutical and research institutes, including GlaxoSmithKline and Partners HealthCare in Boston. The chips are also used at the Harvard Partners Genome Center's new Protein Microarrays Facility. "Just as DNA chips were equivalent to doing thousands of Northerns [experiments monitoring RNA expression], these new protein microarrays are equivalent to doing thousands of Elisa [arrays]," says George Grills, director of the facility.

Although much of its future hinges upon the success of the Protein Profiling Biochip System, Zyomyx is working on several related products. A mouse cytokine chip will be useful in studying mouse models of human disease. MDS Proteomics is teaming with Zyomyx on a protein interaction assay — a sort of solid-state two-hybrid assay. Another product under development is a chip to measure the activity of arrayed ion channels — interesting drug targets, but particularly difficult to work with due to their membrane localization. Although the number of assays on the chemokine chip is only 30, higher-density formats, perhaps into the thousands, are under development.

With the protein chip market estimated to surpass $1 billion annually, the payoff could be dramatic.

Robert M. Frederickson is a biotech writer based in Seattle. He can be reached at rfreder@yahoo.com.