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RESEARCH PROFILE Enzymatically amplified SPR imaging merase and other reagents in the sample aging. Using this approach, multiple To enhance the sensitivity of surface did not have much of an effect on the DNA targets can be detected at femtoplasmon resonance (SPR) imaging for analysis. molar concentrations on a single chip. DNA and RNA microarray analysis, One additional aspect that distin“This is a new paradigm for enzyRobert Corn and colleagues at the Uniguishes this new approach from tradimatic assays, in which the enzymatic versity of California, Irvine (UCI), retional SPR measurements is that the substrate is attached to the surface,” cently developed a new amplification RNA microarrays were created using a says Corn. The new configuration is process based on the enzyme RNase H. single monolayer, rather than the more the opposite of classic enzymatic assays, Now, in this issue of Analytical Chemcommon polymer thin film, says istry (pp 6173–6178), the reCorn. This design allowed the researchers describe the approach RNA/DNA RNA searchers to study surface enzyme in more detail and show that it (a) kinetics. Thin films are typically can be used to detect longer DNA impregnated with binding sites, DNA fragments, including unso “instead of binding to a surpurified PCR products. face, it is more of a partitioning Enzymatically amplified SPR RNase H into a thin film,” says Corn. “You imaging is ~106 more sensitive have to be careful because somethan traditional SPR imaging. times you just measure the kinetThe process uses the enzyme ics of uptake into the film,” he RNase H to repeatedly destroy (b) (c) warns. RNA from RNA–DNA heteroIn the new approach, the SPR duplexes on a gold surface. signal arises from the hydrolysis RNase H selectively degrades and removal of RNA from a gold RNA from these heteroduplexsurface. A series of kinetics experies, but it does not digest singlements showed that this initial rate and double-stranded RNA and of hydrolysis is directly related to DNA. “The enzyme is only ac(a) Complementary DNA binds to an RNA probe, forming an tive when the target [DNA] RNA–DNA heteroduplex. (b) RNase H digests the surface-bound the surface concentration of RNA– DNA heteroduplexes. This conbinds to the substrate [RNA] RNA and releases the DNA. (c) The process repeats itself until centration on the surface can be on the surface,” explains Corn. many RNA probes have been removed by each DNA molecule. linked quantitatively to the conUnlike PCR amplification, in centration of DNA in solution. which multiple copies of DNA are made, such as an ELISA, in which an enzyme The researchers envision all sorts of binds to a target on the surface and enzymatically amplified SPR uses only a applications for enzymatically amplified then turns over a substrate in solution, small number of DNA molecules. “We SPR imaging, including the identificawhich then changes colors or emits are just amplifying the signal by having tion of viruses and bacteria, detection of one DNA chew up lots of surface-bound light, he says. biowarfare agents, DNA expression analThe researchers used the new apRNA,” explains Corn. ysis, and drug discovery. “The hardest proach to detect two different sequences The first step is to create microarrays part for us is making the RNA array,” of DNA at concentrations of 10 fM on in which RNA probes are attached to a says Corn. “We have become very good an array containing three RNA probes. surface that is amenable to SPR, such as at making DNA arrays, and we are just In addition, they showed that the techgold. The researchers then simultanestarting to learn how to make stable nique can be used to analyze longer ously introduce complementary DNA RNA arrays,” he adds. RNA is actually strands of DNA, including a PCR-amand RNase H. The DNA binds to an a relatively stable molecule, but many plified portion of the testis-specific proRNA probe, forming an RNA–DNA enzymes in the environment like to “chew tein Y (TSPY) gene taken from a human heteroduplex, which is then degraded it up,” he says. The key is to keep things DNA sample. A 100 pM solution of the by the RNase H. Once the RNA–DNA very clean. For this reason, the researchpurified PCR product was detected with heteroduplex is destroyed, the DNA can ers look forward to moving into a new negligible nonspecific adsorption using bind with another RNA probe. The building at UCI, equipped with a clean multiple probe sequences. Surprisingly, process is repeated over and over again. when the researchers used the same array room for microfabrication, in the very As RNA molecules are removed from near future. a to test an unpurified TSPY PCR prodthe surface, a change in the reflectivity —Britt E. Erickson uct, they found that the presence of polyof the surface is observed with SPR im386 A
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