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No-glow tags for Raman spectroscopy tions were detectable in the 1-µm viewFluorescent tags are commonly used in In theory, 428 single-stranded DNA Raman spectroscopy, but the utility of molecules could bond with a single gold ing area available with a confocal Raman microscope. “Conventional microarrays nonfluorescent tags has barely been exnanoparticle, the group calculated. This 2 use a 250–400-µm spot to detect just plored. In this issue of Analytical Chemtranslated into 25.1 pmol/cm of DNA one interaction,” Irudayaraj points out. istry (pp 3981–3988), Joseph Irudayaraj on the gold surface. In practice, the reHe predicts that the achievable degree searchers obtained an average coverage and colleagues at Purdue University of multiplexing will be problem-dependof 67%; therefore, a third of the surface have shown that such tags can be used ent. “But we’re thinking that we can remained available for the tags. The to detect up to eight DNA probes at push this to at least 15 tags,” he says. ability to control Raman intensity by one time. “The most immediate potenTags that interfered with the binding altering the probe concentration was tial application is in high-throughput of DNA probes to target screening for genome analyDNA wouldn’t be useful, of sis,” Irudayaraj says. course. To make sure this Nonfluorescent tags have S wasn’t the case, the group several advantages over fluoThiolated oligo sequence obtained a UV–vis spectrum rescent tags. For example, + Au S Au S SH from each tagged probe and many fluorophores tend to S then added complementary photobleach or have broad DNA attached to gold partiemission profiles. And intercles. In every case, the color preting the results is often a changed from red to purple, statistical challenge. MoreNonfluorescent Raman tags S indicating hybridization. over, some molecules in bioAu S S Au Other observations validated logical mixtures can replace this conclusion. The targets fluorescent tags on DNA, givS could be detected at picomoing anomalous Raman signals. lar concentrations, but IrudaIn contrast, nonfluorescent yaraj predicts that femtomotags produce sharp Raman DNA probes with nonfluorescent Raman tags are made by thiolating lar sensitivity could be signals and don’t have to be the probes and allowing them to bind chemically to gold nanoparticles. obtained by optimizing the attached directly to DNA. When the tags are added, they occupy some of the remaining spaces concentrations of the tags “And detection is visual. and probes. (Sensitivity was Therefore, without any statis- on the particle, tagging the probes without binding directly to them. not the focus of this prooftics, we can say whether a pardemonstrated using 4-mercaptopyriof-principle study, however.) ticular probe is present,” Irudayaraj says. dine, one of the eight Raman tags choEncouraged by their results, the The group used molecules with a sen. This feature is a big advantage for group is working to create DNA mithiol group or carboxamide moiety as croarrays with nonfluorescent Raman Raman tags. Instead of joining them di- multiplexing, Irudayaraj says, because tags. “We have demonstrated the princirectly to DNA probes, they attached the the concentration of weaker tags could be increased to give signals that are as ple, but I think the attachment chemprobes to gold nanoparticles, which endetectable as those of stronger tags. istry could be improved,” Irudayaraj hance Raman signals, and allowed the “Therefore, it’s possible to detect DNA says. He adds that the tags used in the tags to occupy the vacancies on the surcurrent study emit in only part of the face. In this way, they avoided the tricky probes at a sensitivity level equal to or –1 Raman spectrum (400–1700 cm ); chemistry needed to directly tag probes; even greater than what you can obtain by labeling probes with fluorescent therefore, many other commercially instead, they relied on the strong covadyes,” Irudayaraj says. available chemicals that give signals in lent bonds that thiol groups and carTo see if multiplexing would be feasi- other areas of the spectrum might also boxamide moieties form with colloidal ble, the researchers mixed two to eight be useful. The group would like to use gold. However, the tags still identified Raman-tagged microarrays to examine the probes, much as an address tag on a DNA probes labeled with different tags and obtained Raman spectra. Because alternative splicing profiles of breast key ring would identify a house key. several sharp peaks characterized the cancer genes. “But getting good, conMoreover, the constructs were very staspectrum of each tag, it was easy to sistent signals is the challenge,” Irudable. After 3 months in the refrigerator, identify the tags from the composite yaraj says. a they produced the same Raman spectra spectra. Moreover, up to eight interac—Linda Sage at the same intensity as before storage. J U N E 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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