Time-resolved spectroscopy plus careful calculations can optimize photoluminescent probes for studying DNA, with results nearly twice as good as standard fluorescence spectroscopy for specific sequences, according to a new paper from Rice University.

The method has been around for a while, at least in the lab of Angel Martí, an assistant professor of chemistry and bioengineering, who said the equations were the commonsense results of years of working with fluorescence spectroscopy. But, he said, when he looked for materials to help teach his students how to use time-resolved techniques to improve probes' resolution, he found none. "I thought there must be some publication out there that would describe the tools we use, but there weren't any," he said. "So we've had to write them."

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A new method using time-resolved spectroscopy optimizes results from photoluminescent probes essential to the study of microscopic structures such as cells, proteins and DNA. The technique doubled the efficiency of a hairpin-shaped probe called a molecular beacon in finding a specific DNA sequence by maximizing the amount of signal pulled from the background noise. (Image: Martí Group/Rice University)

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A long-standing problem with photoluminescent probes, Martí explained, has been that even in an experiment lasting a fraction of a second, a spectrometer can return too much information and obscure the data researchers actually want. "In standard fluorescence spectroscopy, you see noise that overlaps with the signal from your probe, the scattering from your solution or cuvettes, plus the noise from the detector," he said. The saving grace, he said, is that not all those signals last the same amount of time.

Time-resolved spectroscopy provides part of the answer, Martí said. Compared with standard spectroscopy, it's like taking a film instead of a snapshot. "We create a kind of movie that allows us to see a specific moment in the process where photoluminescence is occurring. Then we can filter out the shadows that obscure the measurement or spectra we're looking for," he said.

In an edit of this "movie," which can be captured in real time, they chop off the front and back to narrow the data set to a range that might last only 80 ns, when the probe signal is strongest and the background signals are absent.

But it's critical to know just the right window of time to study, Martí said. That's where the Rice methodology removes any uncertainty. The equations the researchers developed let them analyze all the factors, such as the emission intensity and decay of the specific probe with and without the target and the anticipated level of background noise; they can then maximize the signal-to-background noise ratio. The technique works even with probes that are less than optimal, he said.

To prove their method, Martí and co-author Kewei Huang, a graduate student in his group, tested ruthenium- and iridium-based light-switching probes under standard fluorescence and time-resolved spectroscopy. The hairpin-shaped probes' middles are designed to attach to a specific DNA sequence, while the ends are of opposite natures. One carries the fluorophore, the other a chemical quencher that keeps the fluorescence in check until the probe latches onto the DNA. When that happens, the fluorophore and the quencher are pulled apart, and the probe lights up.

The individual signal is a flash too tiny and quick for the naked eye to see. "But our instruments can," Martí said. "We're trying to show that you can use time-resolved spectroscopy for many applications, but to use it in the right way, you have to do some analysis first," he said. "If you do it in the correct way, then it's a very powerful technique."

Improving a probe's ability to detect ever smaller and harder-to-find targets is important to biologists, engineers and chemists who commonly work on the molecular scale to analyze cell structures, track disease or design tiny machines. In combination with a technique called fluorescence lifetime microscopy, the Rice calculations may improve results from other diagnostic tools that gather data over time, such as MRI machines used in hospitals.

The research was published in the American Chemical Society journal Analytical Chemistry (doi: 10.1021/ac3019894).

For more information, visit: http://amarti.web.rice.edu

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