Enhanced DNA detection using a multiple pulse pumping scheme with time-gating (MPPTG)

Abstract

Fluorescence signal enhancement induced by the binding of intercalators to DNA has been broadly utilized in various DNA detection methods. In most instances the increase in fluorescence intensity is associated with a concomitant increase of fluorescence lifetime. This increase of the fluorescence lifetime presents an additional opportunity to increase detection sensitivity. In this paper, we present a new approach to significantly enhance the sensitivity in detecting minute DNA concentrations. The approach is based on simultaneous use of time-gated detection and multi-pulse pumping. By using a calibrated burst of short pulses we greatly enhance the contribution of long-lived fluorescence species, thus enabling easy time-gated detection. Using a classic DNA intercalator - Ethidium Bromide (EtBr) - as an example with our novel multi-pulse pumping and time-gated detection technique, we were able to increase detection sensitivity over 70-fold with only 3 pulse excitation. This approach is generic and can be used with any analytical probe (exhibiting about 10 times change in lifetime) that shows an increase in fluorescence signal and fluorescence lifetime upon binding to a target.

Förster Resonance Energy Transfer-Enhanced Detection of Minute Amounts of DNA

“…Recently, we also realized that long-lived probes offer the possibility for multipulsed excitation, further improving the relative signal of fluorophores that present longer fluorescence lifetimes. , The highly enhanced signal by multipulsed excitation makes it feasible to utilize typically low brightness yet long-lived probes. Using such technology with time-gated detection, we demonstrated that the detection sensitivity could be enhanced by two orders of magnitude when using one of the earliest used DNA intercalators, ethidium bromide (EtBr). , Additional emission spectra decomposition allowed subtraction of the Raman contribution, further improving detection sensitivity …”

“…The fluorescence lifetime of DAPI is ∼2 ns, much shorter than the fluorescence lifetime of EtBr bound to DNA (∼20 ns). The long fluorescence lifetime of DNA-bound EtBr allows using time-gating for detection. , With such an approach, detection and visualization of less than 100 picograms of DNA in microliters of solution without any sample purification and DNA amplification were achieved.…”

“…Using such technology with time-gated detection, we demonstrated that the detection sensitivity could be enhanced by two orders of magnitude when using one of the earliest used DNA intercalators, ethidium bromide (EtBr). 24,25 Additional emission spectra decomposition allowed subtraction of the Raman contribution, further improving detection sensitivity. 26 This report presents an original approach that utilizes Forster resonance energy transfer (FRET) between intercalating dyes to significantly increase detection sensitivity, limit Raman contribution, and increase specificity for detecting intercalators exclusively bound to DNA.…”

Imaging and detection of long-lived fluorescence probes in presence of highly emissive and scattering background

“…For the majority of typical fluorophores, the fluorescence lifetime will range from sub-nanoseconds to a few nanoseconds, and only a handful of emitters will have a longer fluorescence lifetime. Long-lifetime emitters may have lifetimes in the range of 20 ns like ADOTA 5,6 or ethidium bromide when intercalated to DNA, 7,8 to hundreds of nanoseconds and microseconds like ruthenium metal–ligand complexes 9–12 (MLCs) and some metal clusters, 13,14 or even milliseconds with lanthanide-based emitters. 15–18 Some quantum dot-type emitters will also have long emissive lifetimes over 20 ns.…”

“…When using multipulse pumping and time-gated detection (MPPTGD), even for relatively short probe lifetimes in the range of 20 ns, we increased the detection sensitivity by two orders of magnitude. 7,38…”