Optimizing the Sensitivity of Photoluminescent Probes Using Time-Resolved Spectroscopy: A Molecular Beacon Case Study

Improving probes so that they can perform more sensitive and accurate detections is at the heart of much fundamental and applied research. Within the past few years a considerable amount of effort has been devoted to the study of photoluminescent probes in combination with time-resolved photoluminescence spectroscopy (TRPS). Although TRPS is a powerful and important technique for improving the sensitivity of long-lived probes, there is a lack of a general methodology that would allow one to unambiguously optimize the parameter affecting this technique. In this manuscript it will be shown how parameters that are probe- and technique-specific can affect the effectiveness of TRPS in improving sensitivity. Furthermore, it will be demonstrated that, when TRPS is used, the sensitivity of the probe is strongly dependent on the time window used to generate the time-resolved emission spectra (TRES). A method will be described that will allow one to remove the uncertainty in the selection of the time window that would yield the optimum improvement in probe performance, as well as the experimental parameters that need to be considered. Molecular beacon probes (MBs) were used to demonstrate these points. These probes show signal-to-background ratios (S/B) of less than 9 when SSPS is used, which can be easily enhanced to 17 using TRPS. The detection limits were also improved when TRPS is used allowing detecting target DNA with concentrations as low as 13.6 nM.