B.C. Borders, C.H. Kang, J.A. Brozik
Photon Biosciences, LLC,
Keywords: luminescence, bioimaging, ultrastable probe
Summary:Luminescent probes, such as fluorescent proteins, fluorescent dyes, phosphorescent dyes, and quantum dots, are important tools in biology, biophysics, and medical diagnostics. While these tools are incredibly useful and are suitable for use in many techniques, they all suffer from drawbacks. Fluorescent proteins and dyes photobleach and photoblink [ 1- 3]. Quantum dots blue over time, photoblink, and can disrupt cellular function due to their relatively large sizes [3,4 ]. All three of those probes have high backgrounds resulting from the autofluorescence of biological materials. While phosphorescent dyes do not blink and can be used in time-gated methods to eliminate autofluorescence, they are not as luminous as other probes and they still photobleach . These drawbacks limit the use of these probes, particularly in situations in which trace level detection is necessary. We have developed a new ultrasensitive, ultrastable, genetically expressible bionanoparticle that combines a recombinant protein with a unique biomineralization process. This new bionanparticle is called RECAL®. RECAL® is non-bleaching and non-blinking. Its excitation and emission wavelengths can be tuned in the mineralization step and span much of the visible and near infra-red regions of the electromagnetic spectrum. The excitation and emission bands of RECAL® are narrow and have a large Stokes shift. RECAL® is a phosphorescent probe, but its unique properties result in an overall luminescence intensity that approaches many fluorescent organic molecules. The phosphorescent nature of RECAL® in addition to its narrow excitation and emission bands make it possible to isolate its signal, eliminating the problem of autofluorescence through the use of time-gated techniques and narrow bandpass filters. Because RECAL® doesn’t photoblink or photobleach, it has an unprecedented time-integrated level of detection. This makes it possible to achieve detection at trace levels (20-50 pM) and even possible at ultratrace levels (20-50 fM). This will allow for not only conventional imaging/quantitative tools with increased precision, but also new studies, particularly in the field of single-molecule tracking and detection, that are not currently possible or are impractical with the technology that is currently on the market. Here we show the comparison of two different cell lines expressing RECAL® and GFP. The luminosity of the cells expressing RECAL® was consistent over a long period of time, while the luminosity of the cells expressing GFP decreased over time. The time-integrated levels of detection of conjugated RECAL® probes and conjugated GFP probes were compared. While the luminosity of the GFP probes decreased over time, the luminosity of the RECAL® probes was consistent over time. Tracking experiments show that the individual RECAL® probes do not photoblink or photobleach and are readily detectable at the single-molecule level.