Fluorophore multimerization on a PEG backbone as a concept for signal amplification and lifetime modulation

Abstract

Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels' fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.

Keywords: Brightness; Flow cytometry; Fluorescence lifetime imaging (flim); Fluorescence microscopy; Label; Multimeric dyes; Photostability.

Figure 1

Chemical structures and schematic representation of (a) Fam (5/6-Carboxyfluorescein) succinimidyl ester 1 and PEG intermediates 2 used in this study; (b) antibody conjugates prepared either via conjugation with a monomeric fluorophore or with its multimeric PEG intermediates. The fluorophores are depicted as glowing stars.

Figure 2

Spectroscopic characterization of the monomeric and multimeric Fam and Vio515 dyes and their CD4-antibody conjugates in PBS buffer. (a) and (b) Normalized absorbance and fluorescence spectra of monomeric and multimeric Fam and Vio515 and the respective CD4-conjugated labels. Excitation wavelength used for all fluorescence spectra: 488 nm. Solid lines: absorption spectra, dashed lines: fluorescence spectra. (c) Fluorescence quantum yields (blue) and intensity weighted mean fluorescence lifetimes (red) of the monomeric and multimeric Fam and Vio515 dyes and their CD4-antibody conjugates revealing a bioconjugation-induced drop in fluorescence lifetime and QY of the monomeric dyes and underlining the preservation of the fluorescence properties for the multimeric labels. Fam sodium salt was used as a reference since the 5/6-fluorescein NHS ester shows a strongly pH-dependent QY (see Table 1).

Figure 3

Quantification and comparison of the mean fluorescence intensities (MFI) of the different monomeric and multimeric labels used for the staining of the SUP-T1 cells. (a) Representative flow cytometry dot plots obtained for fixed SUP-T1 cells stained with CD4 conjugates at 10 µg/mL. The y-axis represents the mean fluorescent intensity detected in the B1 channel and the x-axis the forward scatter. (b) Quantification of the fluorescent signals of each conjugate measured in different concentrations on fixed SUP-T1 cells using flow cytometry. In cell staining experiments, the multimeric VioBright dye conjugates (yellow and orange lines) provide a higher fluorescent signal in comparison to their monomeric dye conjugates (black and grey lines). (c) Representative CLSM images of the CD4 conjugates utilized for cell staining at 5 µg/mL targeting the surface receptor CD4 & CD8 of fixed cells and (d) quantification of surface staining at the given conjugate concentrations. The cells were incubated with conjugates for 10 min at room temperature. The scale bar equals 50 µm.

Figure 4

(a) Intensity weighted mean lifetimes of the CD4-conjugated dyes and labels bound to beads and SUP-T1 cells in low and high labeling densities, as calculated from the FLIM measurements (for more details see SI). (b) Representative FLIM measurement results demonstrating the principal feasibility of the reported dyes and labels for lifetime-based analyte discrimination. Top left: Mixture of beads stained with CD4-Fam (low labeling density) and SUP-T1 cells with CD4-VB-Fam (high labeling density). Top right: Mixture of SUP-T1 cells with CD4-Fam and CD8-Vio515 (both with high labeling density). Bottom left: Mixture of SUP-T1 cells with CD4-Fam and with CD4-VB-Vio515 (both with high labeling density) and beads with CD4-VB-Fam (low labeling density). Bottom right: Transmission image for morphological identification in the bottom left image.