Highly cooperative fluorescence switching of self-assembled squaraine dye at tunable threshold temperatures using thermosensitive nanovesicles for optical sensing and imaging

Abstract

Thermosensitive fluorescent dyes can convert thermal signals into optical signals as a molecular nanoprobe. These nanoprobes are playing an increasingly important part in optical temperature sensing and imaging at the nano- and microscale. However, the ability of a fluorescent dye itself has sensitivity and accuracy limitations. Here we present a molecular strategy based on self-assembly to overcome such limitations. We found that thermosensitive nanovesicles composed of lipids and a unique fluorescent dye exhibit fluorescence switching characteristics at a threshold temperature. The switch is rapid and reversible and has a high signal to background ratio (>60), and is also highly sensitive to temperature (10-22%/°C) around the threshold value. Furthermore, the threshold temperature at which fluorescence switching is induced, can be tuned according to the phase transition temperature of the lipid bilayer membrane forming the nanovesicles. Spectroscopic analysis indicated that the fluorescence switching is induced by the aggregation-caused quenching and disaggregation-induced emission of the fluorescent dye in a cooperative response to the thermotropic phase transition of the membrane. This mechanism presents a useful approach for chemical and material design to develop fluorescent nanomaterials with superior fluorescence sensitivity to thermal signals for optical temperature sensing and imaging at the nano- and microscales.

Figure 1

Fluorescence switching of self-assembled squaraine dye (SQR22) in lipid bilayer membrane of nanovesicles (NV) while heating and cooling.

Figure 2

Spectroscopic properties of SQR22. (a) 10 µM SQR22 solutions in a bright field (top) and under UV irradiation (λ = 365 nm, bottom): 1 = hexane, 2 = cyclohexane, 3 = toluene, 4 = benzene, 5 = dichloromethane, 6 = tetrahydrofuran, 7 = chloroform, 8 = ethanol, 9 = methanol, 10 = acetone, 11 = acetonitrile, 12 = N, N-dimethylformamide (DMF), 13 = dimethyl sulfoxide (DMSO), 14 = water. (b) UV-vis-NIR spectra of 10 µM SQR22 solutions. (c) Fluorescence emission spectra of 1 µM SQR22 solutions. (d) Quantum yield and maximum emission wavelength as a function of polarity index.

Figure 3

Reversible fluorescence switching on NV containing SQR22 (NVSQ) during heating and cooling. (a) Photographs showing the fluorescence of the NV consisting of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC₁₆) dispersion (PC₁₆-NVSQ, Lipid/SQR22 = 50 w/w, [SQR22] = 10 µM)) under UV radiation (λ = 365 nm) at 25 °C (fluorescence OFF) and around 50 °C (fluorescence ON). (b) Profiles of fluorescence intensity changes upon heating of PC₁₆-NVSQ at 25 and 50 °C. The arrow indicates the sample (ca. 23 °C) injection point into a quartz cuvette incubated at the target temperature. (c) Repeatability of fluorescence switching for PC₁₆-NVSQ ([SQR22] = 1 µM) through 10 heating/cooling cycles. The fluorescence intensity was measured alternately at 25 (cooling) and 48 °C (heating).

Figure 4

Incorporation of SQR22 into nanovesicles’ lipid bilayer membrane. (a) Scheme of the nanovesicles consisting of SQR22, 1,2-diacyl-sn-glycero-3-phosphocholine (PC), anionic lipid, and PEG-lipid (PC-NVSQ). Five PCs with different acyl chain lengths were used. (b) Size distribution of PC-NVSQ measured by dynamic light scattering. (c) UV-vis-NIR absorbance spectra of PC-NVSQ in phosphate buffered saline (PBS) at 25 °C ([SQR22] = 10 µM). The right photograph shows the PC-NVSQ dispersion ([SQR22] = 80 µM).

Figure 5

Temperature-dependent fluorescence characteristics of PC-NVSQ. (a) PC-NVSQ dispersions ([SQR22] = 10 µM) under UV radiation (λ = 365 nm) at 25 °C. Samples correspond to PC₁₄-, PC₁₅-, PC₁₆-, PC₁₇-, and PC₁₈-NVSQ. (b) Fluorescence emission spectra of PC-NVSQ dispersion (Lipid/SQR22 = 50 w/w) at 40, 45, 50, 55, and 60 °C, respectively for PC₁₄-, PC₁₅-, PC₁₆-, PC₁₇-, and PC₁₈-NVSQ (λ_ex = 570 nm). (c) Change in PC-NVSQ fluorescence intensity as a function of temperature ([SQR22] = 1 µM) during heating and cooling. Data are mean ± standard deviation (SD) acquired from three independent experiments.

Figure 6

Microscopic monitoring of the reversible switching of fluorescence on PC₁₆- multilamellar vesicles (MLV) containing 3.3 mol% SQR22 upon heating and cooling. (a) Schematics of fluorescence switching on vesicles containing SQR22 by heating with 980 nm NIR laser irradiation. (b) Fluorescence images of MLV containing SQR22 fixed in agarose gel; before heating (0 s), during heating (43 s), and after cooling (77 s). (c) Evolution of the SQR22 fluorescence emission. The fluorescence intensity is normalized to the maximum value. Average of the normalized intensity (four dots shown in b) was plotted against time. (d) Time-lapse images of MLV containing SQR22 as a “single dot” during the heating/cooling process. The single dot was chosen from those in (b).

Figure 7

Spectroscopic analysis of fluorescence switching. (a) PC₁₆-NV with different various amounts of SQR22 ([SQR22] = 10 µM) under UV radiation (λ = 365 nm) at 25 °C. (b) Change in fluorescence intensity of SQR22-loaded PC₁₆-NV dispersions as a function of temperature. Data were acquired upon cooling. (c) Change of fluorescence intensity at 25 and 45 °C, and ratio of intensity at 25 (FI₂₅) and 45 °C (FI₄₅) as a function of SQR22 content in PC₁₆-NV ([SQR22] = 1 µM). (d) Change in UV-vis-NIR spectra as a function of temperature in PC₁₆-NV containing 3.3 mol% SQR22 ([SQR22] = 10 µM).

Figure 8

Proposed mechanism for SQR22 fluorescence switching in the lipid bilayer membrane as a response to phase transition. The fluorescence off/on switching is a result of the reversible aggregation-caused quenching/disaggregation-induced emission of SQR22 in the gel to liquid crystalline phase transition temperature (T) of the lipid bilayer membrane.