Ls55 spectrofluorophotometer

Thiourea, urea, cysteine, glycine and ascorbic acid were purchased from Loba Chemie Pvt Ltd (spectrograde), India. All these reagents were used directly by preparing their aqueous solution using distilled water (18.2 MΩ cm, Millipore). The NCDs were synthesized using a Multiwave-300 microwave synthesis reactor (Anton Paar, USA). A LS-55 spectro-fluorophotometer (PerkinElmer) was used to study its optoelectronic properties. The fluorescence lifetime for NCDs in the presence and absence of the analyte was well determined by time-correlated single-photon counting (TCSPC) measurements using a delta-flex modular fluorescence lifetime system (HORIBA Scientific) equipped with a light-emitting diode (LED) light source (340 nm) with an instrument response function (IRF) of ∼200 ps. The synthesized NCDs were also characterized using a high-resolution transmission electron microscope (HRTEM) (TALOS F200S G2, 200 KV, FEG, CMOS Camera 4 K × 4 K, In Column EDS detectors) for the analysis of their surface morphology and particle size using a drop-casting technique. Moreover, Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR, Shimadzu QATR-S model), XPS, X-ray photoelectron microscopy and energy-dispersive X-ray (EDX) spectroscopy techniques were carried out to gain better insights into the NCD surface.

¹O₂ generation from the CEPP monoliths under different conditions was detected using DMA as the ¹O₂ chemical probe. Eighty milligram of CEPP monolith was immersed into a 12-well tissue culture plate filled with 2 mL of DMA solution (20 μM in DMF), followed by a 1 min irradiation at a light power density of 30 mW/cm² using a 635 nm laser source. The fluorescence spectra of DMA (excitation, 360 nm; emission, 430 nm) as a result of the photosensitization reaction were monitored with a Perkin–Elmer LS55 spectrofluorophotometer (Waltham, MA, USA).

The singlet oxygen (¹O₂) generation was measured by an indirect method using a chemical probe [16 (link)]. In this study, ¹O₂ generation from free Ce6 or DC was detected using DMA as the ¹O₂ probe. Free Ce6 or DC (1 mg/mL Ce6) was first dissolved in DMSO to prepare stock solution. This stock solution was dispersed in DPBS (pH 7.4) to obtain a concentration of 4 μg/mL Ce6, and then, DMA stock solution (20 mM in DMF) was added to give a final concentration of 20 μM DMA. Samples containing drug and DMA were irradiated at a light intensity with a 2.5-J/cm², 671-nm laser beam (LVI Technologies, Anyang, Korea). The fluorescence spectra of DMA (excitation, 360 nm; emission, 380–540 nm) as a result of the photosensitization reaction were monitored with a Perkin-Elmer LS55 spectrofluorophotometer (Waltham, MA, USA). The change in DMA fluorescence intensity (F_f − F_s) was plotted after subtracting each sample fluorescence intensity (F_s) from the full DMA fluorescence intensity (without free Ce6 or DC, indicating no singlet oxygen, F_f).

Fluorescence spectra were recorded on a PerkinElmer LS-55 spectrofluorophotometer. Absorption spectra were measured using a Shimadzu UV-2600 spectrophotometer. In a typical titration experiment, the probe solution (25 μM of 5, 10 μM of 8, 5 μM of 9, 10, 11) in 50 mM HEPES buffer (pH = 7.4):MeOH = 1:1 was titrated with aqueous stock solution of metal ion in a quartz cell at 25 °C. The fluorescence and absorption spectra were measured 5 min after the addition of the metal ions at each titration point. In the fluorescence titration, probes 5, 8, 9, 10, and 11 were excited at 365, 410, 488, 578, and 674 nm, respectively. The plot of the fluorescence intensity at the maximum emission wavelength was analyzed by nonlinear least-square curve fitting to obtain the binding constant (K_a, M⁻¹).

Metal ion titration was carried out in an aqueous-methanol solvent (50 mM HEPES buffer (pH = 7.4):methanol = 1:1) containing the set of probes 2 μM of 5, 9, 10 and 4 μM of 11. After the addition of the metal ions, the fluorescence spectra were recorded on a PerkinElmer LS-55 spectrofluorophotometer at 25 °C (λ_ex = 254 nm). PCA was performed with Microsoft Excel 2011 using the data set of the emission intensity change (F/F₀ at 414, 525, 589, and 679 nm) and the emission ratios (F₄₅₀/F₄₁₄, F₅₅₀/F₅₂₅, F₆₁₅/F₅₈₉, and F₆₉₀/F₆₇₉).

The fluorescence spectrum was measured by a PerkinElmer LS-55 spectrofluorophotometer. A quartz cell containing the coumarin probe (5 μM) in HEPES buffer (50 mM, pH = 7.4) was placed at the cell holder and measured the fluorescence (λ_ex = 320 nm) by spectral mode or time-drive mode. For reaction kinetics analysis, plot of the fluorescence intensity at 450 nm was analyzed by nonlinear least squares curve-fitting as a first order reaction to obtain rate constant (k, h⁻¹) and t_1/2 (h).