Fluotime 200

Time-resolved
fluorescence measurements were performed using a time-correlated single-photon
counting (TCSPC) setup (PicoQuant FluoTime 200) at 10 °C. The
concentration of the samples was <0.05 cm^–1 at
the Q_y maximum. A laser diode provided
the pulsed excitation light at a frequency of 10 MHz and a center
wavelength of 466 nm. The instrument response function was determined
to be 92 ps (fwhm) measuring the fluorescence decay of a pinacyanol
iodide dye dissolved in methanol that has a lifetime of 6 ps.^{33 (link)} Power studies were performed to exclude annihilation
effects from the measurements.

The absorbance spectra of the NGQDs were recorded using a UV–vis–near-infrared spectrophotometer (Carry-5000, Agilent Technologies). The PL spectra were recorded at excitation wavelengths (λ_ex) of 379 and 405 nm using a PL spectrophotometer (Darsa, PSI Trading Co. Ltd.) with a Xe lamp. Time-resolved PL decay was measured via the time-correlated single-photon counting (TCSPC) technique using a time-resolved spectroscopy system (TRSS, Fluo Time 200, PicoQuant). A laser with an excitation wavelength of 379 nm was used to excite all of the NGQD samples. A photomultiplier tube (PMT) was used for the detection of the emitted light by the NGQD samples. To study the atomic structure of the NGQDs, HRTEM (HF-3300/NB5000/S-4800, Hitachi) was utilized. The elemental composition of the NGQDs was determined via XPS (ESCALAB 250Xi, Thermo Fisher Scientific). The XPS peaks were calibrated using the C1s peak. The Raman spectra were recorded using a confocal Raman spectrometer (Nicolet Almega XR, Thermo Fisher Scientific) with a λ_ex value of 532 nm.

Fluorescence was measured using an SPEX Flurolog FL 3–22 steady-state fluorescence spectrometer (Jobin Yvon, Edison, NJ, USA) equipped with double grating excitation and emission monochromators. The measurements were made at 25 °C in 2 × 10 mm cuvettes oriented perpendicular to the excitation beam. For the bimane fluorescence measurement, the excitation emission wavelength was 380 nm and the emission spectra were recorded between 395 and 700 nm using excitation and emission spectral slits of 2 and 4 nm, respectively. Solution acidification was achieved by the addition of small amounts of 2.5 M acetic buffer. All the spectra were recorded after 30 min of incubation to ensure the equilibrium of the sample.
The fluorescence lifetime kinetics of bimane-labeled T-domain were measured with a time-resolved fluorescence spectrometer FluoTime 200 (PicoQuant, Berlin, Germany) using a standard time-correlated single-photon counting scheme. Samples were excited at 373 nm by a sub-nanosecond pulsed diode laser LDH 375 (PicoQuant, Berlin, Germany) with a repetition rate of 10 MHz. Fluorescence emission was detected at 480 nm, selected by a Sciencetech Model 9030 monochromator, using a PMA-182 photomultiplier. The fluorescence intensity decay was analyzed using the FluoFit version 2.3 iterative-fitting software based on the Marquardt algorithm (PicoQuant, Berlin, Germany).