NtHzBtd

Operating Instructions
(The following is a recommended experimental protocol for guidance only; adjust it according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: 2 vol% DMSO in 0.2 M HEPES buffer (pH 7.2).
1.2 Concentration recommendation: 10 μM.

2. Working Solution Preparation
2.1 Diluent: HEPES buffer or cell culture system. Spectroscopic experiments in literature use 0.2 M HEPES buffer (pH 7.2), while cell imaging experiments use cell culture systems.
2.2 Working concentration: 10 μM.
2.3 Notes: Adjust the working solution concentration according to the detection system; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Cell samples.
3.1.1 Adherent cells: PC3 cells.
3.2 Incubation conditions:
Add 10 μM NtHzBtd to PC3 cells, incubate under cell culture conditions for approximately 30 min, then perform fluorescence imaging. Subsequently, add Fe³⁺ (0.8 μM or 1.7 μM) and incubate for 1 min to form the NtHzBtd-Fe³⁺ complex and observe fluorescence changes.
3.3 Washing step:
Wash the cells with RPMI solution after staining.
4. Control Setup
4.1 Blank control: PC3 cells without addition of NtHzBtd or Fe³⁺, used to observe the autofluorescence background of cells.
4.2 Fe³⁺ response control:
Observe the fluorescence quenching change of NtHzBtd after adding Fe³⁺.
Results: The fluorescence of NtHzBtd decreases significantly after adding 0.8 μM Fe³⁺; the fluorescence is completely quenched after further adding 1.7 μM Fe³⁺.
4.3 Selectivity control:
Compare NtHzBtd with other metal ions (Mg²⁺, Cr³⁺, Hg²⁺, Ag⁺, Cu²⁺, Zn²⁺, Ca²⁺, Na⁺, Fe²⁺, K⁺, Mn²⁺, Ni²⁺, Pb²⁺, Cd⁺, Al³⁺, Co³⁺).
Results: Except for Fe³⁺, no significant fluorescence change is induced by the other ions, indicating that NtHzBtd has high selectivity for Fe³⁺.

5. Detection and Analysis
5.1 Instruments: Fluorescence spectrophotometer; laser confocal microscope. A spectrofluorometer is used for fluorescence detection, and a Carl Zeiss LSM 710 confocal laser scanning microscope is used for cell imaging.
5.2 Detection wavelengths:
5.2.1 Solution fluorescence detection: Ex = 334 nm; Em = 401 nm.
5.3 Result analysis:
5.3.1 Fluorescence intensity change: Fluorescence quenching occurs after NtHzBtd binds to Fe³⁺. Fe³⁺ coordinates with the imine nitrogen and naphthol oxygen of NtHzBtd to form a 1:1 complex, triggering CHEQ and ICT processes, thereby reducing fluorescence intensity.
5.3.2 Fluorescence localization: NtHzBtd can enter PC3 cells and be used for live-cell imaging of Fe³⁺.
5.3.3 Fluorescence color change: PC3 cells show enhanced green fluorescence after adding 10 μM NtHzBtd; the fluorescence gradually decreases and eventually quenches after adding Fe³⁺.
5.3.4 Detection performance: NtHzBtd binds to Fe³⁺ at a 1:1 ratio, with a binding constant KA = 8.41 × 10⁶ L mol^-1, a quenching constant Ksv = 3.72 × 10⁶ L mol^-1, a limit of detection (LOD) = 0.036 μM, and a response time of approximately 55 s.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.