CR-1

Guidelines (The recommended experimental protocol below is for guidance only and should be adjusted according to specific requirements)

1. Stock Solution Preparation
1.1 Solvent: For most dyes, organic solvents are commonly used for dissolution, such as anhydrous DMSO.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: 10 mM HEPES buffer (containing 10% acetonitrile, pH 7.4).
2.2 Working Concentration: 5-50 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample Type: CR-1 solution is mixed and reacted with Zn²⁺ at different concentrations. Zn²⁺ concentration range: 0-5 μM for absorption spectroscopy, 0-50 μM for PA detection.
3.2 Reaction Conditions: The reaction completes within seconds at room temperature; the solution color changes from light blue to pink.
3.3 Reversibility Verification: After adding excess TPEN (HY-100202) (a Zn²⁺ chelator), the PA₅₃₂/PA₇₁₀ ratio returns to the initial level; the response remains stable after repeating the Zn²⁺/TPEN cycle 5 times.

4. Control Setup
4.1 Blank Control: Replace CR-1 solution with an equal volume of HEPES buffer to subtract background PA signals.
4.2 Negative Control: Zn²⁺: Co-inject 0.25-0.5 mg/kg Zn²⁺ with CR-1.
4.3 Negative Control: Co-inject Zn²⁺ + TPEN.

5. Detection and Analysis
5.1 Instrument Type: UV-visible spectrophotometer, photoacoustic computed tomography (PACT) system.
5.2 Wavelengths: Absorption peaks at 710 nm (free CR-1) and 532 nm (CR-1-Zn²⁺ complex); laser wavelengths of 532 nm and 710 nm for PA imaging.
5.3 Result Analysis
5.3.1 Signal Intensity Changes: As the Zn²⁺ concentration increases, the PA₇₁₀ signal weakens, the PA₅₃₂ signal strengthens, and the PA₅₃₂/PA₇₁₀ ratio shows a linear correlation with Zn²⁺ concentration (0-50 μM, limit of detection 170 nM).
5.3.2 Selectivity: It has high selectivity for Zn²⁺, with no obvious interference from other metal ions; the reaction is reversible, and TPEN can restore the signal.
5.3.3 Color Change: The solution changes from light blue to pink; it remains stable within the pH range of 6.2-8.2.