Design of a comprehensive experiment for the construction of a berberine-based fluorescent probe for mercury ions

The detection of mercury ions (Hg²⁺) is of great significance because they are highly toxic environmental pollutants that pose serious threats to human health and can cause severe diseases. Over the past few decades, fluorescent probe-based detection methods have exhibited immense potential for Hg²⁺ detection owing to their superior characteristics, including high sensitivity, excellent selectivity, rapid response, simple operation, and capability for real-time visual detection. Berberine is a bioactive isoquinoline alkaloid mainly isolated from traditional Chinese medicinal herbs such as Rhizoma Coptidis and Cortex Phellodendri. In addition to its diverse pharmacological activities (e.g., remarkable antibacterial and anti-inflammatory effects), its unique molecular structure provides favorable photophysical properties. Specifically, berberine possesses an extended π-conjugated system that can generate stable fluorescence emission under appropriate excitation conditions, making it an ideal molecular scaffold for fluorescent probe design. Therefore, herein, a fluorescent probe based on the berberine skeleton was designed and synthesized for the rapid and highly sensitive detection of Hg²⁺.

Using berberine hydrochloride as the starting material, a fluorescent probe was constructed by introducing a phenyl carbonothioate group as the recognition unit for Hg²⁺. The molecular structure of the probe was characterized via nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry (HRMS). The sensing performance of the probe toward Hg²⁺ was evaluated using fluorescence spectroscopy, and the recognition mechanism was further investigated using HRMS and infrared spectroscopy. The practical applicability of this probe for Hg²⁺detection was validated through recovery experiments using real water samples.

The fluorescent probe B-PT was synthesized using berberine hydrochloride as the precursor, and its molecular structure was fully confirmed via spectroscopic characterization. In a THF/H₂O (v/v, 9:1) mixed solvent system, B-PT enables the rapid detection of Hg²⁺ with a response time of <3 min. The probe itself exhibits extremely weak fluorescence; however, upon addition of Hg²⁺, a prominent emission peak appears at 550 nm. By contrast, no significant fluorescence changes were observed in the presence of other tested metal ions, including Na⁺, Cu²⁺, Mg²⁺, Cr³⁺, Ca²⁺, K⁺, Al³⁺, Zn²⁺, Ag⁺, Cd²⁺, Fe³⁺, Co²⁺, Ni²⁺, and Pb²⁺, demonstrating the high specificity of the probe toward Hg²⁺. This high specificity was further corroborated by interference experiments, which confirmed the excellent anti-interference capability of B-PT. The calibration curve revealed a good linear correlation between fluorescence intensity and Hg²⁺ concentration in the range of 5.0–60.0 μmol/L, enabling the quantitative determination of Hg²⁺ with a calculated detection limit of 1.6 × 10⁻⁸ mol/L. Mechanistic studies verified that the specific detection of Hg²⁺ originates from Hg²⁺-triggered desulfurization and hydrolysis of the phenyl carbonothioate moiety in B-PT, resulting in the formation of the strongly green-fluorescent product B-OH. Moreover, analysis of real water samples confirmed the stable and reliable sensing performance of B-PT, highlighting its great potential for practical environmental monitoring applications.

This experiment extends the application of berberine, a classic natural product, to the development of the fluorescent probe B-PT for the detection of Hg²⁺ in real environmental samples. The experimental design integrates practicality, scientific interest, and forward-looking value. It helps students deepen their understanding and integration of theoretical knowledge from natural product chemistry, organic chemistry, and analytical chemistry while strengthening their basic experimental skills and cultivating their scientific research literacy and innovative thinking. Teaching practice demonstrates that the integration of cutting-edge research achievements into comprehensive chemistry experiment teaching enhances students’ learning initiative and exploratory enthusiasm. It also promotes the organic integration of theoretical teaching and practical application, facilitates the systematic integration of multidisciplinary knowledge, and provides strong support for cultivating students’ comprehensive innovation abilities.