Jarosova Romana, Kaplan Sam V, Field Thomas M, Givens Richard S, Senadheera Sanjeewa N, Johnson Michael A
Department of Chemistry and R.N. Adams Institute for Bioanalytical Chemistry, University of Kansas, Lawrence, Kansas 66045, United States.
Department of Analytical Chemistry, UNESCO Laboratory of Environmental Electrochemistry, Charles University, Prague 2 12843, Czech Republic.
Anal Chem. 2021 Feb 9;93(5):2776-2784. doi: 10.1021/acs.analchem.0c03452. Epub 2021 Jan 25.
Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using -hydroxyphenyl glutamate (HP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 μM. Moreover, we are able to correct for the presence of residual HP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of HP-Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.
笼形化合物是一类分子，在受到具有足够能量和持续时间的光照射后，会释放出一种保护底物以释放生物活性底物。这种方法的一个显著局限性在于，难以确定组织或不透明溶液中的光活化程度，因为到达所需位置的光会受到阻碍。在这里，我们通过开发一种电化学方法解决了这个问题，在该方法中，笼形分子的光释放量通过碳纤维微电极上的快速扫描循环伏安法（FSCV）来确定。以对羟基苯谷氨酸（HP-Glu）作为我们的模型系统，我们生成了4-羟基苯乙酸（4HPAA）氧化的线性校准曲线，谷氨酸分子从该基团离去，其浓度高达1000 μM。此外，我们能够校正溶液中残留的HP-Glu以及产生的光伪迹的存在。通过在3 μL光反应容器中对HP-Glu进行光活化，随后通过高效液相色谱法进行分析，构建了校正后的校准曲线。这种方法在4HPAA浓度和氧化电流之间产生了线性关系，从而能够独立于到达发色团的光量来测定释放的谷氨酸。此外，我们已经通过在完整的斑马鱼全脑中进行测量成功验证了新开发的方法。这项工作首次证明了用FSCV对脑组织中笼形化合物光化学进行电化学监测，从而促进了对神经元功能的分析。
