Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States.
Anal Chem. 2013 Mar 19;85(6):3095-103. doi: 10.1021/ac302676q. Epub 2013 Feb 26.
We demonstrate an all-electric sampling/derivatization/separation/detection system for the quantitation of thiols in tissue cultures. Extracellular fluid collected from rat organotypic hippocampal slice cultures (OHSCs) by electroosmotic flow through an 11 cm (length) × 50 μm (i.d.) sampling capillary is introduced to a simple microfluidic chip for derivatization, continuous flow-gated injection, separation, and detection. With the help of a fluorogenic, thiol-specific reagent, ThioGlo-1, we have successfully separated and detected the extracellular levels of free reduced cysteamine, homocysteine, and cysteine from OHSCs within 25 s in a 23 mm separation channel with a confocal laser-induced fluorescence (LIF) detector. Attention to the conductivities of the fluids being transported is required for successful flow-gated injections. When the sample conductivity is much higher than the run buffer conductivities, the electroosmotic velocities are such that there is less fluid coming by electroosmosis into the cross from the sample/reagent channel than is leaving by electroosmosis into the separation and waste channels. The resulting decrease in the internal fluid pressure in the injection cross pulls flow from the gated channel. This process may completely shut down the gated injection. Using a glycylglycine buffer with physiological osmolarity but only 62% of physiological conductivity and augmenting the conductivity of the run buffers solved this problem. Quantitation is by standard additions. Concentrations of cysteamine, homocysteine, and cysteine in the extracellular space of OHSCs are 10.6 ± 1.0 nM (n = 70), 0.18 ± 0.01 μM (n = 53), and 11.1 ± 1.2 μM (n = 70), respectively. This is the first in situ quantitative estimation of endogenous cysteamine in brain tissue. Extracellular levels of homocysteine and cysteine are comparable with other reported values.
我们展示了一种用于定量组织培养物中硫醇的全电动采样/衍生化/分离/检测系统。通过电渗流通过 11 厘米（长度）×50 微米（内径）采样毛细管从大鼠器官型海马片培养物（OHSCs）收集的细胞外液被引入到简单的微流控芯片中进行衍生化、连续流门注入、分离和检测。借助硫醇特异性荧光试剂 ThioGlo-1，我们成功地在 23 毫米分离通道中分离和检测了 OHSCs 中外源性还原半胱胺、同型半胱氨酸和半胱氨酸的细胞外水平，检测时间为 25 秒，使用共聚焦激光诱导荧光（LIF）检测器。成功的流门注入需要注意被输送流体的电导率。当样品电导率远高于运行缓冲液电导率时，电渗速度使得通过电渗进入交叉的样品/试剂通道的流体少于通过电渗进入分离和废物通道的流体。由此导致的注入交叉内的流体压力下降会从门控通道中拉动流动。这个过程可能会完全关闭门控注入。使用具有生理渗透压但电导率仅为生理电导率的 62%的甘氨酰甘氨酸缓冲液，并增加运行缓冲液的电导率，解决了这个问题。定量是通过标准添加法进行的。OHSCs 细胞外空间中半胱胺、同型半胱氨酸和半胱氨酸的浓度分别为 10.6±1.0 nM（n=70）、0.18±0.01 μM（n=53）和 11.1±1.2 μM（n=70）。这是首次在原位定量估计脑组织中的内源性半胱胺。细胞外同型半胱氨酸和半胱氨酸的水平与其他报道的值相当。
