Sensitive analysis of multiple low-molecular-weight thiols in a single human cervical cancer cell by chemical derivatization-liquid chromatography-mass spectrometry.

Low-molecular-weight (LMW) thiols are important small molecules that regulate or maintain redox homeostasis in physiological and pathological processes. Assessing the concentrations of LMW thiols in biological systems may provide valuable information regarding physiological processes and the early diagnosis of some diseases. Here, we developed a method to simultaneously determine the concentrations of multiple LWM thiols in single cells by chemical derivatization assisted liquid chromatography-mass spectrometry (LC-MS). In this method, we synthesized a pair of stable isotope reagents, N-(acridin-9-yl)-2-bromoacetamide (AYBA) and N-(1,2,3,4-[2H4]-acridin-9-yl)-2-bromoacetamide ([2H4]AYBA). AYBA was used to derivatize LWM thiols in human cervical cancer (HeLa) cells, while [2H4]AYBA was used to derivatize standard LWM thiols to prepare internal standards for the LC-MS method development. The proposed AYBA derivatization greatly enhanced the detection sensitivity of LWM thiols by LC-MS, and thereby achieved the simultaneous detection of multiple LWM thiols by LC-MS in ∼1000 HeLa cells. Finally, the developed method was successfully utilized for the quantitative analysis of multiple LWM thiols in a single HeLa cell and the content changes of LWM thiols in a single HeLa cell before and after oxidative stress treatment. Accordingly, six LMW thiols were detected, including cysteamine, cysteine, glutathione, homocysteine, hydrogen sulfide, and pantetheine.

Electrochemical Monitoring of Paclitaxel-Induced ROS Release from Mitochondria inside Single Cells.

Mitochondria are believed to be the major source of intracellular reactive oxygen species (ROS). However, in situ, real-time and quantitative monitoring of ROS release from mitochondria that are present in their cytosolic environment remains a great challenge. In this work, a platinized SiC@C nanowire electrode is placed into a single cell for in situ detection of ROS signals from intracellular mitochondria, and antineoplastic agent (paclitaxel) induced ROS production is successfully recorded. Further investigations indicate that complex IV (cytochrome c oxidase, COX) is the principal site for ROS generation, and significantly more ROS are generated from mitochondria in cancer cells than that from normal cells. This work provides an effective approach to directly monitor intracellular mitochondria by nanowire electrodes, and consequently obtains important physiological evidence on antineoplastic agent-induced ROS generation, which will be of great benefit for better understanding of chemotherapy at subcellular levels.

A single nanowire sensor for intracellular glucose detection.

Glucose metabolism plays an important role in cell energy supply, and quantitative detection of the intracellular glucose level is particularly important for understanding many physiological processes. Glucose electrochemical sensors are widely used for blood and extracellular glucose detection. However, intracellular glucose detection cannot be achieved by these sensors owing to their large size and consequent low spatial resolution. Herein, we developed a single nanowire glucose sensor for electrochemical detection of intracellular glucose by depositing Pt nanoparticles (Pt NPs) on a SiC@C nanowire and further immobilizing glucose oxidase (GOD) thereon. Glucose was converted by GOD to an electroactive product H2O2 which was further electro-catalyzed by Pt NPs. The glucose nanowire sensor is endowed with a high sensitivity, high spatial-temporal resolution and enzyme specificity due to its nanoscale size and enzymatic reaction. This allows the real-time monitoring of the intracellular glucose level, and the increase of the intracellular glucose level induced by a novel potential hypoglycemic agent, reinforcing its potential application in lowering the blood glucose level. This work provides a versatile method for the construction of enzyme-modified nanosensors to electrochemically detect intracellular non-electroactive molecules, which is of great benefit for physiological and pathological studies.

Electrochemical Monitoring of ROS/RNS Homeostasis Within Individual Phagolysosomes Inside Single Macrophages.

The existence of a homeostatic mechanism regulating reactive oxygen/nitrogen species (ROS/RNS) amounts inside phagolysosomes has been invoked to account for the efficiency of this process but could not be unambiguously documented. Now, intracellular electrochemical analysis with platinized nanowire electrodes (Pt-NWEs) allowed monitoring ROS/RNS effluxes with sub-millisecond resolution from individual phagolysosomes impacting onto the electrode inserted inside a living macrophage. This shows for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results establish the existence of the long-postulated ROS/RNS homeostasis and allows its kinetics and efficiency to be quantified. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.