ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Xiao-Yuan Wang,Jian Lv,Qin Hong,Ze-Rui Zhou,Da-Wei Li,Ruo-Can Qian
H2O2 is an essential signaling molecule in living cells that can cause direct damage to lipids, proteins, and DNA, resulting in cell membrane rupture. However, current studies mostly focus on probe-based sensing of intracellular H2O2, and these methods usually require sophisticated probe synthesis and instruments. In particular, local H2O2 treatment induces cell membrane rupture, but the level of cell membrane destruction is unknown because the mechanical properties of the cell membrane are difficult to accurately determine. Therefore, highly sensitive and label-free methods are required to measure and reflect mechanical changes in the cell membrane. Here, using an ultrasmall quartz nanopipette with a tip diameter less than 90 nm as a nanosensor, label-free and noninvasive electrochemical single-cell measurement is achieved for real-time monitoring of cell membrane rupture under H2O2 treatment. By spatially controlling the nanopipette tip to precisely approach a specific location on the membrane of a single living cell, stable cyclic membrane oscillations are observed under a constant direct current voltage. Specifically, upon nanopipette advancement, the mechanical status of the cell membrane can be sensibly displayed by continuous current versus time traces. The electrical signals are collected and processed, ultimately revealing the mechanical properties of the cell membrane and the degree of cell apoptosis. This nanopipette-based nanosensor paves the way for developing a facile, label-free, and noninvasive strategy to assay the mechanical properties of the cell membrane during external stimulation at the single-cell level.
研究领域:
生物医学工程
·
细胞生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2018
·
IF 6.042
Ruo-Can Qian,Jian Lv,Yi-Tao Long
Recently, a variety of strategies have been developed for single-cell detection. However, the precise probing of the given area at single-cell level is still a challenge. Here, we put forward a rapid and targeted imaging approach for the mapping of subcelluar domains, which realizes the precise injection of multifluorescence into a single living cell via an ultrasmall quartz capillary nanopipette (∼100 nm) and can successfully transport different fluorescent probe molecules to the pointing subcellullar area around the tip in the cytoplasm within 20 s. This method is also applied for monitoring the loss of intracellular mitochondrial membrane potential under the treatment of metformin in a single MCF-7 breast cancer cell. The major driven force in the nanopipette, electroosmotic flow, is evaluated by a theory calculation method and finite element simulations, and the solution indicates a confined solute distribution profile around the tip within the working range. Overall, the nanopipette approach realizes the precise and simultaneous delivery of multiple probe molecules into the single living cell through the electroosmotically modulated, nondestructive, and one-step injection, which is especially powerful and convenient for multichannel single-cell imaging and monitoring, indicating favorable potential for understanding, monitoring, and controlling the biological processes from the single cell to subcellular organelles.
研究领域:
细胞生物学
·
生物物理学
·
分析化学
✓ 已验证
DOI
PubMed
高影响因子
Nature Protocols
·
2023
·Vol. 18
·期 8
·2579-2599
·
IF 14.8
Jiang Tao,Yi Long,Liu Xu,Ivanov Aleksandar P.,Edel Joshua B.,Tang Longhua
Studying the electrical properties of individual proteins is a prominent research area in the field of bioelectronics. Electron tunnelling or quantum mechanical tunnelling (QMT) probes can act as powerful tools for investigating the electrical properties of proteins. However, current fabrication methods for these probes often have limited reproducibility, unreliable contact or inadequate binding of proteins onto the electrodes, so better solutions are required. Here, we detail a generalizable and straightforward set of instructions for fabricating simple, nanopipette-based, tunnelling probes, suitable for measuring conductance in single proteins. Our QMT probe is based on a high-aspect-ratio dual-channel nanopipette that integrates a pair of gold tunneling electrodes with a gap of less than 5 nm, fabricated via the pyrolytic deposition of carbon followed by the electrochemical deposition of gold. The gold tunneling electrodes can be functionalized using an extensive library of available surface modifications to achieve single-protein–electrode contact. We use a biotinylated thiol modification, in which a biotin–streptavidin–biotin bridge is used to form the single-protein junction. The resulting protein-coupled QMT probes enable the stable electrical measurement of the same single protein in solution for up to several hours. We also describe the analysis method used to interpret time-dependent single-protein conductance measurements, which can provide essential information for understanding electron transport and exploring protein dynamics. The total time required to complete the protocol is ~33 h and it can be carried out by users trained in less than 24 h.
研究领域:
生物电子学
·
纳米技术
·
蛋白质科学
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Yuling Wang,Rongrong Pan,Depeng Jiang,Dechen Jiang,Hong-Yuan Chen
The chemical reaction in a confined space is known to be accelerated due to a high collision probability; however, the study of this confinement effect in a supersmall space down to femtoliter (fL) is seldom reported. Here, an adjustable volume [from picoliter (pL) to fL] of the aqueous phase is retrained at the tip of a nanopipette by an organic solvent so that the confinement effect on the specific activity of glucose oxidase is investigated. The activity is determined by the amount of hydrogen peroxide generated from the reaction between the oxidase and glucose using a nanoelectrode inside the nanopipette. As compared with the activity in bulk solution (82 U/mg), the activity increases up to 7500 U/mg in a 105 fL space. The 2 orders of magnitude increase in the enzymatic activity is the highest amplification in the volume-confined enzyme reaction as reported. A near-exponential drop in the activity is observed with the increase in the space volume, revealing the dominant enhancement in the confined space at the fL level for the first time. The established electrochemical nanopipettes should not only provide a strategy for the study of the enzymatic activity in supersmall confined space but also help understand the confinement effect of enzyme-catalyzed reactions.
研究领域:
生物化学
·
电化学
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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