We have described a compact capillary-based continuous-flow polymerase chain reaction (PCR) microfluidics device, which uses flexible thin film heaters with low thermal mass to construct three isothermal zones. Due to the decreased thermal mass of flexible thin film heater, the low power supply and rapid thermal response was obtained. The energy consumption of a 33-cycle continuous-flow PCR was less than 0.0088 kW h, which is much lower than that of the metal block or liquid bath based capillary continuous-flow PCR microfluidics. Special attention was also paid on the surface passivation of the capillary inner surface based on the competing bovine serum albumin (BSA), and the results showed the effect of dynamic passivation was superior to that of static passivation. With the help of the dynamic passivation, the 249 bp human b-actin gene fragment was amplified in 15 min, which is several times faster than that of the conventional PCR machine. In addition, the effect of initial DNA template concentrations on continuous-flow PCR was also investigated. 1672The concentration limit of DNA template reached 0.18 ng ml 21 , which can satisfy the requirements from different application fields.
Summary We recently developed two quantitative fluorescence resonance energy transfer (FRET) measurement methods based on spectral unmixing of emission spectra (IIem‐spFRET) and excitation–emission spectra (ExEm‐spFRET), respectively. We here evaluated robustness of the two methods by implementing them on a self‐assembled quantitative FRET measurement system using the cells expressing different constructs. For the cells with larger signal‐to‐noise (S/N) ratio (>9), the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 < S/N < 9, IIem‐spFRET obtained bigger RC values than the expected value for VCV construct; for the cells with S/N < 3, although IIem‐spFRET method obtained inaccurate E and RC values for VCV construct, the two methods also obtained consistent E and RC values for all other constructs. Collectively, both ExEm‐spFRET and IIem‐spFRET methods are very applicable for live‐cell FRET measurement, and ExEm‐spFRET has superior robustness especially for the cells with low S/N ratio. Lay Description Fluorescent proteins (FPs)‐based fluorescence resonance energy transfer (FRET) has been widely used as a powerful technique to study protein–protein interaction and stoichiometry of macromolecular complexes in living cells. There are two key issues for quantitative FRET measurement especially in living cells: donor emission crosstalk (donor fluorescence is collected in acceptor detection channel) and acceptor excitation crosstalk (acceptor is excited directly under donor excitation) due to the spectral overlap of FPs. Two‐wavelengths excitation‐based spectral linear unmixing of emission spectra can resolve donor emission crosstalk due to the obvious difference in emission spectra between donor and acceptor, but additional reference is necessary for the correction of acceptor excitation crosstalk. Spectral unmixing of excitation–emission spectra has inherent ability to resolve donor emission crosstalk and acceptor excitation crosstalk simultaneously without additional reference. We recently developed two quantitative FRET measurement methods based on spectral unmixing of emission spectra (IIem‐spFRET) and excitation–emission spectra (ExEm‐spFRET), respectively. We here evaluate robustness of the two methods by implementing them on a self‐assembled quantitative FRET measurement system using the same cells expressing different constructs under different signal‐to‐noise (S/N) ratios. For the cells with S/N > 9, the two methods obtained consistent FRET efficiency (E) values and total concentration ratio (RC) values of acceptor to donor for all constructs; for the cells with 3 < S/N < 9, IIem‐spFRET obtained bigger RC values than the expected value for VCV construct; for the cells with S/N < 3, although IIem‐spFRET method obtained inaccurate E and RC values for VCV construct, the two methods also obtained consistent E and RC values for all other constructs. Collectively, our experimental results demonstrate th...
Quantum yield ratio (Q /Q ) and absorption ratio (K /K ) in all excitation wavelengths used between acceptor and donor are indispensable to quantitative fluorescence resonance energy transfer (FRET) measurement based on linearly unmixing excitation-emission spectra (ExEm-spFRET). We here describe an approach to simultaneously measure Q /Q and K /K values by linearly unmixing the excitation-emission spectra of at least two different donor-acceptor tandem constructs with unknown FRET efficiency. To measure the Q /Q and K /K values of Venus (V) to Cerulean (C), we used a wide-field fluorescence microscope to image living HepG2 cells separately expressing each of four different C-V tandem constructs at different emission wavelengths with 435 nm and 470 nm excitation respectively to obtain the corresponding excitation-emission spectrum (S ). Every S was linearly unmixed into the contributions (weights) of three excitation-emission spectra of donor (W ) and acceptor (W ) as well as donor-acceptor sensitisation (W ). Plot of W /W versus W /W for the four C-V plasmids from at least 40 cells indicated a linear relationship with 1.865 of absolute intercept (Q /Q ) and 0.273 of the reciprocal of slope (K /K ), which was validated by quantitative FRET measurements adopting 1.865 of Q /Q and 0.273 of K /K for C32V, C5V, CVC and VCV constructs respectively in living HepG2 cells.
Summary Acceptor‐sensitised 3‐cube fluorescence resonance energy transfer (FRET) imaging (also termed as E‐FRET imaging) is a popular fluorescence intensity‐based FRET quantification method. Here, an automated E‐FRET microscope with user‐friendly interfaces was set up for dynamical online quantitative live‐cell FRET imaging. This microscope reduces the time of a quantitative E‐FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E‐FRET imaging of the cells can be performed automatically by clicking ‘Capture’ button on interfaces. E‐FRET imaging was performed on the microscope for living cells expressing different FRET tandem constructs. Dynamical E‐FRET imaging on the microscope for live cells coexpressing CFP‐Bax and YFP‐Bax treated by staurosporine (STS) revealed three Bax redistribution stages: Bax translocation from cytosol to mitochondria within 10 min, membrane insertion with conformational change on mitochondrial membrane within about 30 min, and subsequent oligomerisation within about 10 min. Because of excellent user‐friendly interface and stability, the automated E‐FRET microscope is a convenient tool for quantitative FRET imaging of living cell. Lay Description Acceptor‐sensitised 3‐cube fluorescence resonance energy transfer (FRET) imaging (also termed as E‐FRET) is a popular fluorescence intensity‐based FRET quantification methods. E‐FRET measurements are currently performed manually, and a complete FRET measurement takes about 12 s. E‐FRET measurement necessitates not only a skilled operator and specialised equipment but also expertise in the interpretation of FRET signals, a considerable challenge in the application of FRET technology in living cells. Furthermore, manual E‐FRET microscope is hard to perform dynamical quantitative FRET measurement, the ever‐increasing applications in mapping the biochemical signal transduction within cells. Here, an automated E‐FRET microscope with user‐friendly interfaces was set up for dynamical online quantitative live‐cell FRET imaging. This microscope reduces the time of a quantitative E‐FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E‐FRET imaging of the cells can be performed automatically by clicking ‘Capture’ button on interfaces. Because of excellent user‐friendly interface and stability, the automated E‐FRET microscope is a convenient tool for quantitative FRET imaging of living cell.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.