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ABSTRACT: Tracking the folding kinetics of macromolecules under molecular crowding conditions represents a tremendous challenge due to the high viscosity of the solution. In this paper, we report a unique T-type microfluidic mixer with seven consecutive ω-shaped baffles for fast mixing of high-viscosity fluids. Numerical simulations and experimental characterizations proved that the micromixer could achieve a mixing time of 579.4 μs for solutions with viscosities about 33.6 times that of pure water. Over a 1000-fold improvement in mixing dead time was accomplished in comparison to those reported previously. We further used this highly efficient micromixer to track the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions. Results indicated an exponential process in the initial folding phase of G-quadruplex, and the G-quadruplex formed a more compact structure under higher degrees of molecular crowding conditions.
Analytical Chemistry 09/2012; · 5.86 Impact Factor
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ABSTRACT: Analysis of fast biochemical reactions requires rapid mixing of solutions. Micromixers can achieve uniform mixing of solutions in a short time and have been recognized as an attractive tool to analyze fast reactions. However, it is still a challenge to design mixers with simple structure and short dead time. Here, a zigzag turbulent micromixer was developed with a rapid mixing time of 16 μs at sample consumption of 10 μL/s. Numerical simulations and confocal imaging validated this result. Application of the chemiluminescence (CL) reaction demonstrated the use of this mixer in analyzing the kinetic process of the CL reaction. In comparison to the turbulent micromixers reported previously, this zigzag mixer has advantages of short dead time, simple structure and low sample consumption. We anticipate the developed mixer to be a useful tool in studying biochemical kinetics or be integrated to Lab-on-a-chip device as a pretreatment functional unit.
Talanta 01/2012; 88:175-80. · 3.79 Impact Factor
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Dingding Mo,
Lingjia Wu, Youzhi Xu,
Jun Ren,
Long Wang,
Lin Huang,
Qi-Jia Wu,
Penghui Bao,
Mao-Hua Xie,
Ping Yin,
Bi-Feng Liu,
Yi Liang,
Yi Zhang
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ABSTRACT: Folding of large structured RNAs into their functional tertiary structures at high temperatures is challenging. Here we show that I-TnaI protein, a small LAGLIDADG homing endonuclease encoded by a group I intron from a hyperthermophilic bacterium, acts as a maturase that is essential for the catalytic activity of this intron at high temperatures and physiological cationic conditions. I-TnaI specifically binds to and induces tertiary packing of the P4-P6 domain of the intron; this RNA-protein complex might serve as a thermostable platform for active folding of the entire intron. Interestingly, the binding affinity of I-TnaI to its cognate intron RNA largely increases with temperature; over 30-fold stronger binding at higher temperatures relative to 37 °C correlates with a switch from an entropy-driven (37 °C) to an enthalpy-driven (55-60 °C) interaction mode. This binding mode may represent a novel strategy how an RNA binding protein can promote the function of its target RNA specifically at high temperatures.
Biochimie 03/2011; 93(3):533-41. · 3.02 Impact Factor
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ABSTRACT: Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated.
Analytical and Bioanalytical Chemistry 07/2009; 394(7):1911-7. · 3.78 Impact Factor
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ABSTRACT: Chiral recognition of dansyl enantiomeric amino acids by microfluidic open tubular CEC (muOTCEC) with fluorescence detection was demonstrated. Avidin was employed as the chiral selector immobilized on the microchannel wall, which functioned as the chiral stationary phase (CSP) by physical adsorption. The condition of CSP on the glass wall was characterized using field emission SEM. Results indicated that avidin was homogenously distributed on the microchannel surface. Two parameters that played essential roles in muOTCEC for chiral recognition were investigated. Buffer pH could greatly change the amount of adsorption of avidin on the channel wall by altering the electrostatic attraction between them. Methanol, the organic additive to the running buffer, was also found significant for controlling the quality of the muOTCEC chiral separation by regulating the hydrophobic interaction between the enantiomers and the CSP. Under the optimized conditions, four dansyl racemic amino acids were then successfully separated by muOTCEC within 100 s with resolutions of 2.43, 1.88, 3.01 and 2.65 for dansyl-Ser, dansyl-Met, dansyl-Thr and dansyl-Val, respectively. Furthermore, a comparison with microfluidic CZE was investigated demonstrating that muOTCEC was a promising method for rapid chiral recognition.
Journal of Separation Science 02/2009; 32(3):374-80. · 2.73 Impact Factor
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ABSTRACT: In this paper, nonequilibrium capillary electrophoresis (NECE) was attempted for the first time to investigate a dual equilibrium system, where the intramolecular G-quadruplex folding was in competition with the intermolecular duplex formation. Samples of an equilibrium mixture of human telomeric DNA and its complementary strands were separated in capillaries under nonequilibrium conditions without K (+). Polyethylene oxide was added to the running buffer facilitating the separation of single-stranded DNA, duplex, and G-quadruplex. Thus, the folding/unfolding rate constants of the G-quadruplex and the association/dissociation constants of the duplex could be simultaneously derived from the same experiment. Results indicated that the duplex formation induced minimal influence on the G-quadruplex folding. On the basis of the kinetic characterization of the G-quadruplex at varying temperatures, the thermodynamic parameters of the G-quadruplex could also be determined. Thus, the NECE method provided a new avenue for studying the kinetics and thermodynamics of nucleic acids within dual equilibrium systems with significant advantages of extreme-low sample cost (approximately 10 (-18) mol) and high repeatability.
Analytical Chemistry 09/2008; 80(18):6935-41. · 5.86 Impact Factor
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ABSTRACT: Biogenic amines are a group of biological molecules derived from the enzymatic decarboxylation of natural amino acids. They can be found in a variety of foods and some of them are involved in essential cellular pathways regulating cellular functions. To address the issues raised by conventional detection methods for biogenic amines, such as laborious sample preparation and limited sensitivity, a new micellar electrokinetic chromatography scheme was developed based on multiphoton excitation fluorescence (MPEF) detection. Six FITC-labeled biogenic amine species were used for the evaluation of this MEKC-MPEF method in comparison to single photon excitation fluorescence detection. The results indicated that MEKC-MPEF had superior resolution with a detection volume as low as aL. Quantitative analysis of varying concentrations of biogenic amine species has also been achieved suggesting a ymole mass detection limit, a linear dynamic range of about two orders of magnitude, and 95-105% recoveries. Furthermore, the biogenic amine profile of decayed oriental crucian carps was successfully determined and quantified using this new method.
Journal of Separation Science 04/2008; 31(5):824-8. · 2.73 Impact Factor
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ABSTRACT: Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.
Electrophoresis 03/2008; 29(3):734-9. · 3.30 Impact Factor
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ABSTRACT: In this article, it was demonstrated that separation and determination of 20 amino acids were accomplished by micellar electrokinetic chromatography (MEKC) coupling with novel multiphoton excited fluorescence (MPEF) detection method. Different from MPEF achieved by expensive fs laser, continuous wave (CW) diode laser of ultra-low cost was uniquely employed in our MPEF system. Amino acids were fluorescently labeled with fluorescein isothiocyanate (FITC), and were subjected to sodium dodecyl sulfate (SDS)-based MEKC separation and CW-based MPEF detection. The result was compared with that by single photon excited fluorescence (SPEF), which indicated that MPEF had the advantages of better mass detectability and higher separation selectivity over SPEF. Quantitative analysis was performed and revealed linear dynamic range of over 2 orders of magnitude, with mass detection limit down to ymole level. To evaluate the reliability, this method was successfully applied for analyzing a commercial nutrition supplement liquid.
Journal of Chromatography 09/2007; 1162(2):149-53. · 4.53 Impact Factor
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ABSTRACT: Multiphoton excitation is a relatively old concept in quantum optics. But using multiphoton excitation fluorescence (MPEF) for bioanalysis is still in its infancy. Recently, MPEF has been introduced into the microseparation field, particularly CE, as a novel detection method. In this paper, MPEF detection for CE is reviewed, including MPEF fundamentals, approaches to achieving MPEF, detector configurations and applications in biological and environmental analyses. Emphasis will be placed on some recent advances of CE-MPEF in our laboratory. Challenges and future prospects are also discussed.
Journal of Separation Science 05/2007; 30(6):906-15. · 2.73 Impact Factor
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ABSTRACT: Multiphoton-excited fluorescence by diode laser of continuous wave was uniquely developed for capillary electrophoresis to determine aniline species metabolized from pesticides. To achieve 2-photon excitation fluorescence, derivatization procedure was performed using fluorescein isothiocyanate (FITC). The concentration ratio of FITC to the analytes was discussed for quantitative analysis. Several parameters that influenced separation quality of capillary zone electrophoresis were investigated, such as applied voltage, buffer pH value and concentration, etc. Under the optimized conditions, four pesticide residues were completely separated and determined within 4min, with detection limit down to zepptomole level (calculated detection volume: 45.0aL). Quantitative analyses exhibited excellent linear dynamic relationship in the range of about two orders of magnitude. The established method was further validated by testing spiked lake water sample.
Talanta 09/2006; 70(1):63-7. · 3.79 Impact Factor
