Youzhi Xu

Ultrafast Microfluidic Mixer for Tracking the Early Folding Kinetics of Human Telomere G-Quadruplex

The folding of G-quadruplex is hypothesized to undergo a complex process, from the formation of a hairpin structure to a triplex intermediate and to the final G-quadruplex. Currently, no experimental evidence has been found for the hairpin formation, because it folds in the time regime of 10-100 μs, entailing the development of microfluidic mixers with a mixing time of less than 10 μs. In this paper, we reported an ultrarapid micromixer with a mixing time of 5.5 μs, which represents the fastest turbulent micromixer to our best knowledge. Evaluations of the micromixer were conducted to confirm its mixing efficiency for small molecules and macromolecules. This new micromixer enabled us to interrogate the hairpin formation in the early folding process of human telomere G-quadruplex. The experimental kinetic evidence for the formation of hairpin was obtained for the first time.

A microsecond microfluidic mixer for characterizing fast biochemical reactions.

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.

A rapid microfluidic mixer for high-viscosity fluids to track ultrafast early folding kinetics of G-quadruplex under molecular crowding conditions.

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.

A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA–protein interaction

Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

Separation and determination of biogenic amines in fish using MEKC with novel multiphoton excitation fluorescence detection

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.

Analysis of environmental pollutants metabolized from pesticides using capillary electrophoresis with multiphoton-excited fluorescence detection

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.

Kinetic and Thermodynamic Characterization of Telomeric G-Quadruplex by Nonequilibrium Capillary Electrophoresis: Application to G-Quadruplex/Duplex Competition

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.

Open tubular CEC in a microfluidic chip for rapid chiral recognition

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.

On-column detection of multiphoton-excited fluorescence in CE using hyphenated cylindrical-square capillaries

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 μM 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 μM) 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.