Translate this page to:
In JoVE (1)
- Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
Other Publications (11)
Articles by Yonggang Zhu in JoVE
Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming
Wah Chin Boon1, Karolina Petkovic-Duran2, Yonggang Zhu2, Richard Manasseh3, Malcolm K. Horne1, Tim D. Aumann1
1Florey Neuroscience Institutes and Centre for Neuroscience, University of Melbourne, 2Fluid Dynamics Group, CSIRO Materials Science and Engineering, 3Swinburne University of Technology, Faculty of Engineering and Industrial Sciences
We describe a novel method for increasing cDNA yield from single-cell quantities of mRNA in otherwise standard laboratory reverse transcription reactions. The novelty resides in the use of a micromixer, which utilizes the phenomenon of acoustic microstreaming, to mix fluids at microliter scales more effectively than shaking, vortexing or trituration.
Other articles by Yonggang Zhu on PubMed
Effective Deposition Potential Induced Size-dependent Orientation Growth of Bi-Sb Alloy Nanowire Arrays
The Journal of Physical Chemistry. B. Nov, 2006 | Pubmed ID: 17064109
The influence of effective deposition potential on the orientation and diameter of Bi(1-x)Sbx alloy nanowire arrays by pulsed electrodeposition technique was reported. X-ray diffraction, field-emission scanning electron microscopy, and transmission electron microscopy analysis show that the orientation of the Bi(1-x)Sbx nanowires can be turned from the  to the  direction by increasing the effective deposition potential, and the nanowires fully fill in the pores of the AAM in the lower potential region, while in the higher potential region the nanowires partly fill the pores of the AAM. The origin of those phenomena and the growth mechanism of the nanowire are discussed together with composition analysis.
The Journal of Physical Chemistry. B. Dec, 2006 | Pubmed ID: 17181275
The thermal properties of single-crystalline Bi nanowire arrays with different orientations and diameters were studied by differential scanning calorimeter and in situ high-temperature X-ray diffraction. Bi nanowires were fabricated by a pulsed electrodeposition technique within the porous anodic alumina membrane. The relationships between the orientation and diameter of Bi nanowires and the corresponding thermal properties are deduced solely from experimental results. It is shown that the melting point decreases with decreasing nanowire diameter, and there is an anisotropic thermal expansion property of Bi nanowires with different orientations and diameters. The transition of the thermal expansion coefficient from positive at low temperature to negative at high temperature for Bi nanowire arrays was analyzed and discussed.
Detection of 100 AM Fluorophores Using a High-sensitivity On-chip CE System and Transient Isotachophoresis
Analytical Chemistry. Jan, 2007 | Pubmed ID: 17194159
We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.
Advances in Biochemical Engineering/biotechnology. 2008 | Pubmed ID: 18594785
In vitro compartmentalization (IVC) is a powerful tool for studying protein-protein reactions, due to its high capacity and the versatility of droplet technologies. IVC bridges the gap between chemistry and biology as it enables the incorporation of unnatural amino acids with modifications into biological systems, through protein transcription and translation reactions, in a cell-like microdrop environment. The quest for the ultimate chip for protein studies using IVC is the drive for the development of various microfluidic droplet technologies to enable these unusual biochemical reactions to occur. These techniques have been shown to generate precise microdrops with a controlled size. Various chemical and physical phenomena have been utilized for on-chip manipulation to allow the droplets to be generated, fused, and split. Coupled with detection techniques, droplets can be sorted and selected. These capabilities allow directed protein evolution to be carried out on a microchip. With further technological development of the detection module, factors such as addressable storage, transport and interfacing technologies, could be integrated and thus provide platforms for protein studies with high efficiency and accuracy that conventional laboratories cannot achieve.
Electrophoresis. Nov, 2008 | Pubmed ID: 18924102
Contactless conductivity detector technology has unique advantages for microfluidic applications. However, the low S/N and varying baseline makes the signal analysis difficult. In this paper, a continuous wavelet transform-based peak detection algorithm was developed for CE signals from microfluidic chips. The Ridger peak detection algorithm is based on the MassSpecWavelet algorithm by Du et al. [Bioinformatics 2006, 22, 2059-2065], and performs a continuous wavelet transform on data, using a wavelet proportional to the first derivative of a Gaussian function. It forms sequences of local maxima and minima in the continuous wavelet transform, before pairing sequences of maxima to minima to define peaks. The peak detection algorithm was tested against the Cromwell, MassSpecWavelet, and Linear Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometer Peak Indication and Classification algorithms using experimental data. Its sensitivity to false discovery rate curve is superior to other techniques tested.
BioTechniques. Oct, 2009 | Pubmed ID: 19852766
Mixing fluids for biochemical assays is problematic when volumes are very small (on the order of the 10 microL typical of single drops), which has inspired the development of many micromixing devices. In this paper, we show that micromixing is possible in the simple open wells of standard laboratory consumables using appropriate acoustic frequencies that can be applied using cheap, conventional audio components. Earlier work has shown that the phenomenon of acoustic microstreaming can mix fluids, provided that bubbles are introduced into a specially designed microchamber or that high-frequency surface acoustic wave devices are constructed. We demonstrate a key simplification: acoustic micromixing at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a drop in a small well provided an appropriately small radius, and so an introduced bubble was not necessary. Microstreaming showed improvement over diffusion-based mixing by 1-2 orders of magnitude. Furthermore, significant improvements are attainable through the utilization of chaotic mixing principles, whereby alternating fluid flow patterns are created by applying, in sequence, two different acoustic frequencies to a drop of liquid in an open well.
Ultrasonics. Feb, 2010 | Pubmed ID: 19896683
Cavitation microstreaming plays a role in the therapeutic action of microbubbles driven by ultrasound, such as the sonoporative and sonothrombolytic phenomena. Microscopic particle-image velocimetry experiments are presented. Results show that many different microstreaming patterns are possible around a microbubble when it is on a surface, albeit for microbubbles much larger than used in clinical practice. Each pattern is associated with a particular oscillation mode of the bubble, and changing between patterns is achieved by changing the sound frequency. Each microstreaming pattern also generates different shear stress and stretch/compression distributions in the vicinity of a bubble on a wall. Analysis of the micro-PIV results also shows that ultrasound-driven microstreaming flows around bubbles are feasible mechanisms for mixing therapeutic agents into the surrounding blood, as well as assisting sonoporative delivery of molecules across cell membranes. Patterns show significant variations around the bubble, suggesting sonoporation may be either enhanced or inhibited in different zones across a cellular surface. Thus, alternating the patterns may result in improved sonoporation and sonothrombolysis. The clear and reproducible delineation of microstreaming patterns based on driving frequency makes frequency-based pattern alternation a feasible alternative to the clinically less desirable practice of increasing sound pressure for equivalent sonoporative or sonothrombolytic effect. Surface divergence is proposed as a measure relevant to sonoporation.
Lab on a Chip. Jun, 2010 | Pubmed ID: 20480105
This review focuses on the use of Förster Resonance Energy Transfer (FRET) to monitor intra- and intermolecular reactions occurring in microfluidic reactors. Microfluidic devices have recently been used for performing highly efficient and miniaturised biological assays for the analysis of biological entities such as cells, proteins and nucleic acids. Microfluidic assays are characterised by nanolitre to femtolitre reaction volumes, which necessitates the adoption of a sensitive optical detection scheme. FRET serves as a strong 'spectroscopic ruler' for elucidating the tertiary structure of biomolecules, as the efficiency of the non-radiative energy transfer is extremely sensitive to nanoscale changes in the separation between donor and acceptor markers attached to the biomolecule of interest. In this review, we will review the implementation of various microfluidic assays which employ FRET for diverse applications in the biomedical field, along with the advantages and disadvantages of the various approaches. The future prospects for development of microfluidic devices incorporating FRET detection will be discussed.
Management of the Diffusion of 4-methylumbelliferone Across Phases in Microdroplet-based Systems for in Vitro Protein Evolution
Electrophoresis. Sep, 2010 | Pubmed ID: 20803501
Fluorongenic reagents based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase, sulfatase, esterase, lipase and glycosidase activities in conventionally formatted enzyme assay systems. However, the sensitivity of assays based on these substrates is also potentially very useful in the microdroplet formats now being developed for high throughput in vitro evolution experiments. In this article, we report the investigation of diffusion of 4-MU as a model dye from water-in-oil droplets and the internal aqueous phase of water-in-oil-in-water droplets in microfluidics. The effect of BSA in the aqueous phase on the diffusion of 4-MU is also discussed. Based on these results, we provided here proof-of-concept of the reaction of the enzyme OpdA with the substrate coumaphos in water-in-oil-in-water droplets. In this double-emulsion system, the reaction of OpdA and coumaphos was achieved by allowing coumaphos to diffuse from the continuous aqueous phase across the oil phase into the internal aqueous droplets.
Nanoscale Research Letters. 2011 | Pubmed ID: 21711611
ABSTRACT: Temperature and carrier density-dependent spin dynamics for GaAs/AlGaAs quantum wells (QWs) with different structural symmetries have been studied by using time-resolved Kerr rotation technique. The spin relaxation time is measured to be much longer for the symmetrically designed GaAs QW comparing with the asymmetrical one, indicating the strong influence of Rashba spin-orbit coupling on spin relaxation. D'yakonov-Perel' mechanism has been revealed to be the dominant contribution for spin relaxation in GaAs/AlGaAs QWs. The spin relaxation time exhibits non-monotonic-dependent behavior on both temperature and photo-excited carrier density, revealing the important role of non-monotonic temperature and density dependence of electron-electron Coulomb scattering. Our experimental observations demonstrate good agreement with recently developed spin relaxation theory based on microscopic kinetic spin Bloch equation approach.
Sensitive Label-free Oligonucleotide-based Microfluidic Detection of Mercury (II) Ion by Using Exonuclease I
Biosensors & Bioelectronics. Jan, 2012 | Pubmed ID: 22172497
Mercury is a highly toxic metal that can cause significant harm to humans and aquatic ecosystems. This paper describes a novel approach for mercury (Hg(2+)) ion detection by using label-free oligonucleotide probes and Escherichia coli exonuclease I (Exo I) in a microfluidic electrophoretic separated platform. Two single-stranded DNAs (ssDNA) TT-21 and TT-44 with 7 Thymine-Thymine mispairs are employed to capture mercury ions. Due to the coordination structure of T-Hg(2+)-T, these ssDNAs are folded into hairpin-like double-stranded DNAs (dsDNA) which are more difficult to be digested by Exo I, as confirmed by polyacrylamide gel electrophoresis (PAGE) analysis. A series of microfluidic capillary electrophoretic separation studies are carried out to investigate the effect of Exo I and mercury ion concentrations on the detected fluorescence intensity. This method has demonstrated a high sensitivity of mercury ion detection with the limit of detection around 15 nM or 3 ppb. An excellent selectivity of the probe for mercury ions over five interference ions Fe(3+), Cd(2+), Pb(2+), Cu(2+) and Ca(2+) is also revealed. This method could potentially be used for mercury ion detection with high sensitivity and reliability.