Yonggang Zhu

Cavitation microstreaming and stress fields created by microbubbles

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.

A continuous wavelet transform algorithm for peak detection

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.

Management of the diffusion of 4-methylumbelliferone across phases in microdroplet-based systems for in vitro protein evolution

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.

Chaotic micromixing in open wells using audio-frequency acoustic microstreaming

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.

Enzyme synthesis and activity assay in microfluidic droplets on a chip

There is growing demand for high‐throughput measurement of biochemical reactions in drug discovery and directed evolution programs. To meet this need, a powerful platform based on droplet‐based bioreactors manipulated by microfluidic systems is being developed, which can overcome the limitations of scale and power encountered in conventional screening methods. This paper reports our progress in the synthesis of enzymes and assay of their activity within a microfluidic droplet system. The model system we use involves the organophosphorus hydrolase enzyme OpdA from Agrobacterium radiobacter and a robust microchip made from polymethyl methacrylate (PMMA). Synthesis of OpdA from cognate DNA within water‐in‐oil droplets was tested using both in‐house and commercial in vitro transcription and translation (IVTT) kits. OpdA activity was measured using coumaphos as substrate and by monitoring the fluorescence released by its product, chlorferone. OpdA was demonstrated to be synthesized and assayed within the droplets using the commercial in vitro transcription and translation kit, although the activity measured within the droplets diminished over time, apparently due to leakage of chlorferone out of the droplets.

Microfluidic Droplet Technique for In Vitro Directed Evolution

Increasingly over the past two decades, biotechnologists have been exploiting various molecular technologies for high-throughput screening of genes and their protein products to isolate novel functionalities with a wide range of industrial applications. One particular technology now widely used for these purposes involves directed evolution, an artificial form of evolution in which genes and proteins are evolved towards new or improved functions by imposing intense selection pressures on libraries of mutant genes generated by molecular biology techniques and expressed in heterologous systems such as Escherichia coli. Most recently, the rapid development of droplet-based microfluidics has created the potential to dramatically increase the power of directed evolution by increasing the size of the libraries and the throughput of the screening by several orders of magnitude. Here, we review the methods for generating and controlling droplets in microfluidic systems, and their applications in directed evolution. We focus on the methodologies for cell-based assays, in vitro protein expression and DNA amplification, and the prospects for using such platforms for directed evolution in next-generation biotechnologies.

Capillary electrophoresis (CE) peak detection using a wavelet transform technique

Capillary Electrophoresis (CE) is a separation technique that can be used as a sample pre-treatment step in chemical analysis. When coupled with a detection technique, identification of chemical species can be performed on the basis of the elution signals. However, the sensor signals are often complicated by high signal noise, varying baseline and overlapping peaks. There is thus a need for a signal processing technique capable of robustly detecting peaks in acquired sensor data. Here, we report on an algorithm that utilises the Continuous Wavelet Transform (CWT) for the detection of analyte peaks. The algorithm that has been developed makes use of a wavelet equal to the first derivative of a Gaussian function and has been successfully applied to data obtained from a CCD sensor fabricated on a polymer microfluidic separation chip. The algorithm operates by taking the CWT of the sensor response. It then analyses patterns in the local maximum and minimum points evident across scales in the CWT coefficients to find the peaks in the time series data. The performance of two versions of the algorithm have been compared for synthetic data sets each with known baseline, peaks and noise. The improved algorithm has been shown to successfully find peaks with a high sensitivity and low False Discovery Rate within a range of sensitivities.

Joule heating in polymer microfluidic chip

Joule heating is a significant problem for microfluidic chips with electrokinetically driven flows. In this paper, we will present the modeling results of the Joule heating of a Polymethylmethacrylate (PMMA) polymer separation chip using both experimental and computational methods. The temperature distributions on the surface of the chip were measured by an advanced thermograph system. The numerical study was carried out using the multiphysics computational fluid dynamics (CFD) package CFD-Ace+. Different solutions and operating conditions were studied. Both the measurements and CFD data revealed that the heat generation was approximately uniform and the subsequent temperature increase was also uniform along the channel except for regions near the liquid ports. The highest temperature increase was observed along the centerline of the channel and the temperature reduced significantly away from the channel. At an electrical field of 45kV/m, the Joule heating effect was negligible for the solution used, even though at such a high electric field significant heating effect has been observed for micro capillary flows in literature. At a higher electrical field (68-120kV/m), the Joule heating could cause an increase of temperature of up to 40°C.