Identification, characterization and manipulation of Babesia-bovis-infected red blood cells using microfluidics technology

Nowadays numerous microfluidic systems are being developed to address a variety of clinical problems. Latest advances in microfluidic technology are promising to revolutionize the detection of pathogens in vivo through the development of integrated lab-on-chip devices. Such microfabricated systems will undertake all steps in sample analysis from collection and preparation to molecular detection. Micro total analysis systems are suitable candidates for point of care diagnostics due to small size, low cost production and enabled portability. The work here presented aimed the use of microfluidic platforms to identify and manipulate bovine red blood cells infected by the protozoan parasite Babesia bovis. A microfabricated device based on impedance spectroscopy was used for single cell discrimination and its sensitivity and applicability as a diagnostic method for bovine babesiosis was studied. Furthermore, manipulation and sorting of normal and infected red blood cells was performed on a dielectrophoresis based microfabricated cell cytometer. Single cell analysis of normal and B. bovis infected red blood cells was performed by electrorotation and dielectric parameters such as permittivities and conductivities of the cellular membrane and cytoplasm were determined.     

Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery

Skin is an attractive target for delivery of genetic therapies and vaccines. However, new approaches are needed to access this tissue more effectively. Here, we describe a new delivery technology based on arrays of structurally precise, micron-scale silicon projections, which we term microenhancer arrays (MEAs). In a human clinical study, these devices effectively breached the skin barrier, allowing direct access to the epidermis with minimal associated discomfort and skin irritation. In a mouse model, MEA-based delivery enabled topical gene transfer resulting in reporter gene activity up to 2,800-fold above topical controls. MEA-based delivery enabled topical immunization with naked plasmid DNA, inducing stronger and less variable immune responses than via needle-based injections, and reduced the number of immunizations required for full seroconversion. Together, the results provide the first in vivo use of microfabricated devices to breach the skin barrier and deliver vaccines topically, suggesting significant clinical and practical advantages over existing technologies.     

Flow-through polymerase chain reactions in chip thermocyclers

The miniaturization of analytical devices by micromachining technology is destined to have a major impact on medical and bioanalytical fields. To meet the current demands for rapid DNA amplification, various instruments and innovative technologies have been introduced by several groups in recent years. The development of the devices was extended in different directions and adapted to corresponding applications. In this review the development of a variety of devices and components for performing DNA amplification as well as the comparison of batch-process thermocyclers with reaction chambers and flow-through devices for different purposes are discussed. The main attention is turned to a flow device concept for thermocycling using microfabricated elements for local heat flow management, for which simulations and considerations for further improvement regarding design, material choice and applied technology were performed. The present review article mainly discusses and compares thermocycling devices for rapid thermocycling made of silicon or of silicon and glass with a short excursion to the possibility of plastic chip devices. In order to perform polymerase chain reactions (PCRs) in the microreactors, special attention must be paid to the conditions of the internal surfaces. For microchips, surface effects are generally pronounced because the surface to volume ratio increases upon miniaturization. Solutions for solving this problem are presented. We propose an overview of layouts for batch-process thermocyclers with different parallelization of reaction chambers and also of different designs of continuous flow thermocycling chips, paying particular attention to the parameters which influence the efficiency of such chip devices. Finally we point out some recent issues for applications in the field of clinical diagnostics.     

Microfabricated modules for sample handling, sample concentration and flow mixing: application to protein analysis by tandem mass spectrometry

The comprehensive analysis of biological systems requires a combination of genomic and proteomic efforts. The large-scale application of current genomic technologies provides complete genomic DNA sequences, sequence tags for expressed genes (EST's), and quantitative profiles of expressed genes at the mRNA level. In contrast, protein analytical technology lacks the sensitivity and the sample throughput for the systematic analysis of all the proteins expressed by a tissue or cell. The sensitivity of protein analysis technology is primarily limited by the loss of analytes, due to adsorption to surfaces, and sample contamination during handling. Here we summarize our work on the development and use of microfabricated fluidic systems for the manipulation of minute amounts of peptides and delivery to an electrospray ionization tandem mass spectrometer. New data are also presented that further demonstrate the potential of these novel approaches. Specifically, we describe the use of microfabricated devices as modules to deliver femtomole amounts of protein digests to the mass spectrometer for protein identification. We also describe the use of a microfabricated module for the generation of solvent gradients at nl/min flow rates for gradient chromatography-tandem mass spectrometry. The use of microfabricated fluidic systems reduces the risk of sample contamination and sample loss due to adsorption to wetted surfaces. The ability to assemble dedicated modular systems and to operate them automatically makes the use of microfabricated systems attractive for the sensitive and large-scale analysis of proteins.     

构建基于磁珠微流控芯片的iFlora遗传信息高效获取系统

磁珠以其比表面积大、易与生物分子耦联、操控方便等优点,在生命科学中得到了广泛应用。随着微机电系统（MicroElectroMechanicalSystems,MEMS）技术的发展,将磁珠应用到微流控芯片中构建磁珠微流控分析系统,为生物样品分离、检测提供了一种全新方法。新一代植物志iFlora融入现代DNA测序技术．应用高速发展的信息、网络技术及云计算分析平台,收集、整合和管理植物物种相关信息,以实现物种智能鉴定和数据提取,而包括DNA条形码在内的遗传信息及其获取技术在iFlora中的作用至关重要。本文重点概述了基于纳米磁珠的微流控芯片技术及其在分子生物学领域中的应用,提出构建基于纳米磁珠微流控芯片的iFlora遗传信息采集系统,在微芯片上完成从DNA提取到测序全过程,实现物种遗传信息的快速、高效获取。     

One-dimensional acoustic standing waves in rectangular channels for flow cytometry

Flow cytometry has become a powerful analytical tool for applications ranging from blood diagnostics to high throughput screening of molecular assemblies on microsphere arrays. However, instrument size, expense, throughput, and consumable use limit its use in resource poor areas of the world, as a component in environmental monitoring, and for detection of very rare cell populations. For these reasons, new technologies to improve the size and cost-to-performance ratio of flow cytometry are required. One such technology is the use of acoustic standing waves that efficiently concentrate cells and particles to the center of flow channels for analysis. The simplest form of this method uses one-dimensional acoustic standing waves to focus particles in rectangular channels. We have developed one-dimensional acoustic focusing flow channels that can be fabricated in simple capillary devices or easily microfabricated using photolithography and deep reactive ion etching. Image and video analysis demonstrates that these channels precisely focus single flowing streams of particles and cells for traditional flow cytometry analysis. Additionally, use of standing waves with increasing harmonics and in parallel microfabricated channels is shown to effectively create many parallel focused streams. Furthermore, we present the fabrication of an inexpensive optical platform for flow cytometry in rectangular channels and use of the system to provide precise analysis. The simplicity and low-cost of the acoustic focusing devices developed here promise to be effective for flow cytometers that have reduced size, cost, and consumable use. Finally, the straightforward path to parallel flow streams using one-dimensional multinode acoustic focusing, indicates that simple acoustic focusing in rectangular channels may also have a prominent role in high-throughput flow cytometry.     

Profile--Richard A. Mathies. Interview by Suzanne Berry.

Richard A. Mathies (Fig. 1) is a professor of chemistry at the University of California (UC) at Berkeley. His early work at UC was on the use of resonance Raman and time-resolved optical spectroscopy to elucidate the structure and reaction dynamics of energy and information-transducing photoactive proteins called rhodopsins. His work on the Human Genome Project led to the development of high-throughput platform technologies including capillary array electrophoresis and energy transfer fluorescent dye labels for DNA sequencing and analysis. He has also pioneered the development of microfabricated capillary electrophoresis devices, capillary array electrophoresis microplates and microfabriated integrated sample preparation and detection methods. He is the co-founder of the Center for Analytical Biotechnology at UC Berkeley. Mathies was interviewed at the BIOMEMS and Biomedical Nanotechnology conference in Columbus, Ohio, 21-25 September 2001, where he gave a talk about capillary array electrophoresis-based microprocessors. Such devices could be used as point-of-care clinical and genetic analyzers, in integrated microfluidic sequencing chips and in DNA-based computing.     

Nanobiotechnology and biosensor research

Nanobiotechnology is defined as an interdisciplinary field of science that studies the application of fine-sized biological objects (of nanoscale, 1–100 nm) to design the devices and systems of the same size that utilize for new purposes the unusual, known, or previously unknown effects. Analysis demonstrates that the final goals, approaches, solution methods, and applications of nanostructures and biological sensors have much in common. This brief review attempts to systematize a number of the available data and pick out an organic connection of the new research direction with the field of biosensor technology, which have reached the level of sustainable development.     

Nanobiotechnology and biosensor research

Nanobiotechnology is defined as an interdisciplinary field of science that studies the application of fine-sized biological objects (of nanoscale, 1-100 nm) to design the devices and systems of the same size that utilize for new purposes the unusual, known, or previously unknown effects. Analysis demonstrates that the final goals, approaches, solution methods, and applications of nanostructures and biological sensors have much in common. This brief review attempts to systematize a number of the available data and pick out an organic connection of the new research direction with the field of biosensor technology, which have reached the level of sustainable development.     

Affinity separations using microfabricated microfluidic devices:<Emphasis Type="Italic">In situ</Emphasis> photopolymerization and use in protein separations

The use of microfabricated microfluidic devices offers significant advantages over current technologies including fast analysis time and small reagent requirements. In the context of proteomic research, the possibility of using affinity-based separations for prefractionation of samples using microfluidic devices has significant potential. We demonstrate the use of microscale devices to achieve affinity separations of proteins using a device fabricated from borosilicate glass wafers. Photolithography and wet etching are used to pattern individual glass wafers and the wafers are fusion bonded at 650°C to obtain enclosed channels. A polymer has been successfully polymerizedin situ and used either as a frit for packing beads or, when derivatized with Cibacron Blue 3GA, as a separation matrix. Both of these technologies are based onin situ UV photopolymerization of glycidyl methacrylate (GMA) and trimethylolpropane trimethacrylate (TRIM) in channels.     

Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction

During the last few decades in vitro production of mammalian embryos and assisted reproductive technologies such as embryo transfer, cryopreservation, and cloning have been used to produce and propagate genetically superior livestock. However, efficiencies of these technologies remain low. For these technologies to become more commercially viable, the efficiencies must improve. Despite this importance of reproduction for the livestock industry, little progress in decreasing embryonic mortality has been made. The livestock industry has succeeded in achieving large increases in average milk production of dairy cattle, growth rate in beef cattle and leanness in swine but reproductive efficiency has actually decreased. For example, research has provided little progress toward developing an objective method to examine viability of a single living embryo. At the same time, the growth of miniaturization technologies beyond integrated circuits and toward small mechanical systems has created opportunities for fresh examination of a wide range of existing problems. While the investigation and application of miniaturization technologies to medicine and biology is progressing rapidly, there has been limited exploration of microfabricated systems in the area of embryo production. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review the progress in demonstrating microfluidic systems for in vitro embryo production (IVP) and embryo manipulation. Microfluidic technology could have a dramatic impact on the development of new techniques as well as on our basic understanding of gamete and embryo physiology.     

Enzymatic Processing in Microfluidic Reactors

Abstract

Microreaction technology is an interdisciplinary area of science and engineering. It has attracted the attention of researchers from different fields in the past few years and consequently, several microreactors have been developed. Enzymes are organic catalysts used for the production useful substances in an environmentally friendly way, and have high potential for analytical applications. However, relatively few enzymatic processes have been commercialized because of problems in the stability of enzyme molecule, and the cost and efficiency of the reactions. Thus, there have been demands for innovation in process engineering particularly for enzymatic reactions, and microreaction devices can serve as efficient tools for the development of enzyme processes. In this review, we summarize the recent advances of microchannel reaction technologies and focus our discussion on enzyme microreactors. We discuss the manufacturing process of microreaction devices and the advantages of microreactors compared with the conventional reactors. Fundamental techniques for enzyme microreactors and important applications of this multidisciplinary technology in chemical processing are also included in our topics.     

2019生物工程与大健康专刊序言

健康是人类生存和发展的基础,提高人类健康水平是可持续发展的一项重要目标。随着科学技术的发展,生物工程作为一门综合性学科,正日益成为驱动实现这些目标的重要推手。本专刊从工程设计、疾病诊断、基因治疗、细胞治疗等方面阐述了生物工程技术在健康领域的发展现状,展望了未来的发展趋势,为推动生物工程研究应用于人类的生命健康事业提供参考。     

直接心室辅助致动方法的现状与发展

直接心室辅助通过在心脏外侧柔性挤压心脏，帮助虚弱的心脏恢复功能。它能够避免人工器件与血液接触引发的血栓、血感染等问题，是人工心脏辅助器件研究与开发的重要领域之一。直接心室辅助的致动器，决定了器件的结构、形状、及其性能，是整个辅助器件关键中的关键，致动器上的任何突破有可能对直接心室辅助器件产生革命性影响。因此，本文从致动原理的角度，分析、探讨了直接心室辅助的致动方法及其存在问题，这对探索与开发满足要求的下一代直接心室辅助致动器有一定的帮助作用。     

蛋白质即时检测新技术

蛋白质是细胞各类代谢和调控等生命功能的执行者,也是致病因子、药物等对机体作用的重要靶分子.研究蛋白质表达是理解生命现象、疾病进程和药物作用的基础.临床上常规检测方法需要大型仪器支持,但随着医学事业的发展,即时检测(POCT,也称现场检测、床旁检测)成为重要的发展趋势.POCT可以改善患者和医生之间的互动方式,建立一种积...     

Prospects of nanoparticle–DNA binding and its implications in medical biotechnology

Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle–DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle–DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle–DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle–DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.     

Microfabricated Modular Scale-Down Device for Regenerative Medicine Process Development

The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h⁻¹ resulting in a modelled shear stress of 1.1×10⁻⁴ Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.     

综述与专论：蛋白质即时检测新技术

蛋白质是细胞各类代谢和调控等生命功能的执行者,也是致病因子、药物等对机体作用的重要靶分子。研究蛋白质表达是理解生命现象、疾病进程和药物作用的基础。临床上常规检测方法需要大型仪器支持,但随着医学事业的发展,即时检测(POCT,也称现场检测、床旁检测)成为重要的发展趋势。POCT可以改善患者和医生之间的互动方式,建立一种积极的医疗模式。除诊断、治疗疾病外,对从事应急工作的人员来说,POCT在现场和远程检测方面都有优势,因此研发既准确灵敏又简便快捷的蛋白质即时检测方法至关重要。