Abstract
Laboratory Automation and High-Throughput Chemistry Automation Systems
HiTRACE: High-Throughput Robust Analysis for Capillary Electrophoresis
Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics, and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment.
In this study, authors S. Yoon, et al. propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. They illustrate the application of HiTRACE on 13 data sets representing four different RNAs, three chemical modification strategies, and up to 480 single mutant variants; the largest data sets each include 87,360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate data sets. Furthermore, while the smallest of these data sets require 7–10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest data sets herein is achieved in 3–12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands. HiTRACE is available for free download at http://hitrace.stanford.edu (Yoon, S.; et al. Bioinformatics.
Micro Reactor Technology
Bioreactor Technology in Marine Microbiology: From Design to Future Application
Marine microorganisms have been playing highly diverse roles over evolutionary time by defining the chemistry of the oceans and atmosphere. During the last decades, the bioreactors with novel designs have become an important tool to study marine microbiology and ecology in terms of marine microorganism cultivation and deep-sea bioprocess characterization; unique biochemical product formation and intensification; marine waste treatment; and clean-energy generation. In this review, the authors from the laboratory of microbial ecology and technology of Ghent University in Belgium briefly summarize the current status of the bioreactor technology applied in marine microbiology and the critical parameters to take into account during the reactor design. Looking at the growing population and the pollution in the coastal areas of the world, there is an urgency to find sustainable practices that beneficially stimulate both the economy and the natural environment. Here, the authors suggest a few possibilities where innovative bioreactor technology can be applied to enhance energy generation and food production without harming the local marine ecosystem (Zhang, Y.; et al. Biotechnol Adv.
Microfluidic Chip Technology
Droplet Microfluidics for High-Throughput Analysis of Cells and Particles
Droplet microfluidics (DM) is an area of research that combines lab-on-a-chip (LOC) techniques with emulsion compartmentalization to perform high-throughput, chemical and biological assays. The key to this approach lies in the generation, over tens of milliseconds, of thousands of liquid vessels that can be used either as carriers to transport encapsulated particles and cells, or as microreactors, to perform parallel analysis of a vast number of samples. Each compartment composes of a liquid droplet containing the sample, surrounded by an immiscible fluid. This microfluidic technique is capable of generating subnanoliter and highly monodispersed liquid droplets, which offer many opportunities for developing novel single-cell and single-molecule studies, and high-throughput methodologies for the detection and sorting of encapsulated species in droplets. This report by Zagoni and Cooper provides an overview of the features of DM in a broad microfluidic context, and shows the advantages and limitations of the technology in the field of LOC analytical research. Examples are reported and discussed to show how DM can provide novel systems with applications in high-throughput, quantitative cell and particle analysis (Zagnoni, M.; Cooper, J. M. Methods Cell Biol.
Microfluidic Cell-Culture Models for Tissue Engineering
Microfluidic systems have emerged as revolutionary new platform technologies for a range of applications, from consumer products such as inkjet printer cartridges to LOC diagnostic systems. Recent developments have opened the door to a new set of opportunities for microfluidic systems in the field of tissue and organ engineering. Advances in the design of physiologically relevant structures and networks, fabrication processes for biomaterials suitable for in vivo use, and techniques for scaling toward large, three-dimensional constructs are converging toward therapeutic applications of microfluidic technologies in engineering complex tissues and organs. These advances herald a new generation of microfluidic-based approaches designed for specific tissue and organ applications, incorporating microvascular networks, structures for transport and filtration, and a three-dimensional microenvironment suitable for supporting phenotypic cell behavior, tissue function, and implantation and host integration (Inamdar, N. K.; Borenstein, J. T. Curr. Opin. Biotechnol.
Quantitative and Sensitive Detection of Rare Mutations Using Droplet-Based Microfluidics
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment, and follow-up of patients. To have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph, and other patient samples, a simple, sensitive, and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual-probe TaqMan assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of nonmutated DNA from normal cells. Here, D. Pekin et al. describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. The authors demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enables the determination of mutant allelic specific imbalance in several cancer cell lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is limited only by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of the seven different types of droplets, each containing a TaqMan probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it is possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment (Pekin, D.; et al. Lab Chip
Automated Cellular Sample Preparation Using a Centrifuge-on-a-Chip
Centrifuge is one of the most commonly used laboratory instruments in biology and medicine. Without a centrifuge it is impossible for laboratory personnel to perform some of the most basic test procedures, such as concentration, separation, and buffer exchange. Although centrifuge has gone a long way to become what it is today, its basic principle has never changed. It takes advantage of the difference in sample density and size (which is related to the viscous drag of microparticles/cells) to allow the sample to separate. To amplify the effect of minute density difference, high-speed moving parts are needed to create the high Gs. In addition, centrifuge is by nature more of a batch process rather than a continuous process. As a result, it is often challenging to integrate centrifuge in a continuous integrated system.
This problem recently has been addressed by the Dicarlo group at the University of California Los Angeles. In an article recently published in Lab on a Chip by Mach et al., a microfluidic component that replicates the function of a centrifuge is introduced. This “centrifuge” does not compose of any moving parts. The chip uses a series of unique fluid phenomena in microscale to realize cell separation. First, cells are injected into a narrow microfluidic channel, in which the balance between the shear gradient life force and wall effect lift force causes cells to align in a nearly single-file fashion. Subsequently, the aligned cell stream enters a wider flow chamber where vortices are induced because of the sudden widening of the flow channel. Also because of the widening of the channel, the wall effect lift forces dramatically decrease, and larger cells that experience larger gradient lift forces are forced to travel across the streamline where they are trapped in the vortex. At the same time, the smaller cells are still able to continue to follow the streamline and flow out of the system, and the separation of cells by size is completed. This device requires an extremely small footprint, and is simple, continuous, and cost efficient. It is a great example of how novel automation processes can arise from the understanding of unique fluid-flow behavior in microscale. The authors envision that the method may become a promising alternative to the standard benchtop centrifuge and open opportunities in automated, low-cost, and high-throughput sample preparation for cell-based diagnostics and large volume size-based cell separation (Mach et al. Lab Chip
High-Throughput Analytics
Integrating High-Throughput Pyrosequencing and Quantitative Real-Time PCR to Analyze Complex Microbial Communities
New high-throughput technologies continue to emerge for studying complex microbial communities. In particular, massively parallel pyrosequencing enables very high numbers of sequences, providing a more complete view of community structures and a more accurate inference of functions than was possible just a few years ago. In parallel, quantitative real-time PCR (QPCR) allows quantitative monitoring of specific community members over time, space, or different environmental conditions. In this review, the H. Zhang et al. discuss the principles of these two methods and their complementary applications in studying microbial ecology in bioenvironmental systems. The authors explain parallel sequencing of amplicon libraries and using bar codes to differentiate multiple samples in a pyrosequencing run. They also describe best procedures and chemistries for QPCR amplifications and address advantages of applying automation to increase accuracy. H. Zhang et al. provide three examples in which they use pyrosequencing and QPCR together to define and quantify members of microbial communities. These examples include the human large intestine; in a methanogenic digester whose sludge is made more bioavailable by a high-voltage pretreatment; and on the biofilm anode of a microbial electrolytic cell. They highlight key findings in these systems and how both methods are used in concert to achieve those findings. Finally, the authors supply detailed methods for generating PCR amplicon libraries for pyrosequencing, pyrosequencing data analysis, QPCR methodology, instrumentation, and automation (Zhang, H.; et al. Methods Mol. Biol.
Optofluidic Fluorescent Imaging Cytometry on a Cell Phone
Flow cytometer is a powerful high-throughput tool for cell counting and analysis. It is routinely used in biomedical research and clinical diagnostics. One example of its applications in clinical diagnosis is monitoring of HIV disease progression through the counting of CD4+ cells in patient blood. Traditional flow cytometers, however, are bulky, expensive, and require extensive operator training. Therefore, the potential of flow cytometer as a diagnostic tool for HIV diagnosis is limited because of the lack of resource and infrastructure in many developing counties with HIV epidemics.
Recently, the Ozcan research Group at the University of California Los Angeles introduced a simple, cost-effective solution to tackle this problem. In an article published by Zhu et al. in Analytical Chemistry, a device that weighs about half an ounce and costs less than five dollars is introduced. When coupled with a regular cell phone, the device is capable of fluorescent cell-imaging cytometry using a cell phone's CMOS sensor chip. In this cell phone-based imaging cytometry platform, fluorescently labeled cells are injected through a microfluidic channel that is positioned above the cell phone camera. In the flow channel, the injected cell suspension is sandwiched between glass/polymer substrates. The refractive index difference between the fluid and glass/polymer results in an optofluidic waveguide that is able to confine the light in the fluid channel. A fluorescent light from an inexpensive LED is butt-coupled into the channel from the side facets of the microchannel and excites the fluorescent labeled cells. Because of the confinement of the optofluidic waveguide, the excitation light from the LED only propagated along the microfluidic channel, resulting in a close-to-perfect dark background for fluorescent imaging of the cells. Therefore, only a simple plastic adsorption filter is needed in front of the cell phone camera lens to further reduce the background noise for fluorescent imaging. Fluorescent movies of the cell samples are captured by the cell phone camera and further processed to quantify the count and the density of the cells within the sample, a step that also can be potentially realized with the cell phone's processor and software. Authors tested the cell phone-based imaging cytometer with the white blood cell in human blood and achieve a result that is comparable to a commercial hematology analyzer. Further work will be conducted to investigate the potential of this device for applications such as HIV monitoring (Zhu et al. Anal. Chem.
High-Throughput Microfluidic Chip for Single-Cell RT-qPCR
Single-cell analysis holds the key to some of the most fundamental questions of biology, such as differentiation of stem cell and intrinsic noise in gene expression, because information regarding these single-cell events cannot be obtained by studying large cell populations. There have been numerous efforts in developing high-throughput platform for single-cell analysis and the recent achievement of the Hansen group of University of British Columbia is a stellar example of how an integrated microfluidic device allows the mass parallelization of multistep biochemical analysis of singles cells.
White et al. introduce a fully integrated high-throughput microfluidic chip capable of performing high-precision RT-qPCR for several thousand cells simultaneously. The chip is based on a multilayer polydimethylsiloxane microfluidic circuit design. The device composes of hundreds of microfluidic cell manipulation components such as cell traps, valves, mixers to allow the simultaneous execution of various RT-qPCR steps such as cell capture, cell lysis, reverse transcription, and qPCR. The authors demonstrate the capability of the device with experiments such as simultaneous miRNA expression measurement and single nucleotide variant detection for 3300 single cells, simultaneously. In addition to the advantages of high-throughput and lower cost, the authors also claim the benefits of reduced measurement noise and increased sensitivity (White et al. PNAS
Advances in Automation of Optical Measurement and Detection
GelSight: Easy-to-Use Handheld Imaging Sensor for Capturing Microscopic Surface Geometry
Three-dimensional (3D) profiling of microscopic surface structure is needed in numerous applications ranging from dermatology to microelectronics to material science to forensics. Currently, to capture microscopic surface geometry, sophisticated instruments such as optical profilometer or scanning confocal miscoscope are needed. These laboratory instruments often require dedicated handling by lab personnel, tend to be bulky and usually come with large price tags (>$100,000).
A recent article published by Johnson et al. introduces a novel-imaging sensor named GelSight for measuring high-resolution surface geometry. The key component of the GelSight imaging sensor is a slab of clear elastomer covered with a reflective skin. When pressing the elastomer against the object to be measured, the reflective skin distorts to take on the shape of the object's surface, and the surface geometry of the object can then be measured using the photometric stereo techniques. With the improved elastomer material, illumination design and reconstruction algorithm, the authors achieve a spatial resolution of 2 μm and a submicron depth resolution. Compared with the traditional methods such as optical profilemetry, GelSight provides some obvious advantages—it is much easier to use (users simply need to press the sensor against the sample) and it works for glossy or transparent samples. More details on the GelSight are available on authors' webpage at www.mit.edu/∼kimo/gelsight (Johnson et al. Proc. ACM SIGGRAPH
A Tunable 3D Optofluidic Waveguide Dye Laser
Tunable liquid–liquid waveguide dye laser is a great way to provide flexible on-chip tunable light source for a wide variety of lab-on-a-chip applications. In 2D liquid–liquid waveguide lasers, a liquid with a higher refractive index forms the core flow and is sandwiched by liquids with lower refractive indices that form sheath flows. Such co-injection of fluids effectively establishes a liquid–liquid waveguide with the core flow being the core of the waveguide and sheath flows being the cladding of the waveguide. The core flow contains a dye as a gain medium. When pumped with another laser, the dye emits fluorescence that is further amplified by the microcavity formed by micromirrors placed at both ends of channel to realize lasing. The liquid–liquid waveguide laser is deemed as a perfect fit for on-chip applications because it is completely dynamic—its emission wavelength, energy intensity, and nodal content can be easily reconfigured by simply changing the flow injection conditions. There is still a problem, however, with the 2D liquid–liquid waveguide laser, which is that the core flow is not completely wrapped by sheath flows. The liquid core is only confined by the liquid cladding in the horizontal direction (parallel to the chip substrate), and in vertical direction the core flow is exposed to the microfluidic chip materials such as glass and polydimethylsiloxane. Therefore, complete control of the liquid–liquid dye laser cannot be achieved because it is impossible to change the chip materials during lasing operation. In addition, propagation modes can leak in the vertical direction if the refractive index of core flow is close to that of the chip material.
A solution is reported by Yang et al. in which a 3D liquid–liquid dye laser allows the completed confinement of core flow by the liquid sheath in both horizontal and vertical directions. The authors took the advantage of a flow phenomena named “Dean vortex.” Two fluids with different refractive indices are coinjected side by side into a curved microfluidic channel. The Dean vortex induced in the curve causes the higher refractive index fluid to “bulge” toward the lower refractive index fluid. By carefully selecting the flow parameters, when two fluids exit the curve, the higher refractive index fluid is completed isolated from the channel wall and wrapped by the lower refractive index fluid, forming the 3D liquid–liquid waveguide. The dye laser based on the 3D liquid–liquid waveguide shows significant advantages over the 2D liquid–liquid waveguide laser in terms of energy efficiency/output and tunability. This versatile and efficient liquid–liquid waveguide dye laser provides a better solution for on-chip light source for a wide variety of applications (Yang et al. Lab Chip
Advances in Automation of Biomarker Discovery
Immunoaffinity Capillary Electrophoresis: A New Versatile Tool for Determining Protein Biomarkers in Inflammatory Processes
Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long-term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. This, however, can be challenging in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below.
This review highlights the need for state-of-the art methodologies with high-sensitivity and high-throughput capabilities for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology fails to successfully bring this advancement to the patient's bedside. In this report, N. A. Guzman and T. Phillips present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases. In particular, they review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two-dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and posttranslational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, and without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point-of-care biomarker analyzer in translational medicine (Guzman, N. A.; Phillips, T. M. Electrophoresis,
Lectin Magnetic Bead Array for Biomarker Discovery
Alterations in protein glycosylation play an important role in pathophysiology, and much effort has been devoted to detecting glycoprotein biomarkers. D. Loo et al. describe the development of a novel method for monitoring alterations in protein glycosylation. Lectins are used as individual affinity reagents and coupled to magnetic beads (Dynabeads) in a microplate array format for isolation of glycosylated proteins. Isolated glycoproteins are digested with trypsin in-solution followed by LC–MS/MS, allowing a liquid handler-assisted high-throughput workflow. D. Loo et al. demonstrate the specific and reproducible affinity isolation of glycoproteins using the lectin Dynabead array technology. When used with serum, they achieve one-step purification of glycoproteins with minimal coisolation of abundant serum proteins including albumin. They further optimize the proteomics workflow to allow transfer to a liquid handler for automation. In summary, the authors report the development of a high-throughput platform to detect alterations in protein glycosylation that will be useful in glycoproteomics studies, particularly clinical proteomic studies where large sample sizes are required to achieve statistical power (Loo, D.; et al. J. Proteome Res.