Abstract
Laboratory Automation and High-Throughput Chemistry
A Unique Automation Platform for Measuring Low-Level Radioactivity in Metabolite Identification Studies
Generation and interpretation of biotransformation data on drugs (i.e., identification of physiologically relevant metabolites), defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using (14)C or (3)H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo, or clinical study. Metabolite profiling is a very time-intensive activity, particularly for preclinical in vivo or clinical studies that have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies that do not require specialized high-volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification, are a very good example. The current lack of automation for measuring low-level radioactivity in metabolite profiling requires substantial capacity, personal attention, and resources from laboratory scientists. To help address these challenges and improve efficiency, Krauser et al. have innovated, developed, and implemented a novel and flexible automation platform that integrates a robotic plate-handling platform, high-performance liquid chromatography or ultra performance liquid chromatography system, mass spectrometer, and an automated fraction collector. (Krauser, J., et al., PloS One,
The BUME Method: A Novel Automated Chloroform-Free 96-Well Total Lipid Extraction Method for Blood Plasma
Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, Lofgren et al. have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries are linear from 10 to 100 µL plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µL butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µL heptane:ethyl acetate (3:1) using 300 µL 1% acetic acid as buffer. The lipids investigated include the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in the authors’ laboratory and is now used daily. The authors conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources. (Lofgren, L., et al., J. Lipid Res.,
High-Throughput Automated Chromatin Immunoprecipitation as a Platform for Drug Screening and Antibody Validation
Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently, the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low-throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large-scale screening purposes. To address these challenges, Wu et al. report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR, results take only 1 d using HTChIP as compared with several days up to 1 wk for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. The authors perform a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, Wu et al. believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency and further facilitate the use of ChIP as a valuable tool for research and discovery. (Wu, A. R., et al., Lab Chip
High-Throughput Analysis of Therapeutic and Diagnostic Monoclonal Antibodies by Multicapillary SDS Gel Electrophoresis in Conjunction with Covalent Fluorescent Labeling
Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and it is increasingly becoming the method of choice that meets the requirements of the standards of the International Conference of Harmonization. Automated single-channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent detection options, offering general applicability and high detection sensitivity, respectively, but with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies. The methodology reported in this article is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of nonglycosylated heavy chains and separation of possible subunit variations such as truncated light chains (pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multicapillary gel electrophoresis are thoroughly described. Both reducing and nonreducing conditions are applied with and without peptide N-glycosidase F-mediated deglycosylation. (Szekrenyes, A., et al., Anal. Bioanal. Chem.
Automation Systems
Strategies for the Successful Implementation of Viral Laboratory Automation
It has been estimated that more than 70% of all medical activity is directly related to information providing analytical data. Substantial technological advances have taken place recently, which have allowed a previously unimagined number of analytical samples to be processed while offering high-quality results. Concurrently, more new diagnostic determinations have been introduced, all of which lead to a significant increase in the prescription of analytical parameters. This increased workload places great pressure on the laboratory with respect to health costs. Clinical laboratory (CL) managers must examine cost control as well as rationing, meaning that the CL’s focus is not strictly metrological, as if it were purely a system producing results, but instead must include concentrating on efficiency and efficacy. By applying reengineering criteria, an emphasis is placed on improved organization and operating practice within the CL, focusing on the current criteria of the integrated management areas where the technical and human resources are brought together. This reengineering is based on the concepts of consolidating and integrating the analytical platforms while differentiating the production areas (CORE Laboratory) from the information areas. With these present concepts in mind, automation and virological treatment, along with serology in general, follow the same criteria as the rest of the operating methodology in the clinical laboratory. (Avivar, C., Open Virol. J.
Transforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial Strains
Microbial genotyping increasingly deals with large numbers of samples, and data are commonly evaluated by unstructured approaches, such as spreadsheets. The efficiency, reliability, and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage. O’Farrell et al. have developed a medium-throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline is implemented through a combination of four automated liquid-handling systems, a laboratory information management system (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a sample management system, plus numerous Python scripts. All tubes and microwell racks are bar-coded, and their locations and status are recorded in the LIMS. The authors also create a hierarchical set of items that can be used to represent bacterial species, their products, and experiments. The LIMS allows reliable, semiautomated, traceable bacterial genotyping from initial single-colony isolation and subcultivation through DNA extraction and normalization to PCRs, sequencing, and MLST sequence trace evaluation. O’Farrell et al. also describe robotic sequencing to facilitate cherry-picking of sequence dropouts. This pipeline is user-friendly, with a throughput of 96 strains within 10 working days at a total cost of <€25 per strain. Since developing this pipeline, >200 000 items are processed by two to three people. This sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support and provides a general framework for semiautomated bacterial genotyping of large numbers of samples at low cost. (O’Farrell, B., et al., PloS One.
Microfluidic Chip Technology and Micro Reactor Technology
Automated Analysis of Single Stem Cells in Microfluidic Traps
Kobel et al. report a reliable strategy to perform automated image cytometry of single (nonadherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). The authors use this method to quantify successfully the efficiency and spatial distribution of single-cell loading of a microfluidic chip composed of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells faithfully recapitulates the distribution of cells in the G1 and S/G2-M phase of the cell cycle that is measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems. (Kobel, S. A., Lab Chip
Multiplexed Volumetric Bar-Chart Chip for Point-of-Care Diagnostics
Microfluidics has become an enabling technology for point-of-care and personalized diagnostics. Desirable capabilities of microfluidics-based diagnostic devices include simplicity, portability, low cost, and the performance of multiplexed and quantitative measurements, ideally in a high-throughput format. Here, the authors present the multiplexed volumetric bar-chart chip (V-Chip), which integrates all of these capabilities in one device. A key feature of the V-Chip is that quantitative results are displayed as bar-charts directly on the device without the need for optical instruments or any data processing or plotting steps. This is achieved by directly linking oxygen production by catalase, which is proportional to the concentration of the analyte, with the displacement of ink along channels on the device. Song et al. demonstrate the rapid quantification of protein biomarkers in diverse clinical samples with the V-Chip. The development of the V-Chip thus opens up the possibility of greatly simplified point-of-care and personalized diagnostics. (Song, Y., et al., Nat. Commun.
Quantum Dots–Based Immunofluorescent Microfluidic Chip for the Analysis of Glycan Expression at Single Cells
Rich, high-quality single-cell information from rare cell samples is very important for the quantitative systems biology description of cellular function. However, this type of data is often prohibited by conventional analytical technology such as flow cytometry. In this article, the authors describe a microfluidic platform coupled with a quantum dots–based (QDs) immunofluorescence (IF) approach to measure the expression of glycans on the cell surface of single cells or the cell population. Compared with conventional IF staining, the QDs-based IF probe exhibits higher brightness and stability against photo bleaching. With the merits of the novel IF staining protocol and microfluidic platform, high-throughput IF staining is performed to measure the glycan expressions and the changes at single K562 cells after drug treatment. The protocol proposed here shows a high sensitivity on the glycan expression profile owing to the amplification of the signal in indirect IF staining. The size of cell sample is only 4 × 103 cells, which makes the rare cell sample analysis accessible. This method may find widespread application for assessing cell-surface glycoprotein expression as well as analysis of the heterogeneity in cell populations in a high-throughput manner. (Cao, J. T., et al., Anal. Chem.,
High-Throughput Protein Expression Generator Using a Microfluidic Platform
Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint. Glick et al. have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein, protein-RNA, or protein-DNA interactions. The device consists of thousands of reaction chambers, which are individually programmed using a microarrayer. Alignment of these printed microarrays to microfluidics devices programs each chamber with a single spot, eliminating potential contamination or cross-reactivity. Moreover, generating microarrays using standard microarray-spotting techniques is also very modular, allowing for the arraying of proteins, DNA, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics-based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to affect biology. (Glick, Y., et al., J. Vis. Exp.,
Inertial Microfluidics in Parallel Channels for High-Throughput Applications
Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In this study, the authors introduce inertial microfluidics in flows through straight, multiple parallel channels. The scalable, single inlet and two outlet, parallel channel system is enabled by a novel, high-density three-dimensional PDMS microchannel manufacturing technology, mediated via a targeted inhibition of PDMS polymerization. Using single channels, Hansson et al. first demonstrate how randomly distributed particles can be focused into the center position of the channel in flows through low-aspect-ratio channels and can be effectively fractionated. As a proof of principle, continuous focusing and filtration of 10 µm particles from a suspension mixture using 4- and 16-parallel-channel devices with a single inlet and two outlets are demonstrated. A filtration efficiency of 95% to 97% is achieved at throughputs several orders of magnitude higher than previously shown for flows through straight channels. The scalable and low-footprint focusing device requiring neither external force fields nor mechanical parts to operate is readily applicable for high-throughput focusing and filtration applications as a standalone device or integrated with lab-on-a-chip systems. (Hansson, J., et al., Lab Chip,
Breast Cancer Diagnostics Using Microfluidic Multiplexed Immunohistochemistry
A quantitative, reproducible, fast, and inexpensive multiplexed immunohistochemistry (IHC) system might play a locomotive role in drug screening and personalized medicine. Currently, fully automated IHC machines and sequential multiplexed IHC methods based on multiple color reagents have been developed, with the evolution of such methods having revealed novel biological findings over the conventional IHC method, which is time-consuming and labor intensive. Kim et al. describe a novel parallel multiplexed IHC method using a microfluidic multiplexed IHC device for quantitative pathological diagnosis of breast cancer. The key factors for success of parallel multiplexed IHC are the fabrication of a robust microfluidic device, the interface between the device and a tissue slide, and an accurate fluidic control for multiple IHC reagents. To apply conventional thin-section tissues into on-chip systems without any additional modification process, a tissue slide-compatible assembler is developed for optimal compatibility of conventional IHC methods. With this approach, perfect fluid control for various solutions is demonstrated without any leakage, bubble formation, or cross- contamination. The results presented in this chapter indicate that this microfluidic IHC protocol can provide the possibility of tailored cancer treatments as well as precise histopathological diagnosis using numerous specific biomarkers. (Kim M. S., et al., Methods Mol. Biol.
Applications in Emerging Technologies
Next-Generation Sequencing Technologies and Their Impact on Microbial Genomics
Next-generation sequencing technologies have had a dramatic impact in the field of genomic research through the provision of a low-cost, high-throughput alternative to traditional capillary sequencers. These new sequencing methods have surpassed their original scope and now provide a range of utility-based applications, which allow for a more comprehensive analysis of the structure and content of microbial genomes than was previously possible. With the commercialization of a third generation of sequencing technologies imminent, Forde et al. discuss the applications of current next-generation sequencing methods and explore their impact on and contribution to microbial genome research. (Forde, B. M., and O’Toole, P. W., Brief Funct. Genomics,
Mobile Element Biology: New Possibilities with High-Throughput Sequencing
Mobile elements comprise more than half of the human genome, but until recently, their large-scale detection was time-consuming and challenging. With the development of new high-throughput sequencing technologies, the complete spectrum of mobile element variation in humans can now be identified and analyzed. Thousands of new mobile element insertions (MEIs) have been discovered, yielding new insights into mobile element biology, evolution, and genomic variation. Here, Xing et al. review several high-throughput methods with an emphasis on techniques that specifically target MEIs in humans. Xing et al. highlight recent applications of these methods in evolutionary studies and in the analysis of somatic alterations in human normal and tumor tissues. (Xing, J., et al., Trends Genet.,
Next-Generation Sequencing and De Novo Assembly, Genome Organization, and Comparative Genomic Analyses of the Genomes of Two Helicobacter pylori Isolates from Duodenal Ulcer Patients in India
The prevalence of different Helicobacter pylori genotypes in various geographical regions indicates region-specific adaptations during the course of evolution. Complete genomes of H. pylori from countries with high infection burdens, such as India, have not yet been described. In this report, the authors present genome sequences of two H. pylori strains, NAB47 and NAD1, from India. Kumar et al. briefly mention the sequencing and finishing approaches, genome assembly with downstream statistics, and important features of the two draft genomes, including their phylogenetic status. The authors believe that these genome sequences and the comparative genomics emanating thereupon will help to clarify understanding of the ancestry and biology of the Indian H. pylori genotypes, and this will be helpful in solving the so-called Indian enigma, by which high infection rates do not corroborate the minuscule number of serious outcomes observed, including gastric cancer. (Kumar, N., et al., J. Bacteriol.
Next-Generation Sequencing Technology in Clinical Virology
Recent advances in nucleic acid sequencing technologies, referred to as “next-generation” sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represent the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a data set of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harbored by different cell lineages, the dynamics of development of drug resistance, and the reemergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner. (Capobianchi, M. R., et al., Clin. Microbiol. Infect.
Resequencing Rice Genomes: An Emerging New Era of Rice Genomics
Rice is a model system for crop genomics studies. Much of the early work on rice genomics focused on analyzing genome-wide genetic variation to further understand rice gene functions in agronomic traits and to generate data and resources for rice research. The advent of next-generation high-throughput DNA-sequencing technologies and the completion of high-quality reference genome sequences have enabled the development of sequencing-based genotyping and genome-wide association studies that have significantly advanced rice genetics research. This has led to the emergence of a new era of rice genomics aimed at bridging the knowledge gap between genotype and phenotype in rice. These technologies have also led to pyramid breeding through genomics-assisted selection, which will be useful in breeding elite varieties suitable for sustainable agriculture. Here, Huang et al. review the recent advances in rice genomics and discuss the future of this line of research. (Huang, X., et al., Trends Genet.