Abstract
Laboratory Automation and High-Throughput Chemistry
Automated Nucleic Acid Isolation in Viral Molecular Diagnostics: Evaluation of the QIAsymphony SP
The Qiagen QIAsymphony SP is a high-throughput (up to 96 samples per run), fully-automated nucleic acid isolation system. It was implemented in the authors’ laboratory to cope with the high demand for pandemic H1N1 influenza testing in 2009. This study evaluates the QIAsymphony SP for viral nucleic acid isolation from quality control materials, pure cultures, and various clinical specimens. The effect of varying sample volume on detection sensitivity is investigated using serial 10-fold dilutions of pure viral specimens, and target nucleic acids were detected by real-time polymerase chain reaction (PCR) assays. Little variability in detection sensitivity is observed for all the viral targets tested, although variation in cycle threshold values is apparent in some cases. Importantly, pathogens are detectable over a broad concentration range and from diverse clinical specimens. Removal of PCR inhibitors is generally effective, as demonstrated by detection of viral nucleic acids and/or internal controls. The results demonstrate that the QIAsymphony SP is suitable for use in routine virology molecular diagnostics and provides a high-throughput capacity, which is needed in peak seasons of infection or in centralized laboratories. (Parham, N. J., et al., Br. J. Biomed. Sci.
Development of a Fluorescence-Based Microplate Method for the Determination of Volatile Fatty Acids in Anaerobically Digested and Sewage Sludges
This article presents a simple, accurate, and multisample method for the determination of volatile fatty acids (VFAs) thanks to a 96-well microplate technique. A procedure using an activating reagent of the carboxylic function (water-soluble carbodiimide EDC) and a fluorescent amino labeling reagent (N-[1-naphthyl] ethylenediamine, EDAN) allows the formation of an isoindole derivative that needs to be separated from initial fluorescent amine for efficient VFAs determination. Isolation of these fluorescent VFA derivatives is carried out with the use of the fluorescent quenching of EDAN with o-phthaldialdehyde. Quenching is most efficient at a pH of about 7 and by heating at 40 °C within the microplate reader. This optimized procedure has been applied to various carboxylic acids and other organic compounds, demonstrating that VFAs exhibit the highest fluorescence responses with homogeneous results for the main ones (acetic, propionic, and butyric acid, all mass concentration expressed as acetic acid equivalents). This protocol is calibrated against acetic acid, and determination of VFA is thus possible in the range of 3.9 to 2000 mg L(-1) (acetic acid equivalents). Subsequent application to real samples (sewage sludges or anaerobically digested samples) and comparison to gas chromatography analyses give accurate results, proving the great potential of our high-throughput microplate-based technique for the analysis of VFA. (Robert-Peillard, F., et al., Talanta
Salivary Cortisol as a Diagnostic Tool for Cushing’s Syndrome and Adrenal Insufficiency: Improved Screening by an Automatic Immunoassay
Salivary cortisol is increasingly used to assess patients with suspected hypo- and hypercortisolism. This study establishes disease-specific reference ranges for an automated electrochemiluminescence immunoassay (ECLIA). Unstimulated saliva from 62 patients with hypothalamic-pituitary disease is collected at 0800 h. A peak serum cortisol level less than 500 nmol/L during the insulin tolerance test (ITT) is used to identify hypocortisolism. Receiver-operating characteristic (ROC) analysis allows establishment of lower and upper cutoffs with at least 95% specificity for adrenal insufficiency and adrenal sufficiency. Saliva from 40 patients with confirmed hypercortisolism, 45 patients with various adrenal masses, and 115 healthy subjects is sampled at 2300 h and after low-dose dexamethasone suppression at 0800 h. ROC analysis is used to calculate thresholds with at least 95% sensitivity for hypercortisolism. Salivary cortisol is measured with an automated ECLIA.
When screening for secondary adrenal insufficiency, a lower cutoff of 3.2 nmol/L and an upper cutoff of 13.2 nmol/L for unstimulated salivary cortisol allow a highly specific diagnosis (i.e., similar to the ITT result) in 26% of patients. For identification of hypercortisolism, cutoffs of 6.1 nmol/L (sensitivity 95%, specificity 91%, area under the curve [AUC] 0.97) and 2.0 nmol/L (sensitivity 97%, specificity 86%, AUC 0.97) are established for salivary cortisol at 2300 h and for dexamethasone-suppressed salivary cortisol at 0800 h.
The newly established thresholds facilitate initial screening for secondary adrenal insufficiency and allow excellent identification of hypercortisolism. Measurement by an automated immunoassay will allow broader use of salivary cortisol as a diagnostic tool (Deutschbein, T., et al., Eur. J. Endocrinol.
Microfluidic Chip Technology and Micro Reactor Technology
High-Volume Production of Single and Compound Emulsions in a Microfluidic Parallelization Arrangement Coupled with Coaxial Annular World-to-Chip Interfaces
This study describes a microfluidic platform with coaxial annular world-to-chip interfaces for high-throughput production of single and compound emulsion droplets, having controlled sizes and internal compositions. The production module consists of two distinct elements: a planar square chip on which many copies of a microfluidic droplet generator (MFDG) are arranged circularly and a cubic supporting module with coaxial annular channels for supplying fluids evenly to the inlets of the mounted chip, assembled from blocks with cylinders and holes. Three-dimensional (3D) flow is simulated to evaluate the distribution of flow velocity in the coaxial multiple annular channels. By coupling a 1.5 cm × 1.5 cm microfluidic chip with parallelized 144 MFDGs and a supporting module with two annular channels, for example, the authors produce simple oil-in-water emulsion droplets having a mean diameter of 90.7 µm and a coefficient of variation of 2.2% at a throughput of 180.0 mL h(–1). Furthermore, they successfully demonstrate high-throughput production of Janus droplets, double emulsions, and triple emulsions by coupling 1.5 cm × 1.5 cm to 4.5 cm × 4.5 cm microfluidic chips with parallelized 32 to 128 MFDGs of various geometries and supporting modules with three to four annular channels. (Nisisako, T., Lab Chip.
A Cell Electrofusion Microfluidic Chip Using Discrete Coplanar Vertical Sidewall Microelectrodes
A novel cell electrofusion microfluidic chip using discrete coplanar vertical sidewall electrodes is designed, fabricated, and tested. The device contains a serpentine-shaped microchannel with 22 500 pairs of vertical sidewall microelectrodes patterned on two opposing vertical sidewalls of the microchannel. The adjacent microelectrodes on each sidewall are separated by coplanar SiO(2)-polysilicon-SiO(2)/silicon. This design of coplanar discrete vertical sidewall electrodes eliminates the “dead area” present in previous designs using continuous 3D protruding sidewall electrodes and generates a uniform electric field along the height of the microchannel, leading to a lower voltage required for cell fusion compared with designs using 2D thin-film electrodes. This device is tested to fuse NIH3T3 cells under a low voltage (~9 V). Almost 100% cells are aligned to the edge of the discrete microelectrodes, and cell-cell pairing efficiency reaches 70%. The electrofusion efficiency is greater than 40% of the total cells loaded into the device, which is much higher than traditional fusion methods and existing microfluidic devices using continuous 3D protruding sidewall microelectrodes. (Hu, N., et al., Electrophoresis.
Probe Droplet Arrays Generated in the Capillary for Microarray Analysis
Microarray technology is a useful tool for nucleic acid detection and is widely used in biology and related research fields. However, the procedure is labor intensive and time-consuming. Microfluidic chip-based microarrays save time with better performance, but the low spot density and probe number limit its applications. To develop high-performance microarrays with high spot density within a microchannel, a method is reported here for preparing microarrays in a capillary by generating probe droplet arrays. The probes in droplets are immobilized onto the inner wall of the capillary to form a one-dimensional probe array, and then a sample solution is introduced to hybridize with the probe array. The effect of the capillary’s inner diameter is evaluated to realize a high-density probe array. The processes of array generation and probe immobilization are studied to avoid possible cross-contamination. The background from probe immobilization during the array generation and incubation is quantified to ensure sensitivity. Multiple sample detection is also demonstrated within one capillary. The capillary-based microarray assay has high spot density, easy fabrication, fast detection, high sensitivity, and multiple sample capacity. (Qu, X., et al., Biosens. Bioelectron.
Recent Changes in Patenting Behavior in Microprocess Technology and Its Possible Use for Gas-Liquid Reactions and the Oxidation of Glucose
The miniaturization of continuous processes is of increasing interest in the past decade, and microreaction technology and flow chemistry have moved from academic and industrial research to commercial applications. With industry taking up such innovations, this trend is also reflected in the patenting behavior of companies active in this area. This review is a continuation of the review article on microreactor patents published by Hessel et al. and indicates major changes in patenting trends since 2006. Moreover, a different patent database search algorithm is presented, which complements the algorithm explained in the previous review. In addition, the preservation of intellectual property is analyzed for multiphase reactions and particularly solid-catalyzed gas-liquid reactions in microreactors, which play an important role in the chemical and pharmaceutical industries and are reactions that benefit largely from microprocessing. Among other results, Dencic et al. show that the number of patents has increased in this field, with solid-catalyst design and deposition, control of the flow pattern, and ensured stable flow as the main challenges. (Dencic, I., et al., Chem. Sus. Chem.
High-Throughput Analytics
CellMiner: A Web-Based Suite of Genomic and Pharmacologic Tools to Explore Transcript and Drug Patterns in the NCI-60 Cell Line Set
High-throughput and high-content databases are increasingly important resources in molecular medicine, systems biology, and pharmacology. However, the information usually resides in unwieldy databases, limiting ready data analysis and integration. One resource that offers substantial potential for improvement in this regard is the NCI-60 cell line database compiled by the U.S. National Cancer Institute, which has been extensively characterized across numerous genomic and pharmacologic response platforms. In this report, Reinhold et al. introduce a CellMiner (http://discover.nci.nih.gov/cellminer/) Web application designed to improve the use of this extensive database. CellMiner tools allow rapid data retrieval of transcripts for 22 379 genes and 360 microRNAs along with activity reports for 20 503 chemical compounds including 102 drugs approved by the U.S. Food and Drug Administration. Converting these differential levels into quantitative patterns across the NCI-60 clarify data organization and cross-comparisons using a novel pattern match tool. Data queries for potential relationships among parameters can be conducted in an iterative manner specific to user interests and expertise. Examples of the in silico discovery process afforded by CellMiner are provided for multidrug resistance analyses and doxorubicin activity; identification of colon-specific genes, microRNAs, and drugs; microRNAs related to the miR-17-92 cluster; and drug identification patterns matched to erlotinib, gefitinib, afatinib, and lapatinib. CellMiner greatly broadens applications of the extensive NCI-60 database for discovery by creating Web-based processes that are rapid, flexible, and readily applied by users without bioinformatics expertise. (Reinhold, W. C., Cancer Res.
Selective High-Throughput Protein Quantification Based on UV Absorption Spectra
The application of high-throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. A new label-free and noninvasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements is presented. Analytics based on the methodology are shown to increase analytical throughput for selective protein quantification significantly; however, this is demonstrated for only one particular protein combination. In this work, the possibilities and limitations of the analytical method are investigated further. Principal component analysis (PCA) is performed on a broad range of absorption spectra to investigate their common characteristics and differences. The PCA is used both for cluster analysis and to define a measure for spectral similarity. For binary protein combinations, the calibration precision is shown to decrease exponentially with the defined spectral similarity factor. Knowledge of this correlation can be used to determine a priori whether a calibration will be successful or not. Calibration robustness is investigated by applying the analytics to liquid chromatography performed in HTE mode. Further, it is shown that a spectral difference of 0.6% is sufficient to successfully perform a spectral-based calibration of two IgG1 monoclonals. (Hansen, S. K., Hubbuch, J., Biotechnol. Bioeng.
Strategic Assay Selection for Analytics in High-Throughput Process Development: Case Studies for Downstream Processing of Monoclonal Antibodies
During bioprocess development, a potentially large number of analytes require measurement. Selection of the best set of analytical methods to deploy can reduce the analytical requirements for process investigation but currently relies on application of heuristics. This article introduces a generic methodology, strategic assay selection, for screening a large number of analytical methods to produce a subset of analytics that best suit high-throughput studies. The methodology uses a stochastic ranking approach in which analytics are ranked based on their holistic performance in a set of criteria. Strategic assay selection can be used to help minimize the impact of analytics in the generation of bottlenecks often encountered during high-throughput process development studies. This is illustrated by using a typical downstream purification process for a monoclonal antibody product. A list of assays is populated for routinely measured analytes across the different units of operation followed by the calculation of their performances in four criteria. The methodology is then applied to select analytics testing for three analytes, and the results are analyzed to demonstrate how it can lead to the selection of analytical methods with the most favorable features. (Konstantinidis, S., et al., Biotechnol. J.,
Automation Systems
High-Throughput Analysis of Therapeutic and Diagnostic Monoclonal Antibodies by Multicapillary SDS Gel Electrophoresis in Conjunction with Covalent Fluorescent Labeling
Capillary gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry and is increasingly becoming the method of choice that meets the requirements of the standards of 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, also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of bio therapeutics, 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. Reducing and nonreducing conditions are applied both with and without peptide N-glycosidase F mediated deglycosylation. (Szekrenyes, A., et al., Anal. Bioanal. Chem.
One Plate, Two Plates, a Thousand Plates: How Crystallization Changes with Large Numbers of Samples
Turning commercial lab automation into a high-throughput center requires an underlying process, and implementing checks to ensure that the process is working as it should. At the Collaborative Crystallization Centre (C3), protein samples from local, national, and international groups are set up in crystallization screening and optimization experiments with two thousand 96-well plates being set up each year. During its 5 years of operation, the C3 has implemented a series of enabling protocols—from simple “reality checks” to determine if a screen has evaporated during storage to more sophisticated systems such as a sample labeling and tracking system. The most important—and perhaps surprising—lesson has been how much effort is required to effectively communicate between the center and its clients, as well as between the center’s staff members. It is easy to confuse the concept of high throughput in any field with the idea of setting up an experiment quickly. Although automation can be used to set up a single experiment more rapidly than can be done by hand, the distinguishing feature of a high-throughput technology is the sustainability of the increased rate. (Newman, J., Methods
High-Throughput Genotyping Robot-Assisted Method for Mutation Detection in Patients with Hypertrophic Cardiomyopathy
Hypertrophic cardiomyopathy (HCM) is the most frequent autosomal dominant genetic heart muscle disease and the most common cause of sudden cardiac death in young people (younger than 30 years), who are often unaware of their underlying condition. Genetic screening is now considered a fundamental tool for clinical management of HCM families. However, the high genetic heterogeneity of HCM makes genetic screening very expensive. Here, Bortot et al. propose a new high-throughput genotyping method based on an HCM 96-well sequencing plate for the analysis of eight of the most frequent HCM-causing sarcomeric genes by automating several processes required for direct sequencing, using commercially available robotic systems and routinely used instruments. To assess the efficiency of the robot-assisted method, the authors analyze the entire coding sequence and flanking intronic sequences of the eight sarcomeric genes in samples from 18 patients affected by HCM and their relatives, which reveals nine different mutations, three of which are novel. The automated, robot-assisted assembling of PCR, purification of PCR products, and assembly of sequencing reactions result in a substantial savings of time, reagent costs, and reduction of human errors and can therefore be proposed as a primary strategy for mutation identification in HCM genetic screening in many medical genetic laboratories. (Bortot, B., Diagn. Mol. Pathol.
Advances in Robotics
Cell-Based High-Throughput Screens for the Discovery of Chemotherapeutic Agents
With modern advances in robotics and data processing, high-throughput screening (HTS) is playing a growing role in the drug discovery process. The ultimate success of HTS depends on the development of assays that are robust and reproducible in miniaturized formats, have low false-positive rates, and can identify drugs that offer improvements over those currently on the market. One example of such an assay is the ATAD5-luciferase HTS assay, which identifies three antioxidants that could kill cancer cells without inducing mutagenesis. Here Fox discusses the ATAD5-luciferase assay and expands on the value of HTS in identifying other potential cancer drugs, focusing on cell-based assays that involve DNA damage or repair pathways. (Fox, J. T., Oncotarget
Evaluation of a Robotic System for the Recovery of Peripheral Blood Mononuclear Cells
Investigations into immune responses are often based on recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labor intensive and requires a reasonable level of skill by the laboratory technician. Thus, McQuillan et al. set out to determine whether laboratory automation equipment can be used for the recovery of PBMC from blood samples of horses, pigs, and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, and recovery of separated PBMC in RPMI 1640 medium are performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes are tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compare very well to those of the manual one in terms of percentage cell recovery, sterility, and cell viability. Also, a high throughput of samples could be implemented. With the integration of cell counting, it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day. (McQuillan, A., et al., Biologicals