Abstract
Laboratory Automation and High-Throughput Chemistry
The Combinatorial Synthesis of Bioactive Quinoxalines, Quinoxalinones and Quinoxalinols
In the family of biologically active heterocyclic templates, quinoxalines, quinoxalinones, and quinoxalinols have received much attention owing to the wide range of their biological activities. Approaches to the combinatorial synthesis of these compounds are surveyed by H. Xu et al. and in a review that discusses solid-phase, solution-phase, solvent-free, and microwave-assisted parallel synthesis of quinoxalines, quinoxalinones, and quinoxalinols (Comb. Chem. High Throughput Screen.
High-Throughput Discovery of Families of High Activity WGS Catalysts: Part I—History and Methodology
State-of-the-art water gas shift (WGS) catalysts (FeCr for high temperature shifts and CuZn for low temperature shifts) are not active enough to be used in fuel processors for the production of hydrogen from hydrocarbon fuels for fuel cells. The need for drastically lower catalyst volumes has triggered a search for novel WGS catalysts that are more active than current systems. Novel catalytic materials for the high-, medium-, and low-temperature WGS reactions have been discovered by application of combinatorial methodologies. Catalyst libraries are synthesized on 4-inch wafers in 16 × 16 arrays and screened in a high-throughput scanning mass spectrometer in the temperature range of 200-400 °C. More than 200 wafers are screened by C. J. Brooks et al. under various conditions, and more than 250,000 experiments are conducted to comprehensively examine catalyst performance for various binary, ternary and higher-order compositions (Comb. Chem. High Throughput Screen.
Flow Processing of Microwave-Assisted (Heterogeneous) Organic Reactions
A commercially available continuous-flow reactor is adapted by L. A. Hulshof et al. to run three organic reactions; for example, two heterogeneous and one homogeneous mixture under microwave heating. The setup is operated either as a batch-loop reactor for running a biocatalyzed esterification of (R,S)-1-phenylethanol with vinyl acetate and the esterification of (S)-pyroglutamic acid with n-decanol (i.e., Laurydone process) or in a single pass for the aspirin synthesis as a homogeneous mixture. The tubular reactor is characterized to perform with a series of three equally sized, continuously operated stirred tank reactors on average. Although the (operational) cost of the microwave-heated tubular reactor is higher than conventionally heated processes in fine chemical operations, it is demonstrated that the experimental data can be used for process design. Benefits with respect to safety and scalability are expected to enable a fair compensation for the costs when implementing this novel technique (Org. Process Res. Dev.
ReactIR Flow Cell: A New Analytical Tool for Continuous Flow Chemical Processing
A newly developed ReactIR flow cell is reported by S. V. Ley et al. as a convenient and versatile in-line analytical tool for continuous-flow chemical processing. The flow cell is attached directly into a reaction flow stream using standard OmniFit (HPLC) connections and can be used in combination with both meso-and microscale flow chemistry equipment. The IR analysis software enables the monitoring of reagent consumption and product formation, aiding the rapid optimization of procedures. Short-lived reactive intermediates also can be observed in situ, giving further mechanistic insight into complex transformations (Org. Process Res. Dev.
Rapid Identification of a Scalable Catalyst for the Asymmetric Hydrogenation of a Sterically Demanding Aryl Enamide
High-throughput screening (HTS) is used by L. Lefort et al. to find a cost-effective and scalable catalyst for the asymmetric hydrogenation of a sterically demanding enamide as an intermediate toward a new potent melanocortin receptor agonist useful in the treatment of obesity. Lessons drawn from the testing of a first library of 96 chiral monodentate phosphoramidites lead to the design of a second focused
library of 16 chiral ligands, allowing the discovery of a new efficient catalyst. This catalyst is based on rhodium and a bulky monodentate phosphite ligand. The catalyst is scaled up and used in the kilogram production of the desired bulky chiral amide (Org. Process Res. Dev.
Microfluidic Chip Technology and Micro Reactor Technology
Flow Chemistry using Milli-and Microstructured Reactors—From Conventional to Novel Process Windows
The terminology Novel Process Window unites different methods to improve existing processes by applying unconventional and harsh process conditions, such as process routes at much elevated pressure, much elevated temperature, or processing in a thermal runaway regime to achieve a significant impact on process performance. V. Hessel et al. review Novel Process Windows on selected chemical reactions. General characteristics of microreactors are discussed, including excellent mass and heat transfer and improved mixing quality. Different types of reactions are presented in which the use of microstructured devices leads to increased process performance by applying Novel Process Windows. Milli-and microstructured reactors also offer advantages in other areas, for example, HTS of catalysts and better control of size distribution in particle synthesis process by improved mixing (Bioorg. Med. Chem.
Aminolysis of Epoxides in a Microreactor System: A Continuous Flow Approach to β-Amino Alcohols
The use of a continuous-flow microreactor for β-amino alcohol formation by epoxide aminolysis is evaluated by T. F. Jamison et al. When compared with microwave batch reactions, it is revealed that conditions obtainable in the micro-reactor can match or improve yields in many cases. By increasing the pressure of the system, maximum temperatures also can exceed those accessible using a microwave unit. The use of a microreactor for epoxide aminolysis reactions in the synthesis of two pharmaceutical relevant compounds is described (Org. Process Res. Dev.
Expedited Development through Parallel Reaction Screening: Application to PTC-Mediated Knoevenagel Condensation
Parallel microreactor screening reported by Mark B. Mitchell et al. enables rapid identification of effective conditions for a Knoevenagel condensation between an aldehyde and a thiazalone, affording a dramatic reduction in cycle time when compared with traditional conditions. Interesting facets of the reaction mechanism are revealed from kinetic profiling, specifically the operation of an extractive phase transfer catalysis mechanism with a pH optimum for the Knoevenagel condensation, a pKa optimum for the elimination reaction, and the requirement for crystallization of the aldol tautomer to drive the reaction to completion (Org. Process Res. Dev.
The Continuous Flow Synthesis of Butane-2,3-Diacetal Protected Building Blocks using Microreactors
The continuous-flow synthesis of butane-2,3-diacetal protected derivatives is achieved by S. V. Ley et al. using commercially available flow chemistry microreactors in concert with solid-supported reagents and scavengers to provide in-line purification systems. The products are all obtained in superior yield compared with corresponding batch processes (Org. Biomol. Chem.
Microfluidic Cell Culture Systems for Drug Research
In pharmaceutical research, an adequate cell-based assay scheme to efficiently screen and validate potential drug candidates in the initial stage of drug discovery is crucial. To better predict the clinical response to drug compounds, a cell culture model that is faithful to in vivo behavior is required. With recent advances in microfluidic technology, the utilization of a microfluidic-based cell culture has several advantages, making it a promising alternative to conventional cell culture methods. G. B. Lee et al. summarize the role of cell culture in drug research and the characteristics of the cell culture formats commonly used in current microfluidic-based cell-culture practices. Because of the significant differences in several physical phenomena between micro-and macroscale devices, microfluidic technology provides unique functionality that was not previously possible using traditional techniques. The niches for using microfluidic-based cell culture systems for drug research are discussed. Moreover, some critical issues such as cell immobilization, medium pumping, or gradient generation in microfluidic-based cell-culture systems also are reviewed. Practical applications of microfluidic-based cell-culture systems in drug research, particularly those pertaining to drug toxicity testing and those with a high-throughput capability, are highlighted (Lab Chip,
Fully Polymeric Integrated Microreactor/Electrospray Ionization Chip for On-Chip Digestion and Mass Spectrometric Analysis
Integration of several functions such as sample concentration, separation, and analysis on a single microchip has been a goal in microfluidics. In analytic procedures, this integration reduces the laborious manual steps that are typically needed, making the analysis of samples faster and more cost-effective.
The chip, developed by Nissilä et al., integrates a micro-reactor with an electrospray ionization tip for mass spectrometric analysis. The chip is divided in a microreactor spot, a micropillar-filled channel, and an electrospray tip. The spontaneous transportation of the sample from microreactor to electrospray tip happens through strong capillary forces as a result of the hydrophilic micropillar. Although the most common material used in microtechnology is silicon, glass and polymer are often used in microfluidics because of their properties; for example, electrical and thermal insulation or transparency. Furthermore, the use of SU-8 epoxy polymer results in a fabrication process that requires only two lithographic steps, making fabrication simple, cost-effective, rapid, and well suited for mass production.
The chip went through verification with verapamil. The limit of detection is determined and defined as signal-to-noise ratio ≥3, the linearity is tested in the range of 1 nM—10 μM. In contrast to most microchips, there is no need for immobilization of enzymes to the microreactor, which is tested with the digestion of trysin (Sen. Actuators B Chem.
High-Throughput Analytics
Interfacing Microchip Electrophoresis to a Growth Tube Particle Collector for Semicontinuous Monitoring of Aerosol Composition
A wide variety of biogenic, antropogenic, geogenic, and secondary sources may suspend particles in the form of aerosols in the environment, which can react in the atmosphere and produce a high variable distribution of particles. The analysis of aerosol composition with water-soluble fraction is of particular interest for investigation of their effects on human health, environment, and climate. Aerosol analysis is often difficult because of aerosols' ubiquity, large size range, wide compositional spectrum, as well as high temporal and spatial spectrum. Noblitt et al. provide a prototype called aerosol chip electrophoresis (ACE) for semicontinuous monitoring of aerosol chemical composition, which is actually used for determination of inorganic anions such as sulfates and nitrates (Anal. Chem.
Part-per-Trillion Determination of Pharmaceuticals, Pesticides, and Related Organic Contaminants in River Water by Solid-Phase Extraction Followed by Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry
For environmental monitoring, regulatory agencies such as the European Union and United States Environmental Protection Agency have issued dangerous and hazardous contaminant lists for priority substances, which restricts their concentration and occurrence in water. Emerging pollutants such as pharmaceuticals and personal care products are not included in the priority substances category. In this case, the probable enforcement of new regulations on emerging pollutants in surface waters requires suitable and fast analytical techniques for investigation of a large number of these micropollutants. Typical methodologies for analysis of organic contaminants are LC/MS, GC/MS, and LC/MS/MS with a previous solid-phase extraction as a preconcentration-enrichment step. LC/MS methods are limited by the phenomenon of ionic suppression. For the first time, Matamoros et al. demonstrate an analytical method using two-dimensional GC (GC × GC) with coupling to a time-of-flight-MS (Anal. Chem.
Bioautomation and Screening
Miniaturization of a High-Throughput Screening Assay Comparing Air Displacement and Capillary-Based Nanolitre Transfer Technologies
The miniaturization of HTS assays has been a widely debated and researched strategy for reducing screening costs and increasing speed without compromising data quality. In this context, liquid-handling technologies continue to improve. A new and promising development is the emergence of nanoliter dispensers, which are capable of direct compound transfer to assay microplates. C. J. Mageean and F. H. Büttner investigate the miniaturization of an HTS kinase assay and compare real-life performance of current state-of-the-art air displacement transfer technology (MiniTrak V System) and a capillary-based nanoliter dispenser (CyBi-HummingWell). The robustness and effectiveness of the miniaturized assay formats are compared by testing stauro-sporine to generate dose-response curves and 340 previously identified active compounds (Comb. Chem. High Throughput Screen.
High-Throughput Screening of Normal and Neoplastic Tissue Samples
The capacity to rapidly and efficiently elucidate a reliable set of disease-specific biomarkers is paramount to enabling a future of personalized medicine. HTS methods applied to human clinical samples for the discovery of diagnostic, prognostic, and therapeutic targets address this need. Although analyzing either thousands of markers from one sample or one marker from thousands of samples is the current state of HTS, it would be ideal to analyze thousands of markers from thousands of samples to expedite the early discovery phase of biomarkers and their validation. J. W. Gillespie et al. summarize the current state of HTS of tissue specimens and discuss its applications (Comb. Chem. High Throughput Screen.
Miniaturization and Validation of the Ellman's Reaction-Based Acetylcholinesterase Inhibitory Assay into 384-Well Plate Format and Screening of a Chemical Library
P. M. Vuorela et al. report a screen for acetylcholinesterase (AChE) inhibitors from a large chemical library of commercially available compounds. The Ellman's reaction-based assay is miniaturized into a 384-well plate format, and two modifications of the kinetic protocol are studied with the aim of developing a rapid screening platform that can ensure high efficiency in finding true hits. It is proven that when starting the kinetic reaction by adding substrate, better assay performance is achieved, and more practical benefits are obtained. Using the optimized automated protocol, a chemical library of 56,320 compounds is screened. A total of 350 positive hits are identified, and their half maximal inhibitory concentration (IC50) calculated. Three highly active compounds are identified with IC50 values close or even lower to physostigmine (<0.1 μM). The most active hit (IC50[AChE] = 0.019 μM) is identified as a new inhibitor: (N-[3-(3,5-dimethyl-1-piperidinyl)propyl]-5-ethyl-2-methyl-8-oxo-thieno[2′,3′:4,5]pyrrolo[1,2-d] [1,2,4] triazine-7(8H)-acetamid), with no other biological activities reported until now. The miniaturized 384-well plate format of the Ellman's method is proven to be robust and to perform reliably (Comb. Chem. High Throughput Screen.
High-Throughput Bioassays using Nanoparticles
A. Gómez-Hens et al. share an overview of the usefulness of different nanoparticles to improve the features of high-throughput separation and individual and multiplexed detection bioassays. Although the development of microarray and microfluidic systems has expanded the capabilities of these high-throughput assays, the combined use of nanoparticles and these devices provides them with new applications in drug discovery, proteomic and genomic studies, and clinical diagnosis. This article reviews the wide application field of magnetic, gold, silver, semiconductor, and other nanoparticles in high-throughput bioassays (Comb. Chem. High Throughput Screen.
Drug Screening for Hearing Loss: Using the Zebrafish Lateral Line to Screen for Drugs That Prevent and Cause Hearing Loss
Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for HTS. The zebrafish has emerged as a powerful animal model for screening drugs that cause and prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. H. C. Ou et al. use this model to screen and identify multiple drugs that protect hair cells from aminoglycoside-induced death. The identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection (Drug Discov. Today.
Engineering a Polarity-Sensitive Biosensor for Time-Lapse Imaging of Apoptotic Processes and Degeneration
Apoptosis is of central importance to many areas of biological research, but there is a lack of methods that permit continuous monitoring of apoptosis or cell viability in a nontoxic and noninvasive manner. Kim et al. report the development of a tool for live-cell imaging that facilitates the visualization of real-time apoptotic changes without perturbing the cellular environment. They engineer an annexin-based fluorescent biosensor with built-in “on” and “off” fluorescent states by conjugating polarity-sensitive thiol-reactive fluorophores to cysteines introduced at specific sites in annexin. This polarity-sensitive indicator of viability and apoptosis (pSIVA) binds via annexin to phosphatidylserine, a negatively charged lipid exposed on the outer leaflet of the plasma membrane in early apoptotic stages. In this membrane-bound nonpolar state, it fluoresces, whereas in the polar aqueous solution, only negligible fluorescence is detectable.
To test the application of pSIVA to live-cell imaging and its capacity to specifically highlight cells undergoing the apoptotic pathway, the biosensor is directly added to the culture medium of COS-7 cells that are induced to apoptosis, and the cells are monitored under physiological conditions by time-lapse microscopy. A bright pSIVA staining of COS-7 cells in the early stages of apoptosis and a gradual increase in staining concurrent with progression into late-stage cell death detected by propidium iodide staining can be observed. In comparison, no annexin or propidium iodide staining can be observed in COS-7 cells grown under normal conditions. To study pSIVA in a more complex process, the neuronal degeneration of purified dorsal root ganglion sensory neurons is studied. It is shown that pSIVA binding occurs in a specific spatiotemporal order, indicating that phosphatidylserine exposure occurs successively, originating from a particular location in the axon and progressing toward the cell body or the axon terminal. Even the rescue from degeneration in neurons with pSIVA-stained axons but not pSIVA-stained cell bodies is shown. The advantages of pSIVA also extend to imaging apoptotic processes in vivo. After intramuscular injection of pSIVA along a rat's sciatic nerve 3 days after nerve transaction, degenerating nerves are imaged.
Together, current and future technical advances of in vivo imaging methods suggest the utility of pSIVA for examining the spatiotemporal progression of cell death and degeneration in model systems and after injury or disease (Yujin E. Kim et al., Nat. Methods.
Automation Systems and Software
Toward Automated Robotic Nanomanipulation Systems
This article describes the key components that need to be developed and implemented to make full automation on the nanoscale possible. Multiple nanohandling robots are integrated into a flexible robot cell that can quickly adapt to different manipulation scenarios. Thus, the nanorobotic system serves not only industrial processes, where high throughput is essential for profitable manufacturing, but also research work. The author begins by invalidating the fact that microrobotics is simply miniaturized macrorobotics. Finding new solutions for upcoming problems, for example handling the van-der-Waals force or the temperature drift, is of top priority instead. To provide high resolutions down to 1 nm despite a large travel range with up to 40 cm, special actuation technologies are required. Nearly all approaches are based on the inverted piezoelectrical effect combined with a stick-slip principle to reach displacements beyond the maximum piezo deflection.
The next part is determined by the characterization of the control unit. A three-tier low-level controller can effectively move the robot and provide an easy to use, mostly generic interface for higher-level automation. Two aspects are emphasized. The first is the importance of the physical layer, where the signal processing takes place on a digital controller. The signal generation usually has to be done using dedicated hardware because the required signal shapes are very complex. The stable signal amplification is additionally difficult to achieve because the frequently used piezoceramic actuators act as capacitive loads, and actuator principles require high slew rates. Second, the specialties of implementing either open-or closed-loop control architecture for nanorobotic systems are compared. Subsequently, the client/server control architecture with a Common Object Request Broker Architecture (CORBA)-based interprocess communication is displayed. To be independent of a specific scenario, automation sequences can be developed using an architecture-independent scripting language. Furthermore, the structure allows for real-time compliant control loops and the development of specialized methods for collision avoidance and path planning. The architecture is evaluated by real-time capable mobile robot tracking. In the end, the major challenges of performing calibration and adjustment on the nanoscale are highlighted. In conclusion, the flexible automation architecture described, with all its hardware and software components, facilitates efficient and reliable automation even in nanoscale (D. Jasper et al., IEEE/ASME International Conference on Advanced Intelligent Mechatronics, Singapore,
Liquid and Solid Delivery Technologies
Capacitive Nanoliter Droplet Detection on the Fly
Because the application of very small amounts of liquids is becoming a routine task in life sciences and other fields, the need for reliable and monitorable ultralow volume liquid-handling systems is increasing. The widely accepted piezo-based systems on the market lack the capability to monitor ongoing dosing processes. Ernst et al. describe a capacitive sensor that is capable of detecting droplets of 20-60 nL on the fly. To accomplish this, the droplets need to pass through the electric field of an open plate capacitor, where the droplet leads to a change of capacity because of its dielectric constant. The sensor is realized on a printed circuit board to make cost-efficient production possible.
A drawback of this approach is that the sensor is likely to be contaminated when a dosing failure results in an irregular flight path because the droplet has to pass through the plate capacitor at a short distance. The detection of a capacity change is realized by detuning the active high pass filter. Therewith, a change of capacity results in a change of signal amplitude, given a constant sinusoidal input signal. Because an expected change of capacity of 50 fF is very small, a conditioning circuit is developed to make the signal usable.
The described measurement principle is sensitive to droplet-media, volume, and velocity. The effects are briefly described in the article. It is planned to extend the system by a quantitative measurement capability in forthcoming research. To enable a quantitative measurement for a range of different media and flight velocities, these effects must be compensated. Ernst et al. describe a first quantitative experiment, the measurement of water droplets with a resolution of 2 nL in the range of 20-60 nL within a confidence interval of 95%. The promising results show that droplet detection and quantitative measurement can be achieved with the capacitive sensor (Sens Actuators A Phys.
Inkjet Metrology: High-Accuracy Mass Measurements of Microdroplets Produced by a Drop-on-Demand Dispenser
Often, drop-on-demand (DOD) microdispensers use optical methods to monitor droplet ejection and/or to determine droplet volume. Quality assurance procedures for nanoliter and picoliter dispensing technologies are critically important across many advanced and emerging applications. Calibrated microscopic optical imaging and fluorescent techniques are often deployed to measure ejected droplet dimensions. In connection with automated boundary and threshold recognition procedures, these approaches can offer fairly high precision. However, effects such as fluid oscillations, droplet flattening during flight, and refraction may introduce bias as well. Uncertainties in boundary delineations and diameter measurements can translate to volume and mass uncertainties easily exceeding 10%.
The authors describe gravimetric methods for measuring the mass of droplets generated by a DOD microdispenser. Droplets are deposited, either continuously at a known frequency or as a burst of a known number, into a cylinder positioned at a submicrogram balance. Measurements are acquired by the computer, and results are corrected for evaporation. Two calibrated camera systems (a charged-coupled-device—camera with strobed backlight and a high-speed camera with constant backlight illumination) are used for droplet diameter analyses. Capabilities of the prescribed gravimetrical methods are demonstrated using isobutyl alcohol droplets. The gravimetric methods are applicable to a wide range of fluids, including semivolatile solvents. They require either a continuous ejection rate of at least 14 ng/s or a burst aliquot of at least 72 μg. Above these limitations, all gravimetric methods provide consistent results. Total relative uncertainties in average droplet mass are about 1%, whereas repeatabilities are usually less than 0.5%, which is significantly superior to optical methods. Using high-speed videography, individual parameters are measured in droplet bursts, which indicate that the first several droplets emerging from a dispensing device may differ significantly in size from subsequent droplets. This effect is accounted for by pulsed burst gravimetry. Other sources of uncertainty (i.e., the correction for buoyancy, droplet momentum transfer, and droplet evaporation losses during droplet flight) are determined to be insignificant under the operating conditions described (R. Michael Verkouteren and Jennifer R. Verkouteren, Anal. Chem.