Automation Highlights from the Literature

Laboratory Automation and High-Throughput Chemistry

Novel Approach to Optimization of a High-Throughput Semipreparative LC/MS System

Automated semipreparative LC/MS systems are now well established commercially and commonly used for purification of early stage drug discovery compounds. A number of vendors have instruments on the market that are capable of reliably purifying compounds with good water/acetonitrile solubility. However, these systems often fail when the sample has poor solubility, extreme polarity, and/or poor ionization. Even in cases when substantial optimization has been done before purification, a certain percent of failures to recover the desired product is unavoidable. In the past, when most of the samples run on LC/MS semipreparative systems were large combinatorial libraries, some losses in this high-throughput mode were acceptable. However, now the focus shifted to smaller libraries with higher purity requirements and therefore reliability and recovery are more crucial. J. FitzGibbons et al. describe modifications made to customize on MS-triggered semipreparative LC/MS system to ensure improved reliability and recovery of products from traditional medicinal chemistry and combinatorial libraries (J. Comb. Chem. 2009, 11, 592–597).

Parallel Microwave Synthesis of 2-Styrylquinazolin-4(3H)-Ones in a High-Throughput Platform Using HPLC/GC Vials as Reaction Vessels

The application of a high-throughput reaction platform for performing parallel microwave synthesis in sealed HPLC/GC vials contained in a strongly microwave-absorbing silicon carbide plate is described by C.O. Kappe et al. The use of aluminum crimp caps with polytetrafluorethylen coated silicone septa in combination with an appropriate plate sealing mechanism allows processing of reaction volumes from 0.5 to 1.5 mL at temperatures of approximately 250 °C and pressures of up to 20 bar. A library of 39 2-styrylquinazolin-4(3H)-one derivatives is prepared in a two-step/one-pot parallel fashion involving the initial three-component condensation of four anthranilic acids with acetic anhydride and ammonium acetate at 250 °C for 30 min. This is followed by catalyst-free condensation of the resulting 2-methylquinazolinones with a selection of 15 aromatic aldehydes (J. Comb. Chem. 2009, 11, 676–684).

Combinatorial Chemistry on Solid Support in the Search for Central Nervous System Agents

The advent of combinatorial chemistry was one of the most important developments that has significantly contributed to the drug discovery process. Within just a few years, its initial concept, which was aimed at production of libraries containing huge numbers of compounds (thousands to millions), has shifted toward preparation of small- and medium-sized rationally designed libraries. The use of solid supports for the generation of libraries has been important in many cases for enhancing productivity. P. Zajdel et al. discuss in the first part of a review the general premises of combinatorial chemistry and some methods used in the design of primary and focused combinatorial libraries. In the second part, combinatorial chemistry methodologies aimed at discovering bioactive compounds acting on diverse GPCR targets involved in central nervous system disorders are presented (Comb. Chem. High Throughput Screen. 2009, 12, 723–739).

Microfluidic Chip Technology and Microreactor Technology

Facile, Fast, and Safe Process Development of Nitration and Bromination Reactions Using Continuous-Flow Reactors

Chemists working in a pilot plant often face safety issues during scale-up operations. With the help of emerging microfluidic applications and microdevices, running hazardous, highly exothermic, or potentially unstable reactions can be transposed into a safe continuous-flow mode. J. Pelleter and F. Renaud describe how a potentially hazardous pyrazole nitration and the bromination of a variety of electron-rich heteroaromatic substrates are efficiently performed using a cheap and easily available system for bench chemists. Advantages of the continuous-flow mode in organic synthetic chemistry are exemplified by the large-scale production of raw materials under safe and reproducible conditions (Org. Proc. Res. Dev. 2009, 13, 696–705).

Adaptation of an Exothermic and Acylazide-Involving Synthesis Sequence to Microreactor Technology

W. Bannwarth et al. report about the successful adaptation and optimization of a potentially hazardous reaction sequence in a microreactor system. The original three-step batch-wise synthesis of the pharmaceutical intermediate (1R,2S,4S)-(7-oxabicyclo[2.2.1]hept-2-yl)-carbamic acid ethyl ester from (1R,2S,4S)-7-oxabicyclo[2.2.1]heptane-2-carboxylic acid ethyl ester encompasses a highly exothermic hydrazine quenching step and an acylazide intermediate. After appropriately modifying the reaction conditions, all three steps are adapted to a microreactor system and a continuous process that permits the desired carbamate to be prepared under safe operating conditions in yields of 96%, 94%, and 84% for the three individual steps (Org. Proc. Res. Dev. 2009, 13, 747–750).

Development of Fluorination Methods Using Continuous-Flow Microreactors

The safe and reliable use of various fluorination methods, including nucleophilic fluorination, trifluoromethylation, and electrophilic fluorination in a continuous-flow microreactor is reported by S. V. Ley et al. Special attention is given to the use of in-line scavenging procedures to obtain clean products without the need for further purification (Tetrahedron 2009, 65, 6611–6625).

Investigations of Mixing Process in Microfluidic Manifold Designed According to Biomimetic Rule

A main issue in microfluidics is the mixing process because in such small scale, flows are mostly laminar. There are no turbulent propagating eddies, but only diffusive mixing. A paper by Cieslicki et al. focuses on the mechanism of the mixing process in a manifold that mimics the geometrical properties of vascular systems (Lab Chip. 2009, 9, 726–732). Murray discovers the relationship between the diameter of parent and daughter branches in vascular systems that govern their optimum ratios by using the principle of minimum work. The resistance to flow reaches a minimum when the cube of the diameter of parent vessel is equal to the sum of the cubes of the diameters of the daughter vessels (third power law (Hess [1914])). In biological vascular systems, the vessels are circular pipes, and fabricated manifolds are rectangular or trapezoidal with constant depth throughout the device because of fabricating processes, such as photolithography, wet dry edging, or surface micromachining. This article provides comprehensive numerical studies of mixing in such geometry. Furthermore, an analytical estimation is included, how the mixing coefficient depends on the number of generation and the Reynolds number. The focus is set to flows with a very low Reynolds number of <1 where most mixing schemes are completely inefficient. The main goal of Cieslicki et al. was to create a mixer that provides almost perfect mixing at the outlet independent of the Re value with as small as possible number of manifold's generations.

An Integrated Microfluidic Device for Large-Scale In Situ Click Chemistry Screening

H.-R. Tseng et al. describe a newly developed and improved platform for screening an in situ click chemistry library with 1024 reactions carried out in parallel by combination of integrated microfluidic, solid-phase extraction procedure for purification and electrospray–ionization mass spectrometry. The current platform not only accommodates screening of up to 1024 reactions in parallel, but also significantly reduces reagent consumption and screening time (Lab Chip. 2009, 9, 2281–2285).

High-Throughput Analytics

The Influence of Material and Mesh Characteristics on Transmission Mode Desorption Electrospray Ionization

Desorption electrospray ionization (DESI) is a prevalent ambient ionization technique that works with atmospheric pressure for coupling to mass spectrometric analysis. It allows the production of ions by directing charged solvent droplets from an electrospray source toward a sample. The samples can be in their native state or have been deposited from solution to a sampling surface. A special modification of DESI is the transmission mode desorption electrospray ionization (TM-DESI). For this purpose, the sample is deposited onto a sampling mesh and not onto a continuous solid-sampling surface. It allows the analysis of dry and wet samples with similar performance characteristics such as using conventional DESI. Chipuk and Brodbelt investigate the influence of the used mesh material, the open space of the mesh, and the strand diameter of the obtained results by analysis of rhodamine 6G and bradykinin (J. Am. Soc. Mass Spectrom. 2009, 20 (4), 584–592). Special attention is given to the interaction of the electrospray plume with the mesh substrate. Interesting aspects are how various mesh characteristics and materials impact the electrospray transmission as well as desorption and ionization mechanisms of liquid samples. Twenty different mesh substrates consisting of polypropylene, polyetheretherketone, ethylene tetrafluoroethylene, nylon-6,6, and polyethylene terephtalate are investigated. The named analytes are used with meshes of various structural characteristics such as the open space between the strands of the mesh and the diameter of the strands used to compose the mesh. With mass spectrometry and fluorescence microscopy, the significance of the material composition and the mesh characteristics on TM-DESI analysis are reported. The results show the intricate chemistry that exists as the electrospray plume is transmitted through the mesh material. Actual research is directed to develop solvent surface systems to further increase the selectivity of TM-DESI.

Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants

Metabolomics is an experimental methodology used with other approaches for a deeper understanding of biological processes, such as metabolism. In plants, a higher number of metabolites can be found than in animals. More than 200,000 metabolites are assumed. In the field of metabolomics, various metabolites are investigated and they greatly differ in their physical and chemical properties. The analysis of these compounds requires multiple instruments based on different analytical principles. To establish nontargeted analysis procedures, chromatography-coupled MS-based metabolomics and nuclear magnetic resonance-based metabolomics have been developed. With nontargeted metabolomics, broad metabolite profiles can be identified and novel metabolites, intended as biomarkers, can be found. Complex and time-consuming techniques are required to merge the whole data that are obtained from different instruments and to detect the unknown metabolites. The handling of a large number of biological samples is still difficult. Alternative methodologies for targeted metabolomics are required. Sawada et al. present an automated high-throughput methodology for widely targeted metabolomics (Plant Cell Physiol. 2009, 50 (1), 37–47). They construct an authentic compound library with more than 1000 available compounds to obtain wide-ranging reference data. Therefore, a method for automated flow injection analysis is established using ESI-MS and ESI-MS/MS. The analysis of biological samples to obtain metabolite accumulation patterns is conducted with high-throughput ultra performance liquid chromatography tandem quadrupole mass spectrometry. The analytical system is very comprehensive, consistent with high-throughput screenings, and allows the quantification of hundreds of metabolites in thousands of biological samples.

Bioautomation and Screening

Assays for Histone Deacetylases

Epigenetics is defined as heritable changes in gene expression, that is, the presentation of a certain phenotype without alterations to the genetic code. This is a central phenomenon in developmental biology and the pathogenesis of a variety of diseases, for example, for cancer in the case of silencing of tumor suppressor proteins. Reversible histone acetylation is an important player within the epigenetic machinery, and histone deacetylases (HDACs) are the best-studied epigenetic target for drug discovery. A first drug (vorinostat) is already approved for human cancer treatment. The strategies and protocols for monitoring HDAC activity are an integral part of drug discovery approaches toward new HDAC inhibitors and are reviewed by M. Jung et al. The main focus is on biochemical in vitro assays, but cellular hit validation models are also presented (Curr. Top. Med. Chem. 2009, 9, 227–234).

Novel High-Throughput Polymer Biocompatibility Screening Designed for SAR: Application for Evaluating Polymer Coatings for Cardiovascular Drug-Eluting Stents

The development of stents has been a major advancement over balloon angioplasty, improving vessel revascularization in obstructive coronary artery disease. The development of drug-eluting stents (DESs) was the next breakthrough, designed to prevent the development of neointimal hyperplasia (restenosis) after percutaneous coronary interventions. Several DESs are currently in various stages of clinical development. These DESs use different stent platforms, different antiproliferative drugs, and different polymeric coatings that carry the drugs and control their delivery kinetics. After DES implantation, when the entire drug is released, the polymeric coating is still retained on the stent and can influence subsequent tissue response and vascular healing. Therefore, the biocompatibility of the polymeric coatings is an important component of DES safety and needs to be thoroughly evaluated. A. Hezi-Yamit et al. describe the development of a high-throughput screening platform for the evaluation of polymer biocompatibility, assaying whether a polymeric coating triggers inflammation in vascular cells. The data generated by these assays provides a structure–activity relationship that can guide polymer chemists in polymer design. The authors also apply this methodology to evaluate the components of a novel polymer system (BioLinx polymer system, Woodland Hills, CA). In addition, they assay other polymeric coatings similar to those currently used on various DES. The results of this evaluation reveal a remarkable correlation between polymer hydrophobicity and its ability to provoke inflammatory response (Comb. Chem. High Throughput Screen. 2009, 12, 664–676).

High-Throughput Screen for Novel Antimicrobials Using a Whole Animal Infection Model

The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen. F.M. Ausubel et al. describe an automated, high-throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. Using a robot to dispense live, infected animals into 384-well plates and automated microscopy and image analysis, they identify 28 compounds and extracts not previously reported to have antimicrobial properties, including six structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use (ACS Chem. Biol. 2009, 4, 527).

Hydrophilic Fluorescent Nanogel Thermometer for Intracellular Thermometry

The temperature of a living cell is changeable during every cellular event. In mitochondria, surplus energy in ATP production is released as heat, and a consequent rise in temperature coordinates other cellular events. Because of their enhanced metabolic activity, pathological cells are warmer than normal cells. Gota et al. developed a fluorescent nanogel thermometer with which they demonstrate intracellular thermometry. They combine a thermoresponsive polyNIPAM unit with a water-sensitive fluorophore, which are included into a nanogel by an emulsion polymerization technique. At a lower temperature, the gel swells by absorbing water into its interior where the water-sensitive fluorophore units are quenched by the neighboring water molecules. When heated, the gel shrinks with the release of water molecules resulting in fluorescence. By enriching the surface of the nanogel with sulfate groups, it becomes highly hydrophilic and is protected against precipitation at high ionic strength as present in the culture medium of cells, and from localization in the cytoplasmic membrane. The fluorescence response of the thermometer, as shown by Gota et al., is independent of environmental pH and of surrounding protein. To study intracellular thermometry, the fluorescent nanogel thermometer 1 is introduced into COS7 cells by microinjection. It demonstrates that neither photobleaching nor leaking of the fluorescent thermometer are problematic and that the fluorescence dots are dispersed within the cytoplasm but does not move to the nucleus. Over the range of 27–33 °C, the temperature resolution is 0.29–0.50 °C. To show the effects of external chemical stimuli, COS7 cells are exposed to carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation in mitochondria, causing heat production by respiration and to camptothecin. The temperature variation as a result of energy balances in living COS7 cells can be visualized by an increase of fluorescence.

Measuring cellular temperature can contribute to the explanation of intricate biological processes and the development of novel diagnoses (Chie Gota et al. J. Am. Chem. Soc. 2009, 131 (8), 2766–2767).

Pneumatically Driven Microdispenser for Sub-Micro-Liter Pipetting

This article describes a pneumatically driven microdispenser for precise pipetting of sub-micro-liter samples. This microdispenser is fabricated using standard soft lithography of polydimethylsiloxan. It can easily be integrated along with other microfluidic devices for subsequent microfluidic applications.

The key feature of this microdispenser is the incorporation of a pressure-generating unit with a suction membrane to provide a driving force for precise and quick aqueous liquid sampling and pipetting. Different liquid volumes for pipetting are achieved by controlling the release time of the suction membrane. Precise pipetting volumes ranging from 0.05 to 0.45 μL are generated (with 2.9% variation).

From the experimental weighing result, it is revealed that the prototype microdispenser is able to perform liquid dispensing with a high reproducibility. Also, the titration experimental results demonstrate that the performance of the proposed microdispenser is comparable with that of a commercial pipettor.

Therefore, the pneumatically driven microdispenser can dispense variable sub-micro-liter aqueous liquid volumes precisely and quickly, and may be promising for further micrototal-analysis systems in the field of drug dosing or other biomedical applications (Song-Bin Huang, Gwo-Bin Lee. J. Micromech. Microeng. 2009, 19 (3), 035027 (8pp)).

Automation Systems

Open Middleware for Robotics

Despite advances in recent years, autonomous multirobot systems are still considered to be complex and unclear, because the control and coordination of these systems remain a challenging task. This article outlines the features of freely available middleware as a software layer that provides an infrastructure for the integration of applications and data in distributed systems domain.

The authors start with naming the biggest challenges system engineers face when establishing a complex robotic system, whereas the distributed nature and the participation of various fields, such as signal processing, computer science, vision or even artificial intelligence, are stressed most. To accomplish the mentioned challenges, the current trend in robotics research is to design systems based on open robotic software called middleware. This is connectivity software that consists of a set of enabling services that allow multiple modular processes (modules/components) running on board a robot or off board to interact across a network. The modular and independent structure allows for reusability of certain components, and easy extensibility and maintainability lead to reliable overall systems.

Because designing middleware from scratch with a development toolkit such as CARMEN or YARP is an enormous task, a sensible alternative is to adopt existing open middleware. Software packages such as Miro, as a COBRA-based approach, Marie, Orca, and Player have a multilayer system in common. Communication tools, data handling, and operating system-related issues build the core level, which is the basis for the management layer that consists of a framework for implementing new components and the application layer, which offers the interfaces for device integration.

Advantages and drawbacks are discussed in this article. Interprocess communication (Miro, CARMEN) versus client—server communication (Player), cross-platform communication, poor hardware support (Orca), restrictions, for example, to single robot applications (CARMEN), or to certain kind of robots (YARP for humanoid robots) are main catchwords in this respect. Finally, an example of algorithm development for multirobot cooperation using one of the discussed software architectures (Player) is presented. (Proceedings, 15th International Conference on Mechatronics and Machine Vision in Practice, Massey University, Auckland, New Zealand, 2008, 6).

Automation Drives Laboratory Economics

This article deals with the increasing impact of automation technologies in life-science laboratories and underlines this trend by explaining several novel and remarkable approaches, for example, in sample preparation or cell-based screening. Traditionally, because laboratory equipment was expensive and fairly complex to use, it needed a lot of attention and was hard to maintain in the 1980s and early 1990s; today's systems are more reliable and easier to use. It is stated that lower costs of acquisition and the fact that technologies are available for a wider range of applications have increased the assertion of laboratory automation. It is not only applicable for high-throughput screens (drug discovery) in large organizations, but also for more speculative applications, including academic and basic research.

Scientists are now able to use automated tools in novel ways even in the earliest stages of assay development. The author claims that the missing adaptability of the scientist and not of the technology may be the limiting factor in future research. Another point is that automated tools offer exponentially greater efficiency while changing many of the fundamental paradigms of drug discovery research.

A consistent theme in this respect, also discussed at LabAutomation2009 in Palm Springs, CA, is how to adapt the workflow to make the most of automation even when that workflow is radically different from what most people are accustomed to.

To clarify those thoughts, the author continues by delivering insight to some of the new developments and applications of laboratory automation tools exposed at that conference.

One of the most surprising applications uses a robot to take aliquots from frozen samples without thawing the samples and without disturbing the cooling chain. The most highlighted example affects new technologies in cell-based assays. Odyssey Thera, for instance, has developed a high-throughput platform that tracks subcellular protein complexes using a system of automated microscopes and unique cellular probes. Fabrus adapts biologics research to the more common paradigms and technologies that work within the drug discovery reliably by evolving their antibody screening assays.

This reflects the initial statement of rethinking assay development for laboratory automation. The biggest challenge, however, is to merge expertise from the joined disciplines. This is stressed by a citation from Joachim Wiest from Cellasys, which serves as a conclusion at the same time: “The biggest problem to get these systems to run is to solve interdisciplinary problems—to get all this knowledge together. Maybe you can find a good engineer or biologist, but it's hard to find a good bioengineer” (GEN—Genet. Eng. Biotechnol. News 2009, 29).

Information Technology and Systems

Semiautomatic Web Service Composition for the Life Sciences Using the BioMoby Semantic Web Framework

Standard Web services technology combines efficient distributed IT applications. For collaborative networking, remote procedure applications, or information transfer, management of the Web services becomes key to its infrastructure. The discussed framework relates to the ideas of how the semantic Web is able to support the process of composition of Web services for complex IT-network solutions, for example, for biomedical informatics. The article focuses on the problems of interoperability among Web services for life-science applications. The current limitation is addressed by annotating services and their interfaces with semantic information, so that interoperability problems can be reasoned about programmatically. The Moby semantic Web framework is a Web service platform that is used for this purpose. One of the discussed problems is the handling of the services that are available for workflow applications. Some aspects are service selection and composition, including automated service composition. A prototype workflow assembly client reduces the number of choices that users have to make by restricting the overall set of services presented to them and ranking services, so that the most desirable ones are presented first. An evaluation is directed to rank desirable services highly while maintaining interactive response times (J. Biomed. Inform. 2008, 41 (5), 837–847).

A Microarray Information Database

An article J.P. Arrais et al. discusses the IT requirements of microarray technology in genomics research laboratories, especially the computational challenges of the multistep and data-intensive nature of this technology. The full power of microarray technology can only be achieved if researchers are able to efficiently store, analyze, and share their results. In the article, a flexible and extensible platform that can be used to gather the natural workflow of microarray experiments is demonstrated. The presented solution is an open-source Web-based information system. The IT platform integrates a set of statistical and processing methods to analyze and visualize the assay process data. The goal is to speed up the research and discovery processes. The described approaches and the IT solution are adaptable to other user requirements (Proc. Int. Conf. Biocomput. Bioinform. Biomed. Technol. (BIOTECHNO) 2008, art. no. 4561154, 170–175).

Not Electronic—Not a Notebook—Electronic Laboratory Notebooks

Electronic laboratory notebooks (ELNs) have become a hot topic in laboratory IT environments over the last years. Definition, functions, legal aspects including patent issues, FDA 21 CFR Part 11, ISO 17025, and reasons for the trend from paper-based work to ELN are discussed by H. Strass. Based on a reference (Labnotes, UK), possible savings are considered for several standard scenarios. To understand the improvements of an ELN, a lot of problems with paper record books are listed. The article differentiates personal electronic notebook systems and collaborative electronic notebook systems. Market aspects for the ELN technology, and new problems of IT systems integration (ELN, Scientific Data Management Systems, and LIMS) also are included (G.I.T. Lab. J. Europe, 2008, 3–4 (12), 24–27).