Abstract
Laboratory Automation and High-Throughput Chemistry
Multistep Microchemical Synthesis Enabled by Microfluidic Distillation
Complex organic molecules such as active pharmaceutical ingredients are often prepared by multiple reactions that require a workup and isolation of the intermediates. Recent interest in microfluidic systems for continuous-flow synthesis has triggered the integration of multiple chemical reactions and separations in an attempt to streamline the process. Stephen L. Buchwald and Klavs F. Jensen et al. have designed a microfluidic distillation operation that exploits differences in volatility among components in liquid mixtures.
Microfluidic distillation is used to allow the completion of a microchemical synthesis involving multiple reaction and separation steps. A triflate is synthesized in dichloromethane, after which the solvent is switched to either toluene or dimethylforma-mide, and a palladium-catalyzed Heck reaction is performed.
Microfluidic distillation offers new opportunities for continuous-flow chemical syntheses that require a separation between the reaction steps. Strategies for handling solids in microchemical systems will enable multistep synthesis using solid reagents or catalysts, or when solid by-products or products are formed. Furthermore, the integration of multiple distillation stages promises more selective separations (Angew. Chem.
Microfluidic Chip Technology and Micro Reactor Technology
Passive Microfluidic Pumping Using Coupled Capillary/Evaporation Effects
In microfluidics, it is important to create and establish a controlled flow through a microfluidic device. Handling and transport within lab-on-a-chip devices is maybe the most important unit operation. To provide continuous flow with high precision can improve its efficiency and reliability dramatically. This new passive pumping approach creates a stable flow for more than an hour. Traditionally, mechanisms for fluid transport rely on pressure-driven flows from syringe pumps and pressurized chambers or electrokinetic phenomena. These methods provide adjustable volumetric flow while spanning a wide range of flow rates. Biggest disadvantages are the very large dead volume, complicated sample-interface connections, and a minimal sample size of 1 mL.
This new approach does not require an external power source. It uses the large pressure differential arising from a small curved meniscus that is situated along the bottom corners of an outlet reservoir. The system reaches steady state quickly, providing precise volumetric flow for long time periods of more than an hour. To predict fluid and mass transport dynamics, a two-step mathematical model was developed that provides accurate calculations (Lynn and Dandy, Lab Chip,
Methods for Detecting Air Bubble in Piezo Inkjet Dispensers
The most frequently used method for monitoring the jetting performance of inkjet dispensers has been acquiring droplet images via charge-coupled device cameras with strobed light-emitting diodes. This method requires exact alignment of the camera to the specific dispenser nozzle. Therefore, the nozzle must be moved to a designated monitoring position. Typically, these methods are focused on measurement of droplet speed or jetting straightness. However, if there is no jetting from the nozzle, it may be difficult to detect possible malfunction causes.
To detect possible piezo inkjet malfunction, the author proposes two different measurement techniques: meniscus motion and piezo self-sensing signals. Both measurements are related to the pressure wave inside the ink, which causes the ink jetting. Because the measurement of meniscus motion is an optical method, it also requires nozzle movement to a designated monitoring position. The meniscus motion can predict the actual jetting behavior because it is measured at the inkjet nozzle where the actual jetting occurs, but it can be performed without really dispensing droplets.
The second method, piezo self-sensing signals, is based on measuring electric signals only. The use of a piezo as both a sensor and an actuator is principally known from other engineering applications, but the author proposes a new setup, a modified bridge circuit. This approach avoids a backlash from measurement to the driving signal, and in connection with postprocessing, it permits measurement of signals related to pressure waves, whereas other effects are canceled out. The experimental results demonstrate that the air bubbles trapped in the piezo inkjet dispenser, which is one of the major problems in inkjet printing, can be effectively detected during piezoactuation by both methods. A benefit of the self-sensing signals is that the condition monitoring can be performed at any place, as well as at any time, because it can take place at reduced driving signals without really jetting droplets. Measurements also can be taken during nozzle movement from one dispensing position to another. A drawback of measuring self-sensing signals is related to the measuring position. For example, a clogged dispenser nozzle shows no significantly different pressure wave at the piezoele-ment. These malfunctions can be detected by measurement of meniscus motion (Sens. Actuators A Phys.
High-Throughput Analytics
High Resolution Mass Spectrometry for Rapid Characterization of Combinatorial Peptide Libraries
Combinatorial libraries can be characterized in detail by high resolving power mass spectrometry. Palmblad et al. demonstrate this for synthetic decapeptide libraries using a state-of-the-art 15 T Fourier transform ion cyclotron resonance mass spectrometer. Using comparisons between predicted and measured spectra, a number of metrics can be derived that shed light on the library composition and degeneracy of elemental compositions. These techniques can be used for the rapid quality control of combinatorial synthesis products or for following combinatorial libraries in more detail, such as during binding studies or chemical reactions. The methods are, in principle, equally applicable to the analysis of other types of combinatorial libraries (J. Comb. Chem.
Micro-Scaled High-Throughput Digestion of Plant Tissue Samples for Multi-Elemental Analysis
Changing environmental parameters cause various responses from plants. The content and the composition of essential trace metal elements are relevant indicators for these processes. In the field of human nutrition, the diagnosis of nutritional disorders, the development of improved stress-resistant genotypes of crop plants of nutritional quality, and suitable trace metal analysis methods are required for a high number of plant samples. In many cases, the sample quantities are limited, and the concentrations are in a low range. For the subsequent analysis, a time-consuming step is necessary to transfer the samples into adequate analysis vials to allow the use of autosamplers. Hansen et al. have developed a high-throughput microscaled method for sample pretreatment and analysis using small vials (5-mL volume) for microwave digestion and subsequent multielement analysis with inductively coupled plasma-optical emission spectroscopy (ICP-OES) and ICP-MS (Plant Methods
Extractive Electrospray Ionization Mass Spectrometry toward in Situ Analysis without Sample Pretreatment
In situ analysis has become more and more important because it enables a rapid and sensitive detection of trace amounts of compounds such as environmental toxins, explosives, and chemicals, as well as biological warfare agents. Direct field analysis saves time and resources, and it avoids sample contamination in consequence of sample transportation. Li et al. have developed a novel nanoextractive electrospray ionization (nanoEESI) source to perform in situ mass spectrometric analyses of ambient samples without previous sample pretreatment (Anal. Chem.
Bioautomation and Screening
Seamless Bead to Microarray Screening: Rapid Identification of the Highest Affinity Protein Ligands from Large Combinatorial Libraries
Various approaches have been developed in the past for screening combinatorial libraries or collections of synthetic molecules for agonists or antagonists of protein function, each with its own advantages and limitations. Kodadek et al. describe an experimental platform that seamlessly couples massively the parallel bead-based screening of one-bead one-compound combinatorial libraries with microarray-based quantitative comparisons of the binding affinities of many hits isolated from the bead library. The authors claim that combined with other technical improvements, this technique allows the rapid identification of the best protein ligands in combinatorial libraries containing millions of compounds without the need for labor-intensive resynthesis of the hits (Chem. Biol.
Cell-Selective Metabolic Labeling of Proteins
Time-dependent changes in proteomes can be monitored via a variety of electrophoretic and spectroscopic methods, usually using radiolabeled amino acids to label proteins synthesized during an amino acid “pulse.” These methods suffer from limitations when experiments are performed in systems that contain multiple cell types because incorporation of amino acids is nonspecific with respect to cell identity proteins from all cell types. In studies of interactions between different cell types in a single organism, the origin of the identified protein can be difficult to ascertain because the cells share a common genome.
Ngo et al. developed a method with which the cells can be labeled selectively. They use a mutant methionyl-tRNA synthetae (nucleus of the lateral lemniscus [NLL]-MetRS) and amino acids that are excluded by the endogenous protein synthesis machinery. In this approach, cells expressing the mutant synthesis are able to use azidonorleucine as a surrogate for methionine. Because wild type cells cannot change azidonorleucine to tRNA, this amino acid is not used in protein synthesis. In coculture, protein labeling is restricted to mutant cells. To validate the incorporation of azidonorleucine into the proteome of a mutant Escherichia coli strain, the cells are pulse labeled with 1 mM azidonorleucine, lysed, and the lysates are probed for integration of azidonorleucine via Cu(I)-catalyzed azide—alkyne ligation to biotin—FLAG—alkyne followed by western blotting with protein detection by anti-FLAG antibody. In another approach, NLL-MetRS-expressing E. coli cells carry an additional isopropyl β-D-1-thiogalactopyranoside-inducible green fluorescent protein (GFP)-gene, and the Cu(I)-catalyzed labeling is done with dimethylaminocoumarin-alkyne. By using fluorescence microscopy, the coumarin fluorescence can only be detected in cells expressing the inducible GFP. The group also is able to pulse label mammalian cells in a coculture with bacteria. Mouse alveolar macrophages are infected with mutant E. coli cells, and the newly expressed proteins can be shown via con-focal microscopy.
These results show that the use of mutant aminoacyl-tRNA synthetaes enable cell-specific protein labeling with noncanonical amino acids, permitting the additional labeling with affinity reagents or fluorescent dyes for enrichment and visualization of newly synthesized proteins in mixed cellular systems. In a complex multicellular system, this approach can provide new insight into intercellular communication (Nat. Chem. Biol.
Three-Dimensional Cell Culture Array Using Magnetic Force-Based Cell Patterning for Analysis of Invasive Capacity of BALB/3T3/v-src
Invasion and metastasis are the hallmarks of malignant tumors. The invasion process that is accompanied by the degradation of macromolecular components of the extracellular matrix and basement membranes is regulated by the intrinsic properties of tumor cells and by microenvironmental factors. To develop a suitable in vitro model to study cancer cell behavior, Okochi et al. use a three-dimensional (3D) cell culture system in which cells are forced into a microtumor-similar spheroid pattern by applying magnetic force on magnetite nanoparticles in the cells. Via magnetite cationic liposomes, the magnetite nanoparticles are introduced into the target cells, and the cells are seeded on a collagen gel. By using magnetic force, the cells are patterned into an array. A collagen mixture is overlaid, and after gelation, a 3D array of cells is developed in which the cells are surrounded by a type I collagen matrix. The hybrid system for applying the magnetic force consists of an integrated circuit with a microcoil array circuit that produces spatially patterned microscopic magnetic fields and a microfluidic system that provides a biocompatible system with rapid configuration of the field pattern made by the integrated circuit for manipulation of magnetic bead-bound cells. A pin holder device made of electromagnetic soft iron is fabricated with pillars in equal clearances in the order of micrometers, and magnetism can be concentrated on the pillar part by magnetizing this device. In contrast to traditional cell patterning techniques, magnetic cell patterning is not limited by the property of culture surfaces and can be used for any culture surface including biological gels and cell layers.
With this 3D model, Okochi et al. observe the behavior of mouse fibroblast BALB/3T3 cells in comparison with BALB/T3T cells transformed with v-src as the cancer model cells. Elevated src activity can contribute to malignant progression by impacting on multiple receptor signaling centers. They show that in the 3D model the BALB/3T3/v-src cells proliferate at accelerated rates in comparison with BALB/3T3 cells, and the matrix metalloproteinases inhibitor GM6001 suppresses the cell proliferation of BALB/3T3/v-src cells in the 3D culture array, whereas cells in the two-dimensional culture move evenly over the surface, and the inhibitor has no influence on cell growth. Taken together, this 3D cell culture model is simple, easy to handle, and enhances the monitoring of cell morphology, as well as cell behavior (Lab Chip
Automation Systems
Unified Behavior Framework for Reactive Robot Control
Many autonomous controlled multiple mobile robot architectures are developed on a behavior-based system. A well-structured article comprehensively describes how a single software framework, the unified behavior framework (UBF), can be used to represent many existing behavior-based approaches. Initial technologies such as the development of reactive planning in contrary to the sense—plan—act architectures, the application of the common three-layer and the later verified main reactive architectures are introduced. The driving force for research is the structure of these systems, mainly composed of a small number of behaviors with an arbitration element that performs action selection.
The advantage of this robust and compact low-level control is customization for very specific environments. The reuse of the control structures and behaviors for different scenarios or on different robots or the usage of complex approaches with extensions to existing algorithms is barely possible. In contrast, UBF provides for a modular design through well-defined interfaces and strict functionality encapsulation. Object-oriented mechanisms such as polymorphism allow an interchangeable utilization of behaviors from disparate reactive architectures at runtime. The use of different arbiters under a unified interface facilitates the observation of radical differences in external behavior attributes (e.g., robot movements).
Before illustrating this through a Robocode simulation environment and subsequently in a goal-seeking robot implementation with a Pioneer 2AT-8, the mapping of common reactive architectures, such as motor schema, circuit architecture, or utility fusion, into UBF is described. The development of control software for a tank that survives battles as long as it is doing damage to other players is the simulation objective. It is shown that cooperative architectures such as utility fusion achieve better results than that of competitive ones, such as action selection. The result is not surprising because the written base behaviors such as ramming, shooting, and target tracking are designed to support each other, and cooperative approaches or arbiters allow multiple action selection. In the end, the article proves that UBF simplifies development (code reuse, scalability, and composition) and encourages experimentation to verify which architecture offers the best performance. It emphasizes that real-time systems can be of essential importance for future use of behavioral architectures. They ensure responsiveness despite the fact that reactive elements of behavior-based systems grow steadily in complexity (J. Intell. Robotic Syst.