Automation in Optical Analysis and FabricationOn-Chip Continuous Monitoring of Motile Microorganisms on an ePetri PlatformMicroscopic imaging is a commonly used tool in the study of microbiology. Typically, microscopic imaging is performed using a microscope equipped with digital imaging apparatuses. Traditional microscope systems can be bulky and expensive, and many efforts have been focused on developing compact, low-cost alternatives. Recently, Lee et al. reported a complementary metal oxide semiconductor (CMOS) image sensor–based self-imaging Petri dish, or “ePetri” system to allow low-cost, convenient high-resolution imaging of micro-organisms.In this study, the CMOS imaging chip serves as the substrate for cell growths. Because the pixel size of the image sensor and the size of micro-organisms are on the same order of magnitude (micrometers), typically a lens system is required to amplify the image of micro-organisms before it can be resolved by the image sensor. The authors eliminate the need for the lens system by using a subpixel motion microscopy (SPMM) technique.SPMM is based on a pixel super-resolution algorithm, which combines a series of low-resolution images of the same object with subpixel shifts into higher-resolution images. In this work, the authors use the inherent motion of motile micro-organisms to allow the scanning of micro-organisms on the sensor’s pixel grid, generating the subpixel shifts needed to render a higher-resolution image. The ePetri platform was tested for longitudinal study of Euglena gracilis as well as the image analysis of cell motion and morphology. The authors believe the ePetri technology can offer significant cost and size advantage for imaging of motile micro-organisms. (Lee, S. A., et al., Lab Chip 2012, 12, 2385–2390)High-Throughput Single-Microparticle Imaging Flow AnalyzerOptical microscopy is one of the most commonly used laboratory techniques in scientific, industrial, and clinical applications. There has been tremendous progress in the past several decades in microscopy technologies, but Goda et al. argue that although much progress has been made in improving the spatial resolution of the microscopy technique, the temporal resolution of the imaging system has been largely ignored. In this report, they introduce an ultra-fast, ultra high-throughput imaging system for flow cytometry applications. This is made possible by the integration of three different technologies: (1) an ultrafast optical imaging modality named serial time-encoded amplified microscopy (STEAM), (2) inertial microfluidic focusing, and (3) hybrid optoelectronic image-processing circuitry for real-time imaging processing. The combination of these power techniques yields a fully automated real-time imaging and classification tool for high-throughput cell screening based on morphological and biochemical features. Using this system, authors achieve an unprecedented throughput of 100 000 particles/s and a record false-positive rate of 1 per million. (Goda, K., et al., Proc. Natl. Acad. Sci. U. S .A. 2012, 109, 11630–11635)Lithographically Encoded Polymer Microtaggant Using High-Capacity and Error-Correctable QR Code for Anti-counterfeiting of DrugsDrug counterfeiting poses serious challenges for public health and is a major issue for the pharmaceutical industry. Current solutions include packaging-oriented authentications such as printed marks, radiofrequency tags, and so forth. Recently, the pharmaceutical industry began exploring a new form of authentication called on-dose authentication (ODA). The idea of ODA is to tag an individual unit-of-dose form at the drug formation level and is thought to be a stronger authentication method than existing on-package ones.An ODA tag requires microtaggants, which are microscopic and traceable particles or fibers added directly in the drug tablet or capsule during the formulation process. Microtaggants make drug counterfeiting difficult because these micrometer-scale physical identifiers are very difficult to duplicate or replace with counterfeiting ones. Typical microtaggants carry information such as product name, lot number, production/expiration date, and so on.To date, one-dimensional (1D) bar codes have been used on microtaggants, but simple 1D bar codes can carry limited information and data restoration from the damaged microtaggants is difficult due to the lack of an error correction algorithm. This problem is tackled by Han et al., who explore the fabrication of polymer microtaggants with high-capacity encoding (Quick Response Code, or QR code). QR Code is a 2D matrix code that provides high data capacity and hence enables better counterfeiting control. More importantly, the dot-based, 2D QR code enables multilevel coding that allows data from a partially damaged code to be recovered.In their report, the authors describe a novel method for lithographically fabricating QR-coded microtaggants with a single ultraviolet exposure in microfluidic channels. These microtaggants offer features such as high-capacity encoding, error correction capability, and omnidirectional reading. In addition, the complete process of drug authentication from formulation to optical decoding is demonstrated. The authors believe this lithographically QR-coded polymer microtaggant could potentially become a powerful tool to enable future ODA solutions. (Han, S., et al., Adv. Mater. 2012, 24, 5924–5929)Automation in Biodetection and BioanalysisCounting Bacteria Using Functionalized Gold Nanoparticles as the Light-Scattering ReporterRapid detection of foodborne pathogens such as enterohemorrhagic Escherichia coli is critical for food safety and clinical diagnosis. Xu et al. report a simple and rapid bacterial-counting method based on dark-field light-scattering imaging of bacteria and a gold nanoparticle reporter. Dark-field imaging is a highly sensitive imaging method yet is much more cost-effective than fluorescence imaging. The gold nanoparticle is a highly efficient light-scattering probe due to the localized surface plasmon resonance phenomenon. Therefore, the combination of two technologies yields a sensitive, low-cost solution for bacterial detection.In this study, bacteria are first stained with antibody-conjugated gold nanoparticles. These gold nanoparticles bind to the bacteria, and the light-scattering properties of the bacteria increase dramatically. For example, based on the authors’ calculator, the intensity of scattered light of a single gold nanoparticle with the diameter of 60 nm is equivalent to the fluorescence intensity of 2.7 × 105 fluorescent molecules. DH5α E. coli strain is used to demonstrate the performance of this detection method. The nanoparticle cell staining can be completed in a very simple process that takes less than 15 to 30 min, and a detection limit of ~2 × 104 colony-forming units per milliliter (CFU/mL) is achieved. The method is tested with various types of food samples, including water, milk, fruit juice, and more, and shows promise for a simple, fast, and cost-effective method for routine bacteria screening in food samples. (Xu, X., et al., Anal. Chem. 2012, 84, 9721–9728)Colorimetric Paper Bioassay for the Detection of Phenolic CompoundsAlkasir et al. report a paper-based bioassay for the colorimetric detection of phenolic compounds, including phenol, bisphenol A, catechol, and cresols. A paper-based sensor is fabricated using layer-by-layer (LbL) assembly of alternating chitosan and alginate polyelectrolyte layers onto filter papers. In between polyelectrolyte layers, tyrosinase enzymes are entrapped to serve as the biosensing element. During detection, upon contact with the tyrosinase enzyme, phenol compounds generate quinone, which subsequently binds with chitosan to yield a color change. The sensor color change is concentration dependent, visible to naked eyes, and can be further quantified with digital imaging. The authors report a detection limit of 0.86 (±0.1) µg/L and a detection time of less than 20 min. An excellent sensor stability of several months (92% residual activity after 260 days of storage) and good sensor performance in real environmental samples are also demonstrated. The authors also describe a simple procedure for mass production of the biosensors by inkjet printing the LbL layers of polyelectrolyte and enzyme on paper. (Alkasir, R. S. J., et al., Anal. Chem. 2012, 84, 9729–9737).Aptamer-Based Viability Impedimetric Sensor for BacteriaSalmonellosis, or Salmonella enterocolitis, is an infection of the small intestine caused by Salmonella bacteria. It is a serious health concern and cause of many food-poisoning cases worldwide. The traditional gold standard method for Salmonella detection (culturing and plating method) is a complex procedure that includes nonselective preenrichment, selective plating, and biochemical/serological confirmation. It is time-consuming and labor intensive. In addition, certain viable but nonculturable strains of Salmonella may not be detected by this method.Labib et al. introduce a new method for detection of Salmonella using an aptamer-based viability impedimetric sensor. The authors use a technique named systematic evolution of ligands by exponential enrichment (SELEX) to select DNA aptamers that are highly specific to live Salmonella bacteria. SELEX is a combinatorial chemistry technique for producing oligonucleotides that are highly specific to target ligands. In this study, 12 rounds of SELEX iterations are conducted, each including a positive selection against viable Salmonella and a negative selection against dead Salmonella and certain related pathogens. The DNA aptamers obtained via SELEX are integrated into the sensor. The detection results indicate that this aptasensor can specifically detect certain species of Salmonella down to 600 CFU/mL and is insensitive to other related pathogen bacteria. The authors believe this report opens a new venue for an aptamer-based viability sensor for a wide variety of pathogenic bacteria. (Labib, M., et al., Anal. Chem. 2012, 84, 8966–8969)Progress in Electronic Nose and TongueMultifunctionalized Cantilever Systems for Electronic Nose ApplicationsYoo et al. report a multifunctionalized cantilever system for electronic nose applications. In this system, detection is performed by monitoring the change of resonance frequency of receptor functionalized cantilevers as a response of target-receptor bonding. For the electronic nose, or artificial olfactory system, the selectivity of the sensor is essential. Because of the limited selectivity of individual receptors, it is important for the system to be able to simultaneously detect multiple targets using multiple receptors so that techniques such as principle component analysis can be used to further improve the overall selectivity of the detection.A cantilever sensing system typically includes multiple cantilevers; however, simultaneous multitarget detection with individually functionalized cantilevers has not been realized, partially because of the difficulty of functionalizing individual cantilevers considering their size (typically in micrometer scale). Without simultaneous detection of individually functionalized cantilevers, it is very difficult to compensate for the errors caused by environmental changes such as viscosity, density, humidity, and temperature. In this report, the authors address the issue of individually functionalizing cantilevers using micro-reaction chambers.A four-chamber system is used in conjunction with four different groups of cantilevers so each group of cantilevers can be individually functionalized. Four different fictionalizations are performed with 2, 4-dinitrotoluene (DNT) specific peptide, DNA nonspecific peptide, a self-assembled monolayer without receptors, and no treatment (a bare electrode). Upon contact with DNT gases, four different binding signals from four different groups of cantilevers can be simultaneously detected. By comparing the signal differences among four groups of individually functionalized cantilevers, noises caused by temperature fluctuations, mechanical vibration, thermal noise, and humidity changes can be eliminated. The sensor achieves a detection signal of 7.5 Hz resonant shift for 160 ppb DNT gas. (Yoo, Y. K., et al., Anal. Chem. 2012, 84, 8240–8245)Bioelectronic Tongue of Taste Buds on Microelectrode Array for Salt SensingThe electronic tongue systems mimicking the biological taste-working process have attracted a great deal of attention for potential applications in food safety, the pharmaceutical industry, and environment monitoring. The basic principle of electronic tongue is similar to electronic nose described above: it involves an array of sensors that work collaboratively to produce analytically useful signals for multicomponent matrices. Many studies have been conducted based on artificial membranes and electrochemical techniques, but a major gap remains between the electronic tongue and biological taste process, which lies in the biological receptor structures and taste information coding mechanism.Liu et al. present work to close this gap. In this report, the authors functionalize a 36-channel microelectrode array (MEA) with biological tissue-taste receptor cells from rat taste buds. When stimulated by a food ingredient such as salt, electrophysiological activities of taste receptor cells are measured through a multichannel recording system. The authors also establish a computational model to simulate the potential response of salt taste receptor cells to stimuli of NaCl at various concentrations. The multichannel analysis reveals that different groups of MEA channels are activated when responding to salt simulation, suggesting nonoverlapping populations of receptor cells in taste buds involved in salt taste perception. The authors believe this technique provides an effective and reliable biosensor platform for studying salt taste components and may shed light on the biological tasting process. (Liu, Q., et al., Biosens. Bioelectron. 2013, 40, 115–120)Automation in On-Chip AnalysisDigitally Programmable Microfluidic Automaton for Multiscale Combinatorial Mixing and Sample ProcessingProgrammable microfluidic systems have become promising tools for laboratory automation due to their unique capabilities for automating biomolecular assays on microchip formats. Complex microfluidic networks and large-scale micro-reactor arrays fabricated using multilayer soft-lithography techniques have shown great promise for conducting sophisticated, highly parallel bioanalysis.Jensen et al. report efforts to develop a universal language to enable a programmable microfluidic bioanalysis platform based on existing unit operation modules. They develop a digitally programmable microfluidic automaton consisting of a two-dimensional array of pneumatically actuated microvalves. With this system, unit operation such as digital mixing of multiple reagents using combining valve operations, sample storage using external holding reservoirs, and transportation and simultaneous mixing of reagent can be implemented. A language describing the automated protocols is developed and based on basic rules of these automation unit operations. The authors demonstrate the capability of this automated system via automated serial dilution for quantitative analysis as well as the demonstration of on-chip fluorescent derivatization of biomarker targets for microchip capillary electrophoresis on the Mars Organic Analyzer. In addition, the technology is applied to a novel six-input combining valve that enables combinatorial microliter scale mixing operations for large sets (>26 unique combinations) of reagents. (Jensen, E. C., et al., Lab Chip 2013, 10.1039/C2LC40861A)On-Chip Manipulation of Single Microparticles, Cells, and Organisms Using Surface Acoustic WavesDing et al. report a novel method for manipulating single microparticles, cells, and organisms using surface acoustic waves. They name the technology “acoustic tweezers,” as the technology can trap and freely move single micro-objects within a single-layer microfluidic chip. The acoustic tweezers technology uses the standing surface acoustic waves to trap micro-objects at pressure nodes.In this report, the authors use the wide resonance band of chirped interdigital transducers to achieve the real-time position control of the pressure nodes and hence the position of micro-objects trapped in them. Using these novel acoustic tweezers, the authors demonstrate how to move single microparticles, cells, and micro-organisms in an arbitrary fashion in a two-dimensional plane. The power density required for acoustically manipulating micro-objects using acoustic tweezers is significantly lower than previously reported methods such as optical tweezers or dielectrophoresis-based optoelectronic tweezers. In addition, the cost and size of the instrument are significantly lower than previous methods. The low power density also suggests less cell damage. Cell-viability tests performed by the authors shows the technology is very suitable for biological object manipulation. Authors believe this novel acoustic tweezers technology can potentially become the method of choice for manipulating micro-objects in a wide variety of applications. (Ding, X., et al., Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11105–11109)Laboratory Automation and High-Throughput ChemistrySelective High-Throughput Protein Quantification Based on UV Absorption SpectraThe application of high-throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Now, a new label-free and noninvasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements has been presented. Analytics based on the methodology is 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. Furthermore, it is shown that a spectral difference of 0.6% is sufficient to successfully perform a spectral-based calibration of 2 IgG1 monoclonals. (Hansen, S. K., et al., Biotechnol Bioeng. 2012)Strategic Assay Selection for Analytics in High-Throughput Process Development: Case Studies for Downstream Processing of Monoclonal AntibodiesDuring 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 report 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 calculations 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. 2012, 7, 1256–1268)Automation SystemsTransforming Microbial Genotyping: A Robotic Pipeline for Genotyping Bacterial StrainsMicrobial genotyping increasingly deals with large numbers of samples and data are commonly evaluated by unstructured approaches, such as spreadsheets. The efficiency, reliability, and throughput of genotyping would benefit from the automation of manual manipulations within the context of sophisticated data storage.O’Farrell et al. present a medium-throughput genotyping pipeline for MultiLocus Sequence Typing (MLST) of bacterial pathogens. This pipeline is implemented through a combination of four automated liquid-handling systems, a laboratory information management system (LIMS) consisting of a variety of dedicated commercial operating systems and programs, including a sample management system, plus numerous Python scripts. All tubes and microwell racks are bar coded, and their locations and status are recorded in the LIMS.The authors also create a hierarchical set of items that can be used to represent bacterial species, their products, and experiments. The LIMS allows reliable, semiautomated, traceable bacterial genotyping from initial single-colony isolation and subcultivation through DNA extraction and normalization to PCRs, sequencing, and MLST sequence trace evaluation. The authors also describe robotic sequencing to facilitate cherry-picking of sequence dropouts. This pipeline is user friendly, with a throughput of 96 strains within 10 working days at a total cost of <€25 per strain. Since developing this pipeline, >200 000 items were processed by two to three people. This sophisticated automated pipeline can be implemented by a small microbiology group without extensive external support and provides a general framework for semiautomated bacterial genotyping of large numbers of samples at low cost. (O’Farrell, B., et al., PloS One. 2012, 7, e48022)An Automated Workflow for Enhancing Microbial Bioprocess Optimization on a Novel Microbioreactor PlatformHigh-throughput methods are widely used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless, achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the best expression construct, secretion signal peptide, inductor concentration, induction time, temperature, and substrate feed rate in fed-batch operation, to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest.To increase the throughput in bioprocess development, Rohe et al. use a microtiter plate–based cultivation system (Biolector) that is fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform is used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen, and pH in each of the microtiter plate wells triggers sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers.In addition, further automated methods such as media optimization and induction profiling are developed and validated. All biological and bioprocess parameters are exclusively optimized at microtiter plate scale and show perfect scalable results to 1 L and 20 L stirred tank bioreactor scale. The optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation set up embedded into a liquid-handling system, providing more throughput by parallelization and automation. Because of improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared with standard microtiter plate cultivation. (Rohe, P., et al., Microb. Cell Fact. 2012, 11, 144)Current State of Diagnostic Technologies in the Autoimmunology LaboratoryThe methods for detecting and measuring autoantibodies have evolved markedly in recent years, encompassing three generations of analytical technologies. Many different immunoassay methods have been developed and used for research and laboratory practice purposes, from the early conventional (or monoplex) analytical methods able to detect single autoantibodies to the more recent multiplex platforms that can quantify tens of molecules. Although it has been in use for more than 50 years, indirect immunofluorescence remains the standard method for research on many types of autoantibodies due to its characteristics of diagnostic sensitivity and also to recent technological innovations that permit it a greater level of automation and standardization. Recent multiplex immunometric methods, with varying levels of automation, present characteristics of higher diagnostic accuracy but are not yet widely diffused in autoimmunology laboratories because of the limited number of autoantibodies that are detectable and the high cost of reagents and systems. Technological advancements in autoimmunology continue to evolve rapidly, and in the coming years, new proteomic techniques will be able to radically change the approach to diagnostics and possibly also clinical treatment of autoimmune diseases. The scope of this review is to update the state of the art of technologies and methods for the measurement of autoantibodies, with special reference to innovations in indirect immunofluorescence and in multiple proteomic methods. (Tozzoli, R., et al., Clin. Chem. Lab Med. 2012)Microfluidic Chip Technology and Micro Reactor TechnologyMicrofluidic Devices for High-Throughput Proteome AnalysesOver the past decades, microfabricated bioanalytical platforms have gained enormous interest due to their potential to revolutionize biological analytics. Their popularity is based on several key properties, such as high flexibility of design, low sample consumption, rapid analysis time, and minimization of manual handling steps, which are also of particular interest for proteomics analyses. An ideal totally integrated chip-based microfluidic device could allow rapid automated workflows starting from cell cultivation and ending with MS-based proteome analysis. By reducing or eliminating sample handling and transfer steps and increasing the throughput of analyses, these workflows would dramatically improve the reliability, reproducibility, and throughput of proteomic investigations. While these complete devices do not exist for routine use yet, many improvements have been made in the translation of proteomic sample handling and separation steps into microfluidic formats. In this review, the authors focus on recent developments and strategies to enable and integrate proteomic workflows into microfluidic devices. (Chao, T. C., and Hansmeier, N., Proteomics, 2012)An Integrated Microfluidic Device for Rapid Cell Lysis and DNA Purification of Epithelial Cell SamplesIn this report, the authors describe the design and fabrication of a microfluidic device for cell lysis and DNA purification and the results of device tests using a real sample of buccal cells. Cell lysis is thermally executed for 2 min at 80 °C in a serpentine type microreactor (20 µL) using an Au microheater with a microsensor. The DNA is then mixed with other residual products and purified by a new filtration process involving micropillars and 50 to 80 µm microbeads. The entire process of sample loading, cell lysis, DNA purification, and sample extraction is successfully completed in the microchip within 5 min. Sample preparation within the microchip is verified by performing a SY158 gene PCR analysis and gel electrophoresis on the products obtained from the chip. The new purification method enhances DNA purity from 0.93 to 1.62 after purification (Ha, S. M., J. Nanosci. Nanotechnol. 2012, 11, 4250–4253)Multiple and High-Throughput Droplet Reactions via Combination of Microsampling Technique and Microfluidic ChipMicrodroplets offer unique compartments for accommodating a large number of chemical and biological reactions in tiny volume with precise control. A major concern in droplet-based microfluidics is the difficulty to address droplets individually and achieve high throughput at the same time. Wu et al. combine an improved cartridge-sampling technique with a microfluidic chip to perform droplet screenings and aggressive reaction with minimal (nanoliter-scale) reagent consumption. The droplet composition, distance, volume (nanoliter to subnanoliter scale), number, and sequence can be precisely and digitally programmed through the improved sampling technique, while sample evaporation and cross-contamination are effectively eliminated. The combined device provides a simple model to use multiple droplets for various reactions with low reagent consumption and high-throughput. (Wu, J., et al., Anal. Chem. 2012, 84, 9689–9693)High-Throughput Profiling of the Serum N-Glycome on Capillary Electrophoresis Microfluidics SystemsGlycosylation research has gained significant attention in several research fields including immunology, protein production, and biomarker discovery. However, complex and time-consuming protocols are often necessary to obtain suitable samples for analysis.