Abstract
Laboratory Automation and High-Throughput Chemistry
Laboratory automation and high-throughput screening made significant advances over the last two decades. Screening operations involving several thousands of reactions were made possible. Translation of traditional single-tube—based experiments to multiple well plate format allowed collection of numerous data points in a single experiment. Overall, development of laboratory automation went hand in hand with omics efforts in life sciences. The challenge however was to derive meaningful data from the experimental output. Although the last decade optimized most technological aspects of working with several thousands of samples, the trend is slowly but constantly shifting toward using the high-throughput approaches to probe and derive more information from intricate cellular pathways. In a nutshell, this involves combining scientific knowledge gained on basic research over the past five decades with the technological advances in the last decade. Among the below studies, the first two approaches summarize the milestones of high-throughput screening in relation to drug discovery. The latter three studies discuss specific automated applications for analytical measurements.
Novel Trends in High-Throughput Screening
In this report, the authors L.M. Mayr and D. Bojanic discuss the evolution of high-throughput screening (HTS) approaches in Pharma and Biotech and discuss the current state and comment on future trends. HTS is a well-established process for lead discovery in Pharma and Biotech companies and is now also being used for basic and applied research in academia. It comprises the screening of large chemical libraries for activity against biological targets via the use of automation, miniaturized assays, and large-scale data analysis. Since its first advent in the early to mid 1990s, the field of HTS has seen not only a continuous change in technology and processes, but also an adaptation to various needs in lead discovery. HTS has now evolved into a mature discipline that is a crucial source of chemical starting points for drug discovery. Whereas in previous years much emphasis has been put on a steady increase in screening capacity (quantitative increase) via automation and miniaturization, the past years have seen a much greater emphasis on content and quality (qualitative increase). Today, many experts in the field see HTS at a crossroad with the need to decide on either higher throughput/more experimentation or a greater focus on assays of greater physiological relevance, both of which may lead to higher productivity in pharmaceutical R&D. In this article, we describe the development of HTS over the past decade and point out our own ideas for future directions of HTS in biomedical research. We predict that the trend toward further miniaturization will slow down with the balanced implementation of 384-well, 1536-well, and 384 low-volume well plates. Furthermore, we envisage that there will be much more emphasis on rigorous assay and chemical characterization, particularly considering that novel and more difficult target classes will be pursued. In recent years, we have witnessed a clear trend in the drug discovery community toward rigorous hit validation by the use of orthogonal readout technologies, label free and biophysical methodologies. We also see a trend toward a more flexible use of the various screening approaches in lead discovery, that is, the use of both full deck compound screening, and the use of focused screening and iterative screening approaches. Moreover, we expect greater usage of target identification strategies downstream of phenotypic screening and the more effective implementation of affinity selection technologies as a result of advances in chemical diversity methodologies. We predict that, ultimately, each hit finding strategy will be much more project related, tailor made, and better integrated into the broader drug discovery efforts (Curr. Opin. Pharmacol.
Biophysical Techniques for Ligand Screening and Drug Design
The authors J.P. Renaud and M.A. Delsuc outline the involvement of biophysical methods in drug design. In this article, they discuss the two challenges of miniaturization and deriving detailed information: Biophysical methods are currently involved in drug design in two ways: the qualitative detection of small molecule binding to a target (hit identification), and the quantitative determination of physical parameters associated to binding (hit-to-lead progression). In the first case, efforts have been made toward miniaturization, automation, and speed-up of the screening process allowing a higher throughput. In the second one, sophisticated applications have been developed to derive detailed relevant information. Preferably, several methods are used in combination to avoid bias and/or limitations associated with a single one, often together with computational methods. New developments should allow important systems overlooked so far to be studied: membrane proteins, intrinsically unstructured proteins, and in-cell studies (Curr. Opin. Pharmacol.
Fully Automatic Method for the Determination of Fat Soluble Vitamins and Vitamin D Metabolites in Serum
In this study, the authors J.M. Mata-Granados et al. present an automated analytical clinical method for determination of vitamins. They present the step-by-step methodology, significance, and technical details. This type of a complete study serves as an example for other analytical determination studies. Background: Fat-soluble vitamins and vitamin D metabolites are key compounds in bone metabolism. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor vitamin D status, supplementation, and toxicity. Method: Serum (0.5 mL) was mixed with 0.5 mL 60% acetonitrile 150 mM sodium dodecyl sulfate, vortexed for 30 s and injected into an automatic solid-phase extraction (SPE) system for cleanup-preconcentration, then online transferred to a reversed-phase analytical column by a 15% methanolacetonitrile mobile phase at 1.0 mL/min for individual separation of the target analytes. Ultraviolet detection was performed at 265, 325, and 292 nm for vitamin D metabolites, vitamin A, and alpha- and delta-tocopherols, respectively. Results: Detection limits were between 0.0015 and 0.26 μg/mL for the target compounds, the precision (expressed as relative standard deviation) between 0.83% and 3.6% for repeatability and between 1.8% and 4.62% for within-laboratory reproducibility. Recoveries between 97% and 100.2% and 95% and 99% were obtained for low and high concentrations of the target analytes in serum. The total analysis time was 20 min. Conclusions: The online coupling of SPE-HPLC endows the proposed method with reliability, robustness, and user unattendance, making it a useful tool for high-throughput analysis in clinical and research laboratories (Clin. Chim. Acta.
Automated Measurement of Premethylated Serum N-Glycans by MALDI-Linear Ion Trap Mass Spectrometry
This report is another clinical example of using automated measurements of premethylated serum N-glycans in mass spectrometry. Although automation plays an important role in performing experiments, the use of automation in downstream process steps also helps the operational process. In this report, M. Guillard presents an efficient automated data collection method. The use of N-glycan mass spectrometry for clinical diagnostics requires the development of robust high-throughput profiling methods. Still, structural assignment of glycans requires additional information such as MS(2) fragmentation or exoglycosidase digestions. We present a setting that combines a MALDI ionization source with a linear ion trap analyzer. This instrumentation allows automated measurement of samples, thanks to the crystal positioning system, combined with MS(n) sequencing options. 2,5-Dihydroxybenzoic acid, commonly used for the analysis of glycans, failed to produce the required reproducibility because of its nonhomogeneous crystallization properties. In contrast, alpha-cyano-4-hydroxycinnamic acid provided a homogeneous crystallization pattern and reproducibility of the measurements. Using serum N-glycans as a test sample, we focused on the automation of data collection by optimizing the instrument settings. Glycan structures were confirmed by MS(2) analysis. Although sample processing still needs optimization, this method provides a reproducible and high-throughput approach for measurement of N-glycans using a MALDI-linear ion trap instrument (Carbohydr. Res.
Microfluidic Chip Technology and Micro Reactor Technology
There have been several developments in the use of microfluidic technologies in life sciences. The basic principles of microfluidics involve understanding the behavior of small volumes of fluids in a defined space. The field of microfluidics has innumerable applications in life sciences. In several cases of biological studies, microfluidics provided an alternative to routine methods to improve efficiency and minimize cost of the experimental process.
Modular Integration of Electronics and Microfluidic Systems Using Flexible Printed Circuit Boards
This report of integration of electronics into microfluidic systems by A. Wu et al. from Boser's lab is yet another example of achieving economical experimentation. A simple but novel approach of integrating integrated circuits (ICs) with fluid systems has made a significant advancement in the field. Microfluidic systems offer an attractive alternative to conventional wet chemical methods with benefits including reduced sample and reagent volumes, shorter reaction times, high throughput, automation, and low cost. However, most present microfluidic systems rely on external means to analyze reaction products. This substantially adds to the size, complexity, and cost of the overall system. Electronic detection based on submillimeter size ICs has been demonstrated for a wide range of targets including nucleic and amino acids, but deployment of this technology to date has been limited because of the lack of a flexible process to integrate these chips within microfluidic devices. This article presents a modular and inexpensive process to integrate ICs with microfluidic systems based on standard printed circuit board (PCB) technology to assemble the independently designed micro-fluidic and electronic components. The integrated system can accommodate multiple chips of different sizes bonded to glass or polydimethylsiloxane microfluidic systems. Because IC chips and flex PCB manufacturing and assembly are industry standards with low cost, the integrated system is economical for both laboratory and point-of-care settings (Lab Chip.
Scale-Up from Microtiter Plate to Laboratory Fermenter: Evaluation by Online Monitoring Techniques of Growth and Protein Expression in Escherichia coli and Hansenula polymorpha Fermentations
The authors F. Kensy et al. discuss the challenges in comparing small high-throughput scale operations with fermentation scale operations. They present BioLector, with a new monitoring and control system that can provide more information in bioprocess development. Background: In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP), which provides high throughput at minimal expenses in time, money, and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalability of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a microscale. To evaluate the scale-up from a MTP scale (200 μL) to a stirred tank fermenter scale (1.4 L), two standard microbial expression systems, Escherichia coli and Hansenula polymorpha, were fermented in parallel at both scales and compared with regard to the biomass and protein formation. Results: Volumetric mass transfer coefficients (kLa) ranging from 100 to 350 1/h were obtained in 96-well MTPs. Even with a suboptimal mass transfer condition in the MTP compared with the stirred tank fermenter (kLa = 370–600 1/h), identical growth and protein expression kinetics were attained in bacteria and yeast fermentations. The bioprocess kinetics were evaluated by optical online measurements of biomass and protein concentrations exhibiting the same fermentation times and maximum signal deviations below 10% between the scales. In the experiments, the widely applied green fluorescent protein served as an online reporter of protein expression for both strains. Conclusions: The successful 7000-fold scale-up from a shaken MTP to a stirred tank fermenter was demonstrated in parallel fermentations for standard microbial expression systems. This confirms that the very economical and time efficient platform of MTPs can be very easily scaled up to larger stirred tank fermenters under defined engineering conditions. New online monitoring techniques for MTPs, such as the BioLector, provide even more real-time kinetic data from fermentations than ever before and at an affordable price. This paves the way for a better understanding of the bioprocess and a more rational process design (Microb. Cell Fact.
A Microfluidic Platform for Characterization of Protein—Protein Interactions
Study of protein—protein interactions has provided immense knowledge in understanding networks in cellular pathways. Routine biochemical experimental assays include affinity pull down assays followed by mass spectrometric analysis, protein array-based approaches, or genetic studies involving yeast in two hybrid studies. Depending on the goal of the research, protein—protein interaction can be studied at various levels. M. Javanmard et al. report a microfluidic approach of characterizing protein—protein interactions. Moreover, the ability to differentiate strong and weak interactions will help gain further insight into the mode of interaction. Traditionally, expensive and time-consuming techniques such as mass spectrometry and Western Blotting have been used for characterization of protein—protein interactions. In this article, we describe the design, fabrication, and testing of a rapid and inexpensive sensor, involving the use of microelectrodes in a microchannel, which can be used for real-time electrical detection of specific interactions between proteins. We have successfully demonstrated detection of target glycoprotein—glycoprotein interactions, antigen—antibody interactions, and glycoprotein—antigen interactions. We have also demonstrated the ability of this technique to distinguish between strong and weak interactions. Using this approach, it may be possible to multiplex an array of these sensors onto a chip and probe a complex mixture for various types of interactions involving protein molecules (IEEE Sens. J.
High-Throughput Screening of Enzymes by Retroviral Display Using Droplet-Based Microfluidics
Authors L. Granieri et al. discuss the use of microfluidic droplets for display of enzymes for high-throughput screening. In this study, the droplet serves as a mini reaction vessel and amenable to high-throughput screening. They further validate the system using a model enzyme. During the last 25 years, display techniques such as phage display have become very powerful tools for protein engineering, especially for the selection of monoclonal antibodies. However, while this method is extremely efficient for affinity-based selections, its use for the selection and directed evolution of enzymes is still very restricted. Furthermore, phage display is not suited for the engineering of mammalian proteins that require post-translational modifications such as glycosylation or membrane anchoring. To circumvent these limitations, we have developed a system in which structurally complex mammalian enzymes are displayed on the surface of retroviruses and encapsulated into droplets of a water-in-oil emulsion. These droplets are made and manipulated using microfluidic devices and each droplet serves as an independent reaction vessel. Compartmentalization of single retroviral particles in droplets allows efficient coupling of genotype and phenotype. Using tissue plasminogen activator (tPA) as a model enzyme, we show that, by monitoring the enzymatic reaction in each droplet (by fluorescence), quantitative measurement of tPA activity in the presence of different concentrations of the endogenous inhibitor PAI-1 can be made on-chip. On-chip fluorescence-activated droplet sorting allowed the processing of 500 samples per second and the specific collection of retroviruses displaying active wild-type tPA from a model library with a 1000-fold excess of retroviruses displaying a nonactive control enzyme. During a single selection cycle, a more than 1300-fold enrichment of the active wild-type enzyme was demonstrated (Chem. Biol.
Fabrication of a Microfluidic Enzyme Reactor Using Magnetic Beads
Achieving higher enzymatic activity using experimental optimization will significantly improve the yields. In this report, the authors X. Liu et al. demonstrate a novel approach of enzyme-catalyzed microfluidic assay using magnetic microbe-ads. They validate the approach and report their findings. They observed that in their specific case, the microfluidic format resulted in an increase in the efficiency of substrate conversion compared with the batch assay. An enzyme-catalyzed microfluidic assay using magnetic microbeads is described. Here, diaphorase (DI) (E.C. 1.6.99) is covalently attached to the magnetic microbeads (2.7 mum) and integrated into a short section of a microchip fabricated from polydimethylsiloxane. DI converts nonfluorescent resazurin to fluorescent resorufin in the presence of nicotinamide adenine dinucleotide phosphate (NADH). In this work, an embedded magnet holds the microbeads in place within the microchannel, while a solution of resazurin and NADH in buffer is flowed through the beads. Incorporation of the microbeads into the microchannel requires only a few minutes and offers well-defined spatial resolution and reproducibility. At a flow rate of 41.2 μL/h, a stable state for the enzyme reaction in the microfluidic format was achieved within 50 s. The maximum conversion of the reaction was obtained at a concentration of 1.25 mM NADH. The reaction yield is affected by ZnCl(2) and at concentrations in excess of 90.0 mM, the activity of DI was almost double without ZnCl(2). At 5.2 mM potassium chloride, the activity of DI reached its maximum value. Overall, the conversion of resazurin in microfluidic format was more than twice than that in a batch assay (Electrophoresis.
High-Throughput Analytics
The worldwide efforts in genomics, proteomics, and other omics led to numerous high-throughput approaches. Because of the increasing number of high-throughput areas, a classification to define and segregate some studies to families has become necessary. High-throughput analytics is one such family that focuses mostly on measurement and analysis kind of studies. The below articles are some isolated advances in the field of high-throughput analytics.
Screen Printing as Cost-Efficient Fabrication Method for DNA Chips with Electrical Readout for Detection of Viral DNA
The fast development in the field of DNA analytics is driven by the need for cost-effective and high-throughput methods for the detection of biomolecules. The detection of DNA using metal nanoparticles as labels is an interesting alternative to the standard fluorescence technique. Fluorescence is highly sensitive and broadly established, but shows limitations, for example instability of the signal and the requirement for sophisticated and high-cost equipment. A recently developed approach realizes a method for the electrical detection of DNA, based on the induction of silver nanoparticles growth in microelectrode gaps on the surface of a DNA chip. This breakthrough toward robust and cost-effective detection was still hampered by the need for microstructured (and therefore expensive) substrates. We demonstrate that it is possible to use screen printed electrode structures for a chip-based electrical DNA detection. The electrode structures were produced on a glass substrate, which made an additional optical readout possible. The screen printed structures show the required precision and are compatible with the applied biochemical protocols. A comparison with chip substrates produced by standard photolithography showed the same sensitivity and specificity for the screen printed chips. Screen printing of electrode structures for DNA chip with electrical detection offers an interesting and cost-efficient possibility to produce DNA chips with micro-structured electrodes (Schuler, T. et al., Biosens. Bioelectron.
A Predictive High-Throughput Scale-Down Model of Monoclonal Antibody Production in CHO Cells
Multifactorial experimentation is essential in understanding the link between mammalian cell culture conditions and the glycoprotein product of any biomanufacturing process. This understanding is increasingly demanded as bioprocess development is influenced by the Quality by Design paradigm. We have developed a system that allows hundreds of microbioreactors to be run in parallel under controlled conditions, enabling factorial experiments of much larger scope than is possible with traditional systems. A high-throughput analytics workflow was also developed using commercially available instruments to obtain product quality information for each cell-culture condition. The microbioreactor system was tested by executing a factorial experiment varying four process parameters: pH, dissolved oxygen, feed supplement rate, and reduced glutathione level. A total of 180 microbioreactors were run for 2 weeks during this design of experiment to assess this scaled down microbioreactor system as a high-throughput tool for process development. Online measurements of pH, dissolved oxygen, and optical density were complemented by offline measurements of glucose, viability, titer, and product quality. Model accuracy was assessed by regressing the microbioreactor results with those obtained in conventional 3-L bioreactors. Excellent agreement was observed between the microbioreactor and the bench-top bioreactor. The microbioreactor results were further analyzed to link parameter manipulations to process outcomes via leverage plots, and to examine the interactions between process parameters. The results show that feed supplement rate has a significant effect (p 0.05) on all performance metrics with higher feed rates resulting in greater cell mass and product titer. Culture pH impacted terminal integrated viable cell concentration, titer and intact immunoglobulin G titer, with better results obtained at the lower pH set point. The results demonstrate that a microscale system can be an excellent model of larger scale systems, while providing data sets broader and deeper than are available by traditional methods (Legman, et al., Biotechnol. Bioeng.
Performance Evaluation of a Turbidimetric Cystatin C Assay on Different High-Throughput Platforms
Reproducibility of experimental analytical processes across laboratories is critical. In general, the variation for simple manual assays performed in single tubes is more controlled. However, when such assays are translated to high-throughput automated platforms, the variability increases significantly. This increase is because of process variables that include different automation performance features, well-to-well sample variation, variability in readout, etc. Before use of a widely used assay across laboratories, it is best to validate the assay on different platforms. This report by L.O. Hansson et al. is one such analytical experimental method that measures performance of cystatin C immunoassay on different high-throughput platforms. This type of study will serve as a guideline for researchers who want to adapt their assays on multiple platforms.
Abstract—Objective: The goal with this study was to evaluate the analytical performance of a new cystatin C immunoassay (Tina-quant((R)) a Cystatin C, Roche Diagnostics GmbH). The evaluation was carried out at four centers according to a standardized protocol. Material and methods. The Tina-quant((R)) a Cystatin C is a latex particle-enhanced immunoturbidimetric assay. Roche cobas((R)) 6000, MODULAR ANALYTICS SWA and COBAS INTE-GRA((R)) instruments were included in the study. Method comparison studies were carried out against two turbidimetric methods (Dako Cystatin C, Gentian Cystatin C), and one nephelometric method (Siemens N-Latex Cystatin C). Results: Linearity was proven throughout the measuring range from 0.4 to 8 mg/L. Within-run coefficients of variance ranged from 0.7% to 2.8%, and total coefficients of variance from 1.4% to 4.7% (concentration range 0.6–3.9 mg/L). Comparable results were obtained with paired serum and Li-heparinate plasma samples. Good agreement was achieved in the comparisons between the Tina-quant((R)) a Cystatin C assay and the other commercially available cystatin C assays, two different turbidimetric methods (slope range 0.88–1.04, intercept < 0.17 mg/L, r ≥ 0.993) and one nephelometric assay (slope range 0.90–1.05, intercept < 0.21 mg/L, r ≥ 0.986). Conclusions: The Tina-quant ((R)) a Cystatin C assay was shown to be precise and accurate with proven linearity over the measuring range. Good comparability was obtained with other commercially available assays for the determination of cystatin C. The Tina-quant((R)) a Cystatin C assay is very well suited for clinical use on routine clinical chemistry analyzers to detect renal dysfunction with a 24 h availability (Scand. J. Clin. Lab. Invest.
Automation Systems
To Automate or Not to Automate: This is the Question
Traditional protein crystallization projects involve cloning the gene, expressing/purifying protein, setting up and optimizing crystallization trials, subjecting crystals to X-ray diffraction, and finally resolving the structure. Although several advancements to automate every step of the process were made, understanding the realistic need for automation in a given laboratory becomes important. In this article from Cymborowski M, from the laboratories of Minor W, the authors attempt to dissect the steps in the structure determination process and identify extent of need to automate the various individual steps. New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, they show that automation of some (and integration of all) steps of the X-ray structure determination pathway are critical for laboratory productivity. Moreover, they show that the lag period after which the impact of a technology change is observed is longer than expected (J. Struct. Funct. Genomics.
Miniaturization of Molecular Biological Techniques for Gene Assay
An efficient process for diagnosis of genetic disease from sample to result is very much needed. A gist of the current state of the diagnostic process and use of microfluidic approach for rapid detection processes of genetic diseases is reviewed by K.Y. Lien and G.B. Lee in this article. The authors comment on the applicability of the microfluidic systems on preanalysis and analysis of genetic disease diagnosis. The rapid diagnosis of various diseases is a critical advantage of many emerging biomedical tools. Because of advances in preventive medicine, tools for the accurate analysis of genetic mutation and associated hereditary diseases have attracted significant interests in recent years. The entire diagnostic process usually involves two critical steps, namely, sample pretreatment and genetic analysis. The sample pre-treatment processes such as extraction and purification of the target nucleic acids before genetic analysis are essential in molecular diagnostics. The genetic analysis process may require specialized apparatus for nucleic acid amplification, sequencing, and detection. Traditionally, pretreatment of clinical biological samples (e.g., the extraction of deoxyribo-nucleic acid [DNA] or ribonucleic acid [RNA]) and the analysis of genetic polymorphisms associated with genetic diseases are typically a lengthy and costly process. These labor-intensive and time-consuming processes usually result in a high cost per diagnosis and hinder their practical applications. Besides, the accuracy of the diagnosis may be affected owing to potential contamination from manual processing. Alternatively, because of significant advances in microelectro-mechanical systems and microfluidic technology, there are numerous miniature systems used in biomedical applications, especially for the rapid diagnosis of genetic diseases. A number of advantages including automation, compactness, disposability, portability, lower cost, shorter diagnosis time, lower sample and reagent consumption, and lower power consumption can be realized by using these microfluidic-based platforms. As a result, microfluidic-based systems are becoming promising platforms for genetic analysis, molecular biology, and for the rapid detection of genetic diseases. In this review paper, microfluidic-based platforms capable of identifying genetic sequences and diagnosis of genetic mutations are surveyed and reviewed. Some critical issues with the use of microfluidic-based systems for diagnosis of genetic diseases are also highlighted (Analyst.