Abstract
Laboratory Automation and High-Throughput Chemistry
Automated Detection and Quantification of Granular Cell Compartments
Many cellular processes are organized in a compartmentalized and dynamic fashion to ensure effective adaptation to physiological changes. Thus, in response to stress and disease, cells initiate protective mechanisms to restore homeostasis. Among these mechanisms are the arrest of translation and remodeling of ribonucleoprotein complexes into granular compartments in the cytoplasm, known as stress granules (SGs). To date, the analysis of SGs has relied on the manual demarcation and measurement of the compartment, making quantitative studies time-consuming while preventing the efficient use of high-throughput technology.
Mahboubi et al. developed the first fully automated, computer-based procedures that measure the association of fluorescent molecules with granular compartments. Their methods quantify automatically multiple granule parameters and generate data at the level of single cells or individual SGs. These techniques detect simultaneously in an automated fashion proteins and RNAs located in SGs. The effectiveness of the protocols is demonstrated by studies that reveal several of the unique biological and structural characteristics of SGs. In particular, the authors show that the type of stress determines granule size and composition, as illustrated by the concentration of poly(A)-RNA and a specific SG marker protein. Furthermore, they take advantage of the computer-based and automated methods to design assays suitable for high-throughput screening. (Mahboubi, H., et al., Microsc. Microanaly.
A Quantitative Liposome Microarray to Systematically Characterize Protein-Lipid Interactions
Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds, and signaling molecules, but they are still largely underrepresented in known biological networks. Here, Saliba et al. describe a liposome microarray-based assay, a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed, and high-throughput manner. (Saliba, A. E., et al., Nat. Methods
Screening for Protein-DNA Interactions by Automatable DNA-Protein Interaction ELISA
DNA-binding proteins (DBPs), such as transcription factors, constitute about 10% of the protein-coding genes in eukaryotic genomes and play pivotal roles in the regulation of chromatin structure and gene expression by binding to short stretches of DNA. Despite their number and importance, only for a minor portion of DBPs has the binding sequence has been disclosed. Methods that allow the de novo identification of DNA-binding motifs of known DBPs, such as protein binding microarray technology or SELEX, are not yet suited for high-throughput and automation.
To close this gap, Brand et al. report an automatable DNA-protein interaction (DPI)-ELISA screen of an optimized double-stranded DNA (dsDNA) probe library that allows the high-throughput identification of hexanucleotide DNA-binding motifs. In contrast to other methods, this DPI-ELISA screen can be performed manually or with standard laboratory automation. Furthermore, output evaluation does not require extensive computational analysis to derive a binding consensus. The authors show that the DPI-ELISA screen discloses the full spectrum of binding preferences for a given DBP. As an example, AtWRKY11 is used to demonstrate that the automated DPI-ELISA screen reveals the entire range of in vitro binding preferences. In addition, protein extracts of AtbZIP63 and the DNA-binding domain of AtWRKY33 are analyzed, which leads to a refinement of their known DNA-binding consensi. Finally, the authors perform a DPI-ELISA screen to disclose the DNA-binding consensus of a yet uncharacterized putative DBP, AtTIFY1. A palindromic TGATCA consensus is uncovered, and the authors show that the GATC-core is compulsory for AtTIFY1 binding.
This specific interaction between AtTIFY1 and its DNA-binding motif is confirmed by in vivo plant one-hybrid assays in protoplasts. Thus, the value and applicability of the DPI-ELISA screen for de novo binding site identification of DBPs, also under automatized conditions, is a promising approach for a deeper understanding of gene regulation in any organism of choice. (Brand, L. H., et al. PLoS One
Digital PCR on an Integrated Self-Priming Compartmentalization Chip
An integrated on-chip valve-free and power-free microfluidic digital PCR device is for the first time developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize “divide and conquer” for single DNA molecule detection. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The life span of its self-priming capability is about 2 weeks, tested using an air-tight packaging bottle sealed with a small amount of petroleum jelly, which is significant for a practical platform. The SPC chip contains 5120 independent 5 nL microchambers, allowing the samples to be compartmentalized completely.
Using this platform, three different abundances of lung cancer–related genes are detected to demonstrate the feasibility and flexibility of the microchip for amplifying a single nucleic acid molecule. For maximal accuracy, within less than 5% of the measurement deviation, the optimal number of positive chambers is between 400 and 1250, evaluated by the Poisson distribution, which means one panel can detect an average of 480 to 4804 template molecules. This device without world-to-chip connections eliminates the constraint of the complex pipeline control and is an integrated on-chip platform, which would be a significant improvement to digital PCR automation and be more user friendly. (Zhu, Q., Lab Chip
High-Throughput Analysis of Antimalarial Susceptibility Data by the WorldWide Antimalarial Resistance Network (WWARN) In Vitro Analysis and Reporting Tool
Assessment of in vitro susceptibility is a fundamental component of antimalarial surveillance studies, but wide variations in the measurement of parasite growth and the calculation of inhibitory constants make comparisons of data from different laboratories difficult. Here, C. J. Woodrow describes a Web-based, high-throughput in vitro analysis and reporting tool (IVART) generating inhibitory constants for large data sets.
Fourteen primary data sets examining laboratory-determined susceptibility to artemisinin derivatives and artemisinin combination therapy partner drugs are collated from 11 laboratories. Drug concentrations associated with half-maximal inhibition of growth (IC50s) are determined by a modified sigmoid Emax model-fitting algorithm, allowing standardized analysis of 7350 concentration-inhibition assays involving 1592 isolates. Examination of concentration-inhibition data reveal evidence of apparent paradoxical growth at high concentrations of nonartemisinin drugs, supporting amendment of the method for calculating the maximal drug effect in each assay. Criteria for defining more reliable IC50s based on estimated confidence intervals and growth ratios improve correlation coefficients for the drug pairs mefloquine-quinine and chloroquine-desethylamodiaquine in 9 of 11 and 8 of 8 data sets, respectively. Further analysis shows that maximal drug inhibition is higher for artemisinins than for other drugs, particularly in enzyme-linked immunosorbent assay (ELISA)–based assays, a finding consistent with the earlier onset of action of these drugs in the parasite life cycle.
This is the first high-throughput analytical approach to apply consistent constraints and reliability criteria to large, diverse antimalarial susceptibility data sets. The data also illustrate the distinct biological properties of artemisinins and underline the need to apply more sensitive approaches to assessing the in vitro susceptibility to these drugs. (Woodrow, C. J., Antimicrob. Agents Chemother.
Microfluidic Chip Technology and Micro Reactor Technology
A 1024-Sample Serum Analyzer Chip for Cancer Diagnostics
Garcia-Cordero and Maerkl present a platform that combines microarrays and microfluidic techniques to measure four protein biomarkers in 1024 serum samples for a total of 4096 assays per device. Detection is based on a surface fluorescence sandwich immunoassay with a limit of detection of ~1 pM for most of the proteins measured: PSA, TNF-α, IL-1β, and IL-6. To validate the utility of the platform, the authors measure these four biomarkers in 20 clinical human serum samples, 10 from prostate cancer patients and 10 from female and male controls. The authors compare the results of the platform to a conventional ELISA and find a good correlation between them. However, compared with a classical ELISA, the device reduces the total cost of reagents by four orders of magnitude while increasing throughput by two orders of magnitude. Overall, they demonstrate an integrated approach to performing low-cost and rapid quantification of protein biomarkers from more than 1000 serum samples. This new high-throughput technology will have a significant impact on disease diagnosis and management. (Garcia-Cordero, J. L., and Maerkl, S. J., Lab Chip
Advances in Coupling Microfluidic Chips to Mass Spectrometry
Microfluidic technology has shown advantages of low sample consumption, reduced analysis time, high throughput, and potential for integration and automation. Coupling microfluidic chips to mass spectrometry (chip-MS) can greatly improve the overall analytical performance of MS-based approaches and expand their potential applications.
In this article, Feng et al. review the advances of chip-MS in the past decade, covering innovations in microchip fabrication, microchips coupled to electrospray ionization–MS, and matrix-assisted laser desorption/ionization–MS. Development of integrated microfluidic systems for automated MS analysis will be further documented, as well as recent applications of chip-MS in proteomics, metabolomics, cell analysis, and clinical diagnosis. (Feng, X., et al., Mass Spectrom. Rev.
Micro-a-Fluidics ELISA for Rapid CD4 Cell Count at the Point of Care
HIV has become one of the most devastating pathogens in human history. Despite fast progress in HIV-related basic research, antiretroviral therapy (ART) remains the most effective method to save AIDS patients’ lives. Unfortunately, ART cannot be universally accessed, especially in developing countries, because of the lack of effective treatment-monitoring diagnostics. Here, the authors present an inexpensive, rapid, and portable micro-a-fluidic platform, which can streamline the process of an ELISA in a fully automated manner for CD4 cell count.
The micro-a-fluidic CD4 cell count is achieved by eliminating operational fluid flow via “moving the substrate,” as opposed to “flowing liquid” in traditional ELISA or microfluidic methods. This is the first demonstration of capturing and detecting cells from unprocessed whole blood using the ELISA in a microfluidic channel. Combined with cell phone imaging, this micro-a-fluidic ELISA platform holds great promise for offering rapid CD4 cell count to scale up much needed ART in resource-constrained settings. The developed system can be extended to multiple areas for ELISA-related assays. (Wang, S., et al., Sci. Rep.
High-Throughput Analytics
Automated Disposable Small-Scale Reactor for High-Throughput Bioprocess Development: A Proof-of-Concept Study
The acceleration of bioprocess development for biologics and vaccines can be enabled by automated high-throughput technologies. This will alleviate the significant resource burden from the multifactorial statistical experimentation required for controlling product quality attributes of complex biologics. Recent technology advances have improved clone evaluation and screening but have struggled to combine the scale-down criteria required for both high-cell-density cell culture and microbial processes, with sufficient automation and disposable technologies to accelerate process development.
This article describes the proof-of-concept evaluations of an automated disposable small-scale reactor for high-throughput upstream process development. Characterization studies establish the small-scale stirred tank disposable 250 mL reactor as similar to those of lab and pilot scale. The reactor generates equivalent process performance for industrial biologics processes for therapeutic protein and monoclonal antibody production using CHO cell culture, Pichia pastoris and Escherichia coli. This includes similar growth, cell viability, product titer, and product quality. The technology is shown to be robust across multiple runs and meets the requirements for the ability to run high-cell-density processes (>400 g/L wet cell weight) with exponential feeds and sophisticated event-triggered processes. Combining this reactor into an automated array of reactors will ultimately be part of a high-throughput process development strategy. This will combine upstream, small-scale purification with rapid analytics that will dramatically shorten timelines and costs of developing biological processes. (Bareither, R., et al., Biotechnol. Bioeng.
Determination of Residual Enrofloxacin in Food Samples by a Sensitive Method of Chemiluminescence Enzyme Immunoassay
A chemiluminescence enzyme immunoassay (CLEIA) based on the HRP-luminol-H2O2 chemiluminescence system for highly sensitive detection of enrofloxacin (ENR) is proposed in this study. Key factors that affect the precision and accuracy for the determination of ENR residues are optimized. Under optimal conditions, the proposed method shows excellent performance. The linearity range for the method developed for determination of ENR is 0.35 to 1.0 ng/mL, with a correlation coefficient greater than 0.994. The limit of detection is 0.03 ng/mL, and the relative standard deviations are less than 9.4% and 13.0% for intraday and interday assays. The proposed method is satisfactorily applied to determine ENR in milk, eggs, and honey samples at three spiked levels (0.4, 0.7, and 1.0 ng/mL), and the recoveries range from 92.4% to 104.2% for milk, 93.8% to 103.2% for eggs, and 94.1% to 105.0% for honey, respectively. When the results of CLEIA are compared with those of ELISA and high-performance liquid chromatography, the advantages of the CLEIA are further confirmed. Moreover, one 96-well microtiter plate coated with anti-ENR can be used to detect multiple samples at the same time, which indicates that the CLEIA using the HRP-luminol-H2O2 system is a sensitive, high-throughput, and real-time method for analysis of ENR residues. (Yu, F., et al., Food Chem.
Optimization of a Paper-Based ELISA for a Human Performance Biomarker
Monitoring aspects of human performance during various activities has recently become a highly investigated research area. Many new commercial products are available now to monitor human physical activity or responses while performing activities ranging from playing sports to driving and even sleeping. However, monitoring cognitive performance biomarkers, such as neuropeptides, is still an emerging field because of the complicated sample collection and processing, as well as the need for a clinical lab to perform analysis.
ELISAs provide specific detection of biomolecules with high sensitivity (picomolar concentrations). Even with the advantage of high sensitivity, most ELISAs need to be performed in a laboratory setting and require about 6 h to complete. Transitioning this assay to a platform where it reduces cost, shortens assay time, and is able to be performed outside a lab is invaluable. Recently developed paper diagnostics provide an inexpensive platform on which to perform ELISAs; however, the major limiting factor for moving out of the laboratory environment is the measurement and analysis instrumentation. Using something as simple as a digital camera or camera-enabled Windows- or Android-based tablets, the authors are able to image paper-based ELISAs, perform image analysis, and produce response curves with high correlation to target biomolecule concentration in the 10 pM range. Neuropeptide Y detection is performed. In addition, silver enhancement of Au NPs conjugated with IgG antibodies shows a concentration-dependent response to IgG, thus eliminating the need for an enzyme-substrate system. Automated image analysis and quantification of antigen concentrations are able to be performed on Windows- and Android-based mobile platforms. (Murdock, R. C., et al., Anal. Chem.
Loss of Connectivity in Cancer Co-Expression Networks
Differential gene expression profiling studies have led to the identification of several disease biomarkers. However, the oncogenic alterations in coding regions can modify the gene functions without affecting their own expression profiles. Moreover, posttranslational modifications can modify the activity of the coded protein without altering the expression levels of the coding gene but elicit variations to the expression levels of the regulated genes. These considerations motivate the study of the rewiring of networks’ co-expressed genes as a consequence of the aforementioned alterations in order to complement the informative content of differential expression.
Anglani et al. analyze 339 mRNAomes of five distinct cancer types to find single genes that present co-expression patterns strongly differentiated between normal and tumor phenotypes. Their analysis of differentially connected genes indicates the loss of connectivity as a common topological trait of cancer networks and unveils novel candidate cancer genes. Moreover, their integrated approach that combines the differential expression together with the differential connectivity improves the classic enrichment pathway analysis by providing novel insights on putative cancer gene biosystems not still fully investigated. (Anglani, R., et al., PLoS One
Advances in Automated Biomarker Discovery Research
Antibody-Based Proteomics and Biomarker Research: Current Status and Limitations
Antibody-based proteomics play a very important role in biomarker discovery and validation, facilitating the high-throughput evaluation of candidate markers. Nowadays, most proteomics-driven discovery is based on the use of mass spectrometry. Mass spectrometry has many advantages, including its suitability for hypothesis-free biomarker discovery, because information on the protein content of a sample is not required prior to analysis. However, mass spectrometry presents one main caveat, which is the limited sensitivity in complex samples, especially for body fluids, where protein expression covers a huge dynamic range. Antibody-based technologies remain the main solution to address this challenge because they reach higher sensitivity.
In this article, Solier and Langen review the benefits and limitations of antibody-based proteomics in preclinical and clinical biomarker research for discovery and validation in body fluids and tissue. The combination of antibodies and mass spectrometry, using the best of both worlds, opens new avenues in biomarker research. (Solier, C., and Langen, H., Proteomics,
Long-Term Dry Storage of an Enzyme-Based Reagent System for ELISA in Point-of-Care Devices
Lateral flow devices are commonly used for many point-of-care (POC) applications in low-resource settings. However, they lack the sensitivity needed for many analytes relevant in the diagnosis of diseases. One approach to achieving higher sensitivity is signal amplification, which is commonly used in laboratory assays but uses reagents that require refrigeration and inherently requires multiple assay steps not normally compatible with POC settings. Enzyme-based signal amplification, such as the one used in ELISA, could greatly improve the limit of detection if it were translated to a format compatible with POC requirements. A signal-amplified POC device not only requires the reagents to be stored in a stable form but also requires automation of the multiple sequential steps of signal amplification protocols.
Here, S. Ramachandran et al. describe a method for the long-term dry storage of ELISA reagents: horseradish peroxidase (HRP)–conjugated antibody label and its colorimetric substrate diaminobenzidine (DAB). The HRP conjugate retains ~80% enzymatic activity after dry storage at 45 °C for more than 5 months. The DAB substrate is also stable at 45 °C and exhibits no detectable loss of activity over 3 months. These reagents are incorporated into a two-dimensional paper network device that automates the steps of ELISA for the detection of a malarial biomarker. These results demonstrate the potential of enzyme-based signal amplification for enhanced sensitivity in POC devices for low-resource settings. (Ramachandran, S., et al., Analyst