A cost-effective maize ear phenotyping platform enables rapid categorization and quantification of kernels.

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.

Advancing automation in Compound Management: a novel industrial process underpinning drug discovery.

Faced with ageing infrastructure and ever-increasing demands from hit discovery and lead optimisation functions, AstraZeneca has chosen to develop innovative technologies and process solutions to support the future of drug discovery. These include the miniaturisation of compound storage tubes for high-density storage and rapid access to the corporate collection for feeding samples to the predicted tripling number of high throughput screening (HTS) campaigns. The acoustically- compatible tubes also enable the first fully-acoustic plate production process for faster sample supply to screening with less waste and continued high quality. Operating at a smaller scale reduces compound synthesis, storage, and consumption, prompting miniaturisation of upstream chemistry and downstream biological assays, while offering a transformative and sustainable solution to many drug discovery issues applicable across the industry.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays.

Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

Low-cost multi-core inertial microfluidic centrifuge for high-throughput cell concentration.

We developed a low-cost multi-core inertial microfluidic centrifuge (IM-centrifuge) to achieve a continuous-flow cell/particle concentration at a throughput of up to 20 mL/min. To lower the cost of our IM-centrifuge, we clamped a disposable multilayer film-based inertial microfluidic (MFIM) chip with two reusable plastic housings. The key MFIM chip was fabricated in low-cost materials by stacking different polymer-film channel layers and double-sided tape. To increase processing throughput, multiplexing spiral inertial microfluidic channels were integrated within an all-in-one MFIM chip, and a novel sample distribution strategy was employed to equally distribute the sample into each channel layer. Then, we characterized the focusing performance in the MFIM chip over a wide flow-rate range. The experimental results showed that our IM-centrifuge was able to focus various-sized particles/cells to achieve volume reduction. The sample distribution strategy also effectively ensured identical focusing and concentration performances in different cores. Finally, our IM-centrifuge was successfully applied to concentrate microalgae cells with irregular shapes and highly polydisperse sizes. Thus, our IM-centrifuge holds the potential to be employed as a low-cost, high-throughput centrifuge for disposable use in low-resource settings.

High throughput bar adsorptive microextraction: A novel cost-effective tool for monitoring benzodiazepines in large number of biological samples.

In this work, we propose an innovative high throughput (HT) apparatus using the bar adsorptive microextraction (BAµE) technique, which enables the simultaneous enrichment of up to 100 samples. This novel configuration was combined with microliquid desorption and high-performance liquid chromatography-diode array detection to monitor trace levels of eight benzodiazepines (diazepam, prazepam, bromazepam, oxazepam, lorazepam, alprazolam, temazepam and loflazepate) in biological samples. The proposed methodology was fully developed, optimized and validated, resulting in suitable intraday and interday precision (RSD ≤ 15%), with recovery yields ranging from 33.0% to 104.5%. The lower limits of quantification were between 20.0 and 100.0 µg L-1, using 1.0 mL of urine and 0.5 mL of plasma or serum samples. The application of the proposed methodology to real matrices resulted in average sample preparation time of around 2 min per sample, demonstrating that it is user-friendly, cost-effective and a rapid decision-making tool, whenever large number of samples are involved.

High-Throughput Assessment of Mechanistic Toxicity of Chemicals in Miniaturized 3D Cell Culture.

High-content imaging (HCI) assays on two-dimensional (2D) cell cultures often do not represent in vivo characteristics accurately, thus reducing the predictability of drug toxicity/efficacy in vivo. On the other hand, conventional 3D cell cultures are relatively low throughput and possess difficulty in cell imaging. To address these limitations, a miniaturized 3D cell culture has been developed on a micropillar/microwell chip platform with human cells encapsulated in biomimetic hydrogels. Model compounds are used to validate human cell microarrays for high-throughput assessment of mechanistic toxicity. Main mechanisms of toxicity of compounds can be investigated by analyzing multiple parameters such as DNA damage, mitochondrial impairment, intracellular glutathione level, and cell membrane integrity. IC50 values of these parameters can be determined and compared for the compounds to investigate the main mechanism of toxicity. This paper describes miniaturized HCI assays on 3D-cultured cell microarrays for high-throughput assessment of mechanistic profiles of compound-induced toxicity. © 2018 by John Wiley & Sons, Inc.

A 96-well microplate bioreactor platform supporting individual dual perfusion and high-throughput assessment of simple or biofabricated 3D tissue models.

Traditional 2D monolayer cell cultures and submillimeter 3D tissue construct cultures used widely in tissue engineering are limited in their ability to extrapolate experimental data to predict in vivo responses due to their simplistic organization and lack of stimuli. The rise of biofabrication and bioreactor technologies has sought to address this through the development of techniques to spatially organize components of a tissue construct, and devices to supply these tissue constructs with an increasingly in vivo-like environment. Current bioreactors supporting both parenchymal and barrier tissue constructs in interconnected systems for body-on-a-chip platforms have chosen to emphasize study throughput or system/tissue complexity. Here, we report a platform to address this disparity in throughput and both system complexity (by supporting multiple in situ assessment methods) and tissue complexity (by adopting a construct-agnostic format). We introduce an ANSI/SLAS-compliant microplate and docking station fabricated via stereolithography (SLA), or precision machining, to provide up to 96 samples (Ø6 × 10 mm) with two individually-addressable fluid circuits (192 total), loading access, and inspection window for imaging during perfusion. Biofabricated ovarian cancer models were developed to demonstrate the in situ assessment capabilities via microscopy and a perfused resazurin-based metabolic activity assay. In situ microscopy highlighted flexibility of the sample housing to accommodate a range of sample geometries. Utility for drug screening was demonstrated by exposing the ovarian cancer models to an anticancer drug (doxorubicin) and generating the dose-response curve in situ, while achieving an assay quality similar to static wellplate culture. The potential for quantitative analysis of temporal tissue development and screening studies was confirmed by imaging soft- (gelatin) and hard-tissue (calcium chloride) analogs inside the bioreactor via spectral computed tomography (CT) scanning. As a proof-of-concept for particle tracing studies, flowing microparticles were visualized to inform the design of hydrogel constructs. Finally, the ability for mechanistic yet high-throughput screening was demonstrated in a vascular coculture model adopting endothelial and mesenchymal stem cells (HUVEC-MSC), encapsulated in gelatin-norbornene (gel-NOR) hydrogel cast into SLA-printed well inserts. This study illustrates the potential of a scalable dual perfusion bioreactor platform for parenchymal and barrier tissue constructs to support a broad range of multi-organ-on-a-chip applications.

A multi-column plate adapter provides an economical and versatile high-throughput protein purification system.

Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting.

A microfluidics platform for combinatorial drug screening on cancer biopsies.

Screening drugs on patient biopsies from solid tumours has immense potential, but is challenging due to the small amount of available material. To address this, we present here a plug-based microfluidics platform for functional screening of drug combinations. Integrated Braille valves allow changing the plug composition on demand and enable collecting >1200 data points (56 different conditions with at least 20 replicates each) per biopsy. After deriving and validating efficient and specific drug combinations for two genetically different pancreatic cancer cell lines and xenograft mouse models, we additionally screen live cells from human solid tumours with no need for ex vivo culturing steps, and obtain highly specific sensitivity profiles. The entire workflow can be completed within 48 h at assay costs of less than US$ 150 per patient. We believe this can pave the way for rapid determination of optimal personalized cancer therapies.

High throughput detection of deamidation using S-(5'-adenosyl)-l-homocysteine hydrolase and a fluorogenic reagent.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

Assessment of the scalability of a microtiter plate system for screening of oleaginous microorganisms.

Recent developments in molecular biology and metabolic engineering have resulted in a large increase in the number of strains that need to be tested, positioning high-throughput screening of microorganisms as an important step in bioprocess development. Scalability is crucial for performing reliable screening of microorganisms. Most of the scalability studies from microplate screening systems to controlled stirred-tank bioreactors have been performed so far with unicellular microorganisms. We have compared cultivation of industrially relevant oleaginous filamentous fungi and microalga in a Duetz-microtiter plate system to benchtop and pre-pilot bioreactors. Maximal glucose consumption rate, biomass concentration, lipid content of the biomass, biomass, and lipid yield values showed good scalability for Mucor circinelloides (less than 20% differences) and Mortierella alpina (less than 30% differences) filamentous fungi. Maximal glucose consumption and biomass production rates were identical for Crypthecodinium cohnii in microtiter plate and benchtop bioreactor. Most likely due to shear stress sensitivity of this microalga in stirred bioreactor, biomass concentration and lipid content of biomass were significantly higher in the microtiter plate system than in the benchtop bioreactor. Still, fermentation results obtained in the Duetz-microtiter plate system for Crypthecodinium cohnii are encouraging compared to what has been reported in literature. Good reproducibility (coefficient of variation less than 15% for biomass growth, glucose consumption, lipid content, and pH) were achieved in the Duetz-microtiter plate system for Mucor circinelloides and Crypthecodinium cohnii. Mortierella alpina cultivation reproducibility might be improved with inoculation optimization. In conclusion, we have presented suitability of the Duetz-microtiter plate system for the reproducible, scalable, and cost-efficient high-throughput screening of oleaginous microorganisms.

High-Throughput Screening to Identify Anesthetic Ligands Using Xenopus laevis Tadpoles.

General anesthetics are considered among the most significant advances in modern medicine; however, they are also some of the most dangerous commonly administered drugs. Despite this, the discovery of novel anesthetics has been slow, with few clinically used agents regardless of their nearly 200-year history. Xenopus laevis frogs have a long history as a model organism and provide a vital bridge between in vitro and preclinical mammalian assays. The provided protocols are efficient and cost-effective and therefore readily amendable for high-throughput evaluation of novel anesthetic ligands. By using the X. laevis bioassay, a researcher is capable of determining relative general anesthetic tolerance and/or cross-tolerance for candidate nonvolatile and/or volatile ligands.

An impedance-based cell contraction assay using human primary smooth muscle cells and fibroblasts.

INTRODUCTION: Many cell types (including muscle cells and fibroblasts) can contract at physiological conditions and their contractility may change during tissue injury and repair or other diseases such as allergy and asthma. The conventional gel contraction assay is commonly used to monitor the cellular contractility. It is a manual assay and the experiment usually takes hours even days to complete. As its readout is not always accurate and reliable, the gel contraction assay is often used to qualitatively (but not quantitatively) characterize cellular contractility under various conditions. METHOD: To overcome the limits of the gel contraction assay, we developed an impedance-based contraction assay using the xCELLigence RTCA MP system. This technology utilizes special 96-well E-plates with gold microelectrode arrays printed in individual wells to monitor cellular adhesion by recording the electrical impedance in real time. The impedance change (percentage vs. control) can be used as the readout for cellular contraction. RESULTS: We demonstrated that the impedance-based contraction assay can be performed within 2h. Using this new method, we quantitatively characterized the effects of several contractile stimulators and inhibitors on human primary bronchial smooth muscle cells and primary lung fibroblasts. DISCUSSION: The impedance-based contraction assay can be applied to both basic research and drug discovery for characterizing cellular contraction quantitatively. Because it has high throughput capacity and high reproducibility, the impedance-based contraction assay is useful for high throughput functional screening in drug industry.

Monitoring enzymatic degradation of emerging contaminants using a chip-based robotic nano-ESI-MS tool.

Up to now, knowledge of enzymes capable of degrading various contaminants of emerging concern (CEC) is limited, which is especially due to the lack of rapid screening methods. Thus, a miniaturized high-throughput setup using a chip-based robotic nanoelectrospray ionization system coupled to mass spectrometry has been developed to rapidly screen enzymatic reactions with environmentally relevant CECs. Three laccases, two tyrosinases, and two peroxidases were studied for their ability to transform ten pharmaceuticals and benzotriazole. Acetaminophen was most susceptible to enzymatic conversion by horseradish peroxidase (HRP), laccase from Trametes versicolor (LccTV), and a tyrosinase from Agaricus bisporus (TyrAB). Diclofenac and mefenamic acid were converted by HRP and LccTV, whereas sotalol was solely amenable to HRP conversion. Benzotriazole, carbamazepine, gabapentin, metoprolol, primidone, sulfamethoxazole, and venlafaxine remained persistent in this study. The results obtained here emphasize that enzymes are highly selective catalysts and more effort is required in the use of fast monitoring technologies to find suitable enzyme systems. Despite the methodological limitations discussed in detail, the automated tool provides a routine on-line screening of various enzymatic reactions to identify potential enzymes that degrade CECs. Graphical abstract A chip-based robotic nano-ESI-MS tool to rapidly monitor enzymatic degradation of environmentally relevant emerging contaminants.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics.

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-µLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by µLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the µLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor.

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.

Development of a method for efficient cost-effective screening of Aspergillus niger mutants having increased production of glucoamylase.

OBJECTIVES: To develop an efficient cost-effective screening process to improve production of glucoamylase in Aspergillus niger. RESULTS: The cultivation of A. niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates, which increased the throughput of the samples compared to traditional flask cultivation. There was a close negative correlation between glucoamylase and its pH of the fermentation broth. A novel high-throughput analysis method using Methyl Orange was developed. When compared to the conventional analysis method using 4-nitrophenyl α-D-glucopyranoside as substrate, a correlation coefficient of 0.96 by statistical analysis was obtained. CONCLUSION: Using this novel screening method, we acquired a strain with an activity of 2.2 × 103 U ml-1, a 70% higher yield of glucoamylase than its parent strain.

A multichannel smartphone optical biosensor for high-throughput point-of-care diagnostics.

Current reported smartphone spectrometers are only used to monitor or measure one sample at a time. For the first time, we demonstrate a multichannel smartphone spectrometer (MSS) as an optical biosensor that can simultaneously optical sense multiple samples. In this work, we developed a novel method to achieve the multichannel optical spectral sensing with nanometer resolution on a smartphone. A 3D printed cradle held the smartphone integrated with optical components. This optical sensor performed accurate and reliable spectral measurements by optical intensity changes at specific wavelength or optical spectral shifts. A custom smartphone multi-view App was developed to control the optical sensing parameters and to align each sample to the corresponding channel. The captured images were converted to the transmission spectra in the visible wavelength range from 400nm to 700nm with the high resolution of 0.2521nm per pixel. We validated the performance of this MSS via measuring the concentrations of protein and immunoassaying a type of human cancer biomarker. Compared to the standard laboratory instrument, the results sufficiently showed that this MSS can achieve the comparative analysis detection limits, accuracy and sensitivity. We envision that this multichannel smartphone optical biosensor will be useful in high-throughput point-of-care diagnostics with its minimizing size, light weight, low cost and data transmission function.