Liquid Handling Research Articles

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

Highly scalable and standardized organ-on-chip platform with TEER for biological barrier modeling

ABSTRACT The development of new therapies is hampered by the lack of predictive, and patient-relevant in vitro models. Organ-on-chip (OOC) technologies can potentially recreate physiological features and hold great promise for tissue and disease modeling. However, the non-standardized design of these chips and perfusion control systems has been a barrier to quantitative high-throughput screening (HTS). Here we present a scalable OOC microfluidic platform for applied kinetic in vitro assays (AKITA) that is applicable for high, medium, and low throughput. Its standard 96-well plate and 384-well plate layouts ensure compatibility with existing laboratory workflows and high-throughput data collection and analysis tools. The AKITA plate is optimized for the modeling of vascularized biological barriers, primarily the blood-brain barrier, skin, and lung, with precise flow control on a custom rocker. The integration of trans-epithelial electrical resistance (TEER) sensors allows rapid and repeated monitoring of barrier integrity over long time periods. Together with automated liquid handling and compound permeability testing analyses, we demonstrate the flexibility of the AKITA platform for establishing human-relevant models for preclinical drug and precision medicine’s efficacy, toxicity, and permeability under near-physiological conditions.

Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

A Novel, Fully Automated, and Reagent-Agnostic Transient Transfection Protocol.

Transfection is a potent technique to introduce foreign nucleic acids into eukaryotic cells. The capacity of the technique to alter the genetic content of host cells means it is useful for a wide range of applications, including the study of typical cellular processes, disease molecular mechanisms, and gene therapy effects. Here, we discuss a highly reliable and fully automated transient transfection protocol that utilizes an open-source liquid handler and accompanying HEPA Module. Two commonly used transfection reagents are employed to study the transfection efficiency in two cell lines with a GFP plasmid construct. The detailed method of the protocol, image acquisition, and analysis for evaluating transfection efficacy is provided. With HeLa cells, the transfection efficiency of the reagents ranges from 40.92% to 73.26%, while with the difficult-to-transfect A549 cells, the transfection efficiency is between 42.15% and 54%. The efficiency achieved is comparable to similar experiments performed manually. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Seeding of adherent cells (A549 and HeLa) for transient transfection on a Costar 6-well plate using a liquid handler on Day 0 Basic Protocol 2: Transfection of the cell lines using the transfection reagents Lipofectamine 3000 and FuGENE HD on Day 1 Support Protocol: Image acquisition and semi-quantitative analysis of transfection after 24 hr to calculate the transfection efficiency.

A Modular Robotic Platform for Biological Research: Cell Culture Automation and Remote Experimentation

Robotic arms are now commonplace in diverse settings and are poised to play a crucial role in automating laboratory tasks. However, biological experiments remain challenging for automation due to their dependence on human factors, such as researchers’ skills and experience. This article introduces robotic automation and remote control for both general and biological research tasks through a modularized platform comprising a robotic arm, auxiliary tools, and software. This platform facilitates fully automated or remote execution of key experiments in chemistry and biology, including liquid handling, mixing, cell seeding, culturing, and genetic manipulation. The robot interfaces seamlessly with standard laboratory equipment and operates remotely in real time through an online program. Integration of a vision system via robotic arm webcams ensures precise positioning and object localization, enhancing accuracy. This modularized robotic platform signifies a substantial advancement in lab automation, promising enhanced efficiency, reproducibility, and scientific progress compared to human‐led experiments.

Screening and confirmation of psilocin, mitragynine, phencyclidine, ketamine and ketamine metabolites by liquid chromatography-tandem mass spectrometry.

A safe and productive workplace requires a sober workforce, free from substances that impair judgment and concentration. Although drug monitoring programs already exist, the scope and loopholes of standard workplace testing panels are well known, allowing other substances to remain a source of risk. Therefore, a high-throughput urine screening method for psilocin, mitragynine, phencyclidine, ketamine, norketamine and dehydronorketamine was developed and validated in conjunction with a urine and blood confirmation method. There are analytical challenges to overcome with psilocin and mitragynine, particularly when it comes to drug stability and unambiguous identification in authentic specimens. Screening and confirmation methods were validated according to the American National Standards Institute/Academy Standards Board (ANSI/ASB) Standard 036, Standard Practices for Method Validation in Forensic Toxicology. An automated liquid handling system equipped with dispersive pipette extraction tips was utilized for preparing screening samples, whereas an offline solid-phase extraction method was used for confirmation sample preparation. Both methods utilized liquid chromatography-tandem mass spectrometry to achieve limits of detection between 1-5 ng/mL for the screening method and 1 ng/mL for the confirmation method. Automation allows for faster throughput and enhanced quality assurance, which improves turnaround time. Compared to previous in-house methods, specimen volumes were substantially decreased for both blood and urine, which is an advantage when volume is limited. This screening technique is well suited for evaluating large numbers of specimens from those employed in safety-sensitive workforce positions. This method can be utilized by workplace drug testing, human performance and postmortem laboratories seeking robust qualitative screening and confirmation methods for analytes that have traditionally been challenging to routinely analyze.

Automated self-optimization, intensification, and scale-up of photocatalysis in flow.

The optimization, intensification, and scale-up of photochemical processes constitute a particular challenge in a manufacturing environment geared primarily toward thermal chemistry. In this work, we present a versatile flow-based robotic platform to address these challenges through the integration of readily available hardware and custom software. Our open-source platform combines a liquid handler, syringe pumps, a tunable continuous-flow photoreactor, inexpensive Internet of Things devices, and an in-line benchtop nuclear magnetic resonance spectrometer to enable automated, data-rich optimization with a closed-loop Bayesian optimization strategy. A user-friendly graphical interface allows chemists without programming or machine learning expertise to easily monitor, analyze, and improve photocatalytic reactions with respect to both continuous and discrete variables. The system's effectiveness was demonstrated by increasing overall reaction yields and improving space-time yields compared with those of previously reported processes.

Enzyme-Mediated Exponential Glucose Release: A Model-Based Strategy for Continuous Defined Fed-Batch in Small-Scale Cultivations.

Miniaturized cultivation systems offer the potential to enhance experimental throughput in bioprocess development. However, they usually lack the miniaturized pumps necessary for fed-batch mode, which is commonly employed in industrial bioprocesses. An alternative are enzyme-mediated glucose release systems from starch-derived polymers, facilitating continuous glucose supply. Nevertheless, while the glucose release, and thus the feed rate, is controlled by the enzyme concentration, it also strongly depends on the type of starch derivative, and the culture conditions as well as pH and temperature. So far it was not possible to implement controlled feeding strategies (e.g., exponential feeding). In this context, we propose a model-based approach to achieve precise control over enzyme-mediated glucose release in cultivations. To this aim, an existing mathematical model was integrated into a computational framework to calculate setpoints for enzyme additions. We demonstrate the ability of the tool to maintain different pre-defined exponential growth rates during Escherichia coli cultivations in parallel mini-bioreactors integrated into a robotic facility. Although in this case study, the intermittent additions of enzyme and dextrin were performed by a liquid handler, the approach is adaptable to manual applications. Thus, we present a straightforward and robust approach for implementing defined continuous fed-batch processes in small-scale systems, where continuous feeding was only possible with low accuracy or high technical efforts until now.

An LC-MS/MS method for serum cystatin C quantification and its comparison with two commercial immunoassays.

The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking. The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated. No significant matrix effect or interference wasobserved with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method. The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.

Reaping the benefits of liquid handlers for high-throughput gene expression profiling in a marine model invertebrate

BackgroundModern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures.ResultsHere, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing.ConclusionsThis work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.

Optimization of a Hydrogen Exchange-Mass Spectrometry Robotic Liquid Handler Using Tracers.

Hydrogen exchange-mass spectrometry (HX-MS) is a valuable analytical technique that can provide insight into protein interactions and structure. The deuterium labeling necessary to gain this insight is affected by many physical and chemical factors, making it challenging to achieve high reproducibility. Poor precision during dispensing, transfer, and mixing of solutions during the experiment contributes substantially to the overall variability. While the use of a robotic liquid handler can potentially improve precision, its operation must be optimized. We observed poor precision in data collected using a robotic liquid handler to perform HX-MS. In this work, we describe how we were able to improve that system's precision considerably based on tracking performance using caffeine, caffeine-d3, and caffeine-d9 as tracers for the sample, label, and quench to report on each operation of the liquid handling workflow. The insights gained about liquid handler performance and the three-tracer approach can aid in optimizing HX-MS workflow operations, whether performed manually or when using a liquid handling system. Additionally, these tracers can be incorporated as internal tracers during an experiment to report on the labeling and quench operations of each sample throughout the run and, if desired, be used to implement an uptake correction described previously.

Robotics-Driven Manufacturing of Cartilaginous Microtissues for Skeletal Tissue Engineering Applications.

Automated technologies are attractive for enhancing the robust manufacturing of tissue-engineered products for clinical translation. In this work, we present an automation strategy using a robotics platform for media changes, and imaging of cartilaginous microtissues cultured in static microwell platforms. We use an automated image analysis pipeline to extract microtissue displacements and morphological features as noninvasive quality attributes. As a result, empty microwells were identified with a 96% accuracy, and dice coefficient of 0.84 for segmentation. Design of experiment are used for the optimization of liquid handling parameters to minimize empty microwells during long-term differentiation protocols. We found no significant effect of aspiration or dispension speeds at and beyond manual speed. Instead, repeated media changes and time in culture were the driving force or microtissue displacements. As the ovine model is the preclinical model of choice for large skeletal defects, we used ovine periosteum-derived cells to form cartilage-intermediate microtissues. Increased expression of COL2A1 confirms chondrogenic differentiation and RUNX2 shows no osteogenic specification. Histological analysis shows an increased secretion of cartilaginous extracellular matrix and glycosaminoglycans in larger microtissues. Furthermore, microtissue-based implants are capable of forming mineralized tissues and bone after 4 weeks of ectopic implantation in nude mice. We demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous microtissues and anticipate the progressive substitution of manual operations with automated solutions for the manufacturing of microtissue-based living implants.

Thermal segment microwell plate control for automated liquid handling setups.

Automated high-throughput liquid handling operations in biolabs necessitate miniaturised and automatised equipment for effective space utilisation and system integration. This paper presents a thermal segment microwell plate control unit designed for enhanced microwell-based experimentation in liquid handling setups. The development of this device stems from the need to move towards geometry standardization and system integration of automated lab equipment. It incorporates features based on Smart Sensor and Sensor 4.0 concepts. An enzymatic activity assay is implemented with the developed device on a liquid handling station, allowing fast characterisation via a high-throughput approach. The device outperforms other comparable devices in certain metrics based on automated liquid handling requirements and addresses the needs of future biolabs in automation, especially in high-throughput screening.

Optimization of liquid handling parameters for viscous liquid transfers with pipetting robots, a “sticky situation”

Multi-objective optimization of aspiration and dispense rates enables automated liquid handling platforms to accurately transfer viscous liquids.

Vibrational manipulation of dry granular materials in lab-on-a-chip devices.

We present vibrational techniques to pump, mix, and separate dry granular materials using multifrequency vibrations applied to a solid substrate with a standard audio system. The direction and velocity of the granular flow are tuned by modulating the sign and amplitude, respectively, of the vibratory waveform, with typical pumping velocities of centimeters per second. Different granular materials are mixed by combining them at Y-shaped junctions, and mixtures of granules with different friction coefficients are separated along straight channels by judicious choice of the vibratory waveform. We demonstrate that the observed velocities accord with a theory valid for sufficiently large or fast vibrations, and we discuss the implications for using vibrational manipulation in conjunction with established microfluidic technologies to combine liquid and dry solid handling operations at sub-millimeter length scales.

A Fast‐Forward Dilute‐and‐Shoot Multielement Method for Analysis of 33 Elements in Human Whole Blood, Serum, and Urine by Inductively Coupled Plasma Mass Spectrometry: A Streamlined Approach for Clinical Diagnostic and Biomonitoring

The analysis of toxic and essential elements in human matrices is used in clinical diagnostics and for biomonitoring of different populations to study related health outcomes. This work aimed to develop fast and reliable methods for the analysis of a broad range of elements in liquid human matrices, such as whole blood, serum, and urine, with a similar setup for the three matrices and different analysis needs. An easy and fast‐forward dilute‐and‐shoot method for 33 elements (i.e., Ag, Al, As, B, Ba, Be, Bi, Cd, Ce, Co, Cr, Cu, Hg, I, Li, Mn, Mo, Ni, Pb, Pd, Pt, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Zn, and Zr) was developed. 200 µL of either sample material was diluted with an alkaline reagent to a volume of 4 mL in total. Sample dilution and preparation of matrix‐matched calibration standards were performed in 48‐well plates by an automated liquid handler. Diluted samples were analyzed by inductively coupled plasma mass spectrometry on a Perkin Elmer NexIon 300D ICP‐MS instrument equipped with an ESI‐FAST SC2DX autosampler in kinetic energy discrimination mode with helium as cell gas at either 4.8 mL or 5.7 mL and 1600 W RF generator power. The method validation results showed good accuracy for fresh human samples from an external quality assessment scheme with measured concentrations within the assigned concentration ranges. Good precision and reproducibility for most elements were demonstrated with variation coefficients below or far below 8% and 15% for whole blood, 8% and 10% for serum, and 10% and 10% for urine, respectively. The developed reagent and instrumental setup were applicable to all three matrices. This minimizes the risk of human errors when switching between analyses of the different sample matrices and allows a rapid and easy analysis of whole blood, serum, and urine within one day if needed. The method demonstrated robustness over time, withstanding minor changes in the preparation of working solutions and samples, instrumental analysis, and setup. Analysis of human real samples showed the method’s applicability for 33 toxic and essential elements in whole blood, serum, and urine and at concentrations relevant to clinical diagnostics as well as biomonitoring.

Supported gel slab scaffolds as a three-dimensional cell-based assay platform.

Cell-based assays are heavily relied on in the drug discovery pipeline, quickly pairing down large compound libraries to a manageable number of drug candidates for further characterization and evaluation. Monolayer cultures in which cells are deposited onto the bottom of well plates are the workhorse of many of these screens despite continued evidence of their inability to predict in vivo responses. Three-dimensional (3D) culture platforms can generate tissue-like environments with more representative cellular phenotypes than monolayers but have proven challenging to incorporate into already-developed workflows. Scaffold-based approaches are a tractable means of generating tissue-like environments, supporting cell-laden gels whose preparation is analogous to depositing cells in a well plate. Here, we describe supported gel slab (SGS) scaffolds prepared from commercially available materials, an adhesive spray, and a laser cutter. These cell-containing scaffolds can readily fit into well plates, providing a format compatible with current liquid handlers and analytical instrumentation. The scaffolds enable the evaluation of cellular responses in individual or stacked structures, which contain extracellular matrix-rich microenvironments. With a series of demonstrations, we highlight the utility of the readily assembled SGS scaffolds to quantify cellular responses. These readouts include confocal microscopy, quantifying cellular invasion in Transwell-like and stacked formats, generating multilayered spheroid-on-demand structures capable of providing spatially resolved maps of drug responses, and identifying potential chemotherapies in a screening application.

Drug Screening Using Normal Cell and Cancer Cell Mixture in an Automated 3D Cell Culture System.

Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.

A high-throughput LC-MS/MS assay for piperaquine from dried blood spots: Improving malaria treatment in resource-limited settings

BackgroundMalaria is a parasitic disease that affects many of the poorest economies, resulting in approximately 241 million clinical episodes and 627,000 deaths annually. Piperaquine, when administered with dihydroartemisinin, is an effective drug against the disease. Drug concentration measurements taken on day 7 after treatment initiation have been shown to be a good predictor of therapeutic success with piperaquine. A simple capillary blood collection technique, where blood is dried onto filter paper, is especially suitable for drug studies in remote areas or resource-limited settings or when taking samples from children, toddlers, and infants. MethodsThree 3.2 mm discs were punched out from a dried blood spot (DBS) and then extracted in a 96-well plate using solid phase extraction on a fully automated liquid handling system. The analysis was performed using LC-MS/MS with a calibration range of 3 – 1000 ng/mL. ResultsThe recovery rate was approximately 54–72 %, and the relative standard deviation was below 9 % for low, middle and high quality control levels. The LC-MS/MS quantification limit of 3 ng/mL is sensitive enough to detect piperaquine for up to 4–8 weeks after drug administration, which is crucial when evaluating recrudescence and drug resistance development. While different hematocrit levels can affect DBS drug measurements, the effect was minimal for piperaquine. ConclusionA sensitive LC-MS/MS method, in combination with fully automated extraction in a 96-well plate format, was developed and validated for the quantification of piperaquine in DBS. The assay was implemented in a bioanalytical laboratory for processing large-scale clinical trial samples.

Pitfalls and Considerations in Determining the Potency and Mutant Selectivity of Covalent Epidermal Growth Factor Receptor Inhibitors.

Enzyme inhibitors that form covalent bonds with their targets are being increasingly pursued in drug development. Assessing their biochemical activity relies on time-dependent assays, which are distinct and more complex compared with methods commonly employed for reversible-binding inhibitors. To provide general guidance to the covalent inhibitor development community, we explored methods and reported kinetic values and experimental factors in determining the biochemical activity of various covalent epidermal growth factor receptor (EGFR) inhibitors. We showcase how liquid handling and assay reagents impact kinetic parameters and potency interpretations, which are critical for structure-kinetic relationships and covalent drug design. Additionally, we include benchmark kinetic values with reference inhibitors, which are imperative, as covalent EGFR inhibitor kinetic values are infrequently consistent in the literature. This overview seeks to inform best practices for developing new covalent inhibitors and highlight appropriate steps to address gaps in knowledge presently limiting assay reliability and reproducibility.