Abstract
Laboratory Automation and High-Throughput Chemistry
High-Throughput Screening Using Acoustic Droplet Ejection to Combine Protein Crystals and Chemical Libraries on Crystallization Plates at High Density
Teplitsky et al. describe a high-throughput method for screening up to 1728 distinct chemicals with protein crystals on a single microplate. Acoustic droplet ejection (ADE) is used to co-position 2.5 nL of protein, precipitant, and chemicals on a MiTeGen in situ 1 crystallization plate for screening by co-crystallization or soaking. ADE-transferred droplets follow a precise trajectory that allows all components to be transferred through small apertures in the microplate lid. The apertures are large enough for 2.5-nL droplets to pass through them but small enough so that they do not disrupt the internal environment created by the mother liquor.
Using this system, thermolysin and trypsin crystals are efficiently screened for binding to a heavy-metal mini-library. Fluorescence and X-ray diffraction are used to confirm that each chemical in the heavy-metal library is correctly paired with the intended protein crystal. A fragment mini-library is screened to observe two known lysozyme ligands using both co-crystallization and soaking. A similar approach is used to identify multiple, novel thaumatin binding sites for ascorbic acid. This technology pushes toward a faster, automated, and more flexible strategy for high-throughput screening of chemical libraries (such as fragment libraries) using as little as 2.5 nL of each component (Teplitsky, E.; et al. J. Struct Biol.
Current Status and Future Prospects of Point-of-Care Testing around the Globe
In the past half-century, routine central laboratory testing has become increasingly automated and efficient. The majority of clinical chemistry, immunochemistry, and hematology testing is performed using high-throughput instrumentation with sophisticated automation. Microbiology, immunohematology, and molecular diagnostic testing are also becoming increasingly automated. Recent challenges in health care demand new diagnostic solutions worldwide. Point-of-care testing (POCT) offers considerable advantages over central laboratory testing, such as fast and simple specimen handling and simpler sample requirement (no additives and mostly blood from finger stick and urine). No transportation is required, and POCT delivers short turnaround time of approximately 5 to 15 min. In recent years, POCT has gained ground worldwide. In advanced health care systems, POCT may be beneficial if health or cost-benefits can be established. In resource-poor countries, POCT may be the only means of delivering advanced testing for epidemiologically important diseases, such as tuberculosis or human immunodeficiency virus infection (Abel, G. Expert Rev. Mol. Diagn.
Quantification of Serum Apolipoproteins A-I and B-100 in Clinical Samples Using an Automated SISCAPA-MALDI-TOF-MS Workflow
A fully automated workflow developed and validated for simultaneous quantification of the cardiovascular disease risk markers apolipoproteins A-I (apoA-I) and B-100 (apoB-100) in clinical sera is presented in this report by Van Den Broek et al. By coupling stable-isotope standards and capture by antipeptide antibodies (SISCAPA) for enrichment of proteotypic peptides from serum digests to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS detection, the standardized platform enables rapid, liquid chromatography–free quantification at a relatively high throughput of 96 samples in 12 h. The average imprecision in normo- and triglyceridemic serum pools is 3.8% for apoA-I and 4.2% for apoB-100 (four replicates over 5 days). If stored properly, the MALDI target containing enriched apoA-1 and apoB-100 peptides can be reanalyzed without any effect on bias or imprecision for at least 7 days after initial analysis.
Validation of the workflow reveals excellent linearity for daily calibration with external, serum-based calibrators (R2 of 0.984 for apoA-I and 0.976 for apoB-100 as average over 5 days) and absence of matrix effects or interference from triglycerides, protein content, hemolysates, or bilirubins. Quantification of apoA-I in 93 normo- and hypertriglyceridemic clinical sera shows good agreement with immunoturbidimetric analysis (slope = 1.01, R2 = 0.95, mean bias = 4.0%). Measurement of apoB-100 in the same clinical sera using both methods, however, reveals several outliers in SISCAPA-MALDI-TOF-MS measurements, possibly as a result of the lower MALDI-TOF-MS signal intensity (slope = 1.09, R2 = 0.91, mean bias = 2.0%). The combination of analytical performance, rapid cycle time, and automation potential validates the SISCAPA-MALDI-TOF-MS platform as a valuable approach for standardized and high-throughput quantification of apoA-I and apoB-100 in large sample cohorts (Van Den Broek, I.; et al. Methods.
Microfluidics and Microbioreactors
Building Bio-Assays with Magnetic Particles on a Digital Microfluidic Platform
Digital microfluidics (DMF) has emerged as a promising liquid handling technology for a variety of applications, demonstrating great potential in terms of miniaturization and automation. DMF is based on the manipulation of discrete, independently controllable liquid droplets, which make it highly reconfigurable and reprogrammable. One of its most exclusive advantages, compared to microchannel-based microfluidics, is its ability to precisely handle solid nano- and microsized objects, such as magnetic particles.
Magnetic particles have become very popular in the past decade, since their high surface-to-volume ratio and the ability to magnetically separate them from the matrix make them perfectly suitable as a solid support for bioassay development. The potential of magnetic particles in DMF-based bioassays has been demonstrated for various applications.
In this review, Kokali et al. discuss the latest developments of magnetic particle–based DMF bioassays with the aim to present, identify, and analyze the trends in the field. The authors also discuss the state of the art of device integration, current status of commercialization, and issues that still need to be addressed. With this article, the authors intend to stimulate researchers to exploit and unveil the potential of these exciting tools, which will shape the future of modern biochemistry, microbiology, and biomedical diagnostics (Kokali, T. N. Biotechnol.
High-Throughput Screening Approaches and Combinatorial Development of Biomaterials Using Microfluidics
From the first microfluidic devices used for analysis of single metabolic by-products to highly complex multicompartmental co-culture organ-on-chip platforms, efforts of many multidisciplinary teams around the world have been invested in overcoming the limitations of conventional research methods in the biomedical field.
Close spatial and temporal control over fluids and physical parameters, integration of sensors for direct readout, and the possibility to increase throughput of screening through parallelization, multiplexing, and automation are some of the advantages of microfluidic over conventional, 2D tissue culture in vitro systems. Moreover, small volumes and relatively small cell numbers used in experimental setups involving microfluidics can potentially decrease research cost. On the other hand, these small volumes and numbers of cells also mean that many of the conventional molecular biology or biochemistry assays cannot be directly applied to experiments that are performed in microfluidic platforms. Development of different types of assays and evidence that such assays are indeed a suitable alternative to conventional ones is a step that needs to be taken to have microfluidics-based platforms fully adopted in biomedical research.
In this review, rather than providing a comprehensive overview of the literature on microfluidics, the authors aim to discuss developments in the field of microfluidics that can aid advancement of biomedical research, with emphasis on the field of biomaterials. Three important topics are discussed: screening, in particular high-throughput and combinatorial screening; mimicking of a natural microenvironment ranging from 3D hydrogel-based cellular niches to organ-on-chip devices; and production of biomaterials with closely controlled properties. While important technical aspects of various platforms are discussed, the focus is mainly on their applications, including the state of the art, future perspectives, and challenges.
Microfluidics, being a technology characterized by the engineered manipulation of fluids at the submillimeter scale, offers some interesting tools that can advance biomedical research and development. Screening platforms based on microfluidic technologies that allow high-throughput and combinatorial screening may lead to breakthrough discoveries in basic research and also are relevant to clinical applications. This is further strengthened by the fact that reliability of such screens may improve, since microfluidic systems allow close mimicking of physiological conditions. Finally, microfluidic systems are also very promising as microfactories of a new generation of natural or synthetic biomaterials and constructs, with finely controlled properties (Barata, D.; et al. Acta Biomater.
A Versatile Microfluidic Device for Automating Synthetic Biology
New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, bio-based chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field.
Shih et al. present an innovative microfluidic platform for assembling DNA fragments with 10 times lower volumes (compared to current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to those with conventional methods, which require multiple pipetting steps.
For assembling DNA fragments, the authors implement three commonly used DNA assembly protocols on their microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). Shih et al. demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. The authors anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process (Shih, S. C.; et al. ACS Synth. Biol.
Evaluation of Pre-Induction Temperature, Cell Growth at Induction and IPTG Concentration on the Expression of a Leptospiral Protein in E. coli Using Shaking Flasks and Microbioreactor
Leptospirosis is a zoonose that is increasingly endemic in built-up areas, especially where there are communities living in precarious housing with poor or nonexistent sanitation infrastructure. Leptospirosis can kill because its symptoms are easily confused with those of other diseases. As such, a rapid diagnosis is required so it can be treated effectively. A test for leptospirosis diagnosis using Leptospira immunoglobulin-like (Lig) proteins is currently at final validation at Fiocruz.
In this work, the process for expression of LigB (131–645aa) in Escherichia coli BL21 (DE3)Star/pAE is evaluated. No significant difference is found for the experiments at two different preinduction temperatures (28 °C and 37 °C). Then, the strain is cultivated at 37 °C until IPTG addition, followed by induction at 28 °C, thereby reducing the overall process time. Under this condition, expression is assessed using a central composite design for two variables: cell growth at which LigB (131–645aa) is induced (absorbance at 600 nm between 0.75 and 2.0) and inducer concentration (0.1–1 mM IPTG). Both variables influence cell growth and protein expression. Induction at the final exponential growth phase in shaking flasks with Abs(ind) = 2.0 yielded higher cell concentrations and LigB (131–645aa) productivities. IPTG concentration has a negative effect and could be 10-fold lower than the concentration commonly used in molecular biology (1 mM), while keeping expression at similar levels and inducing less damage to cell growth. The expression of LigB (131–645aa) is associated with cell growth. The induction at the end of the exponential phase using 0.1 mM IPTG at 28 °C for 4 h is also performed in microbioreactors, reaching higher cell densities and 970 mg/L protein. LigB (131–645aa) is purified by nickel affinity chromatography with 91% homogeneity. It is possible to assess the effects and interactions of the induction variables on the expression of soluble LigB (131–645aa) using an experimental design, with a view to improving process productivity and reducing the production costs of a rapid test for leptospirosis diagnosis (Larentis, A. L. BMC Res. Notes.
Instrument Platforms for Nano Liquid Chromatography
The history of liquid chromatography started more than a century ago, and miniaturization and automation are two leading trends in this field. Nanocolumn liquid chromatography (nano LC) and largely synonymous capillary liquid chromatography (capillary LC) are the most recent results of this process where miniaturization of column dimensions and sorbent particle size play crucial role. Very interesting results achieved in the research of extremely miniaturized LC columns at the end of the last century lacked distinctive raison d’être and only advances in mass spectrometry brought a real breakthrough.
Configuration of nano LC-electrospray ionization mass spectrometry (LC-ESI-MS) has become a basic tool in bioanalytical chemistry, especially in proteomics. This review discusses and summarizes past and current trends in the realization of nano LC platforms. Special attention is given to the mobile phase delivery under nanoflow rates (isocratic, gradient) and sample injection to the nanocolumn. Available detection techniques applied in nano LC separations are also briefly discussed. The authors follow up the key themes from the original scientific reports over gradual improvements to the contemporary commercial solutions (Sestak, J.; et al. J. Chromatogr. A.
Piezoelectric-Driven Droplet Impact Printing with an Interchangeable Microfluidic Cartridge
Microfluidic impact printing has been recently introduced, using its nature of simple device architecture, low cost, noncontamination, and scalable multiplexability and high throughput. In this article, Li et al. introduce an impact-based droplet printing platform using a simple plug-and-play microfluidic cartridge driven by piezoelectric actuators. Such a customizable printing system allows for ultrafine control of droplet volume from picoliters (∼23 pL) to nanoliters (∼10 nL), a 500-fold variation. The high flexibility of droplet generation can be simply achieved by controlling the magnitude of actuation (e.g., driving voltage) and the waveform shape of actuation pulses, in addition to nozzle size restrictions. Detailed printing characterizations on these parameters are conducted consecutively. A multiplexed impact printing system is prototyped, and it demonstrates it can provide the functions of single-droplet jetting and droplet multiplexing as well as concentration gradient generation. Moreover, a generic biological assay is tested and validated on this printing platform. Therefore, the microfluidic droplet printing system could be of potential value to establish multiplexed microreactors for high-throughput life science applications (Li, B.; et al. Biomicrofluidics,
Automation Systems
Impact of Automation on Mass Spectrometry
Mass spectrometry coupled to liquid chromatography (LC-MS and LC-MS/MS) is an analytical technique that has rapidly grown in popularity in clinical practice. In contrast to traditional technology, mass spectrometry is superior in many respects, including resolution, specificity, and multiplex capability, and has the ability to measure analytes in various matrices. Despite these advantages, LC-MS/MS remains high cost and labor intensive, and it has limited throughput. This specialized technology requires highly trained personnel and therefore has largely been limited to large institutions, academic organizations, and reference laboratories. Advances in automation will be paramount to break through this bottleneck and increase its appeal for routine use.
This article reviews these challenges, shares perspectives on essential features for LC-MS/MS total automation, and proposes a stepwise and incremental approach to achieve total automation through reducing human intervention, increasing throughput, and eventually integrating the LC-MS/MS system into the automated clinical laboratory operations (Zhang, Y. V.; Rockwood, A. Clin. Chim. Acta.
Mass Spectrometric Sampling of a Liquid Surface by Nanoliter Droplet Generation from Bursting Bubbles and Focused Acoustic Pulses: Application to Studies of Interfacial Chemistry
The complex chemistry occurring at the interface between liquid and vapor phases contributes significantly to the dynamics and evolution of numerous chemical systems of interest, ranging from damage to the human lung surfactant layer to the aging of atmospheric aerosols.
This work presents two methods to eject droplets from a liquid water surface and analyze them via mass spectrometry. In bursting bubble ionization (BBI), droplet ejection is achieved via the formation of a jet following bubble rupture at the surface of a liquid to yield 250-µm diameter droplets (10 nL volume). In interfacial sampling by an acoustic transducer (ISAT), droplets are produced by focusing pulsed piezoelectric transducer-generated acoustic waves at the surface of a liquid, resulting in the ejection of droplets of 100 µm in diameter (500 pL volume).
In both experimental methods, ejected droplets are aspirated into the inlet of the mass spectrometer, resulting in the facile formation of gas-phase ions. Thomas et al. demonstrate the ability of this technique to readily generate spectra of surface-active analytes, and they compare the spectra to those obtained by electrospray ionization. Charge measurements indicate that the ejected droplets are near neutral (
Finally, the authors present the oxidation of oleic acid by ozone as an initial demonstration of the ability of ISAT-MS to monitor heterogeneous chemistry occurring at a planar water-air interface (Thomas D. A.; et al. Anal. Chem.
Advances in Clinical Diagnostics
A Review of the Current State of Digital Plate Reading of Cultures in Clinical Microbiology
Digital plate reading (DPR) is increasingly being adopted as a means to facilitate the analysis and improve the quality and efficiency within the clinical microbiology laboratory. This review discusses the role of DPR in the context of total laboratory automation and explores some of the platforms currently available or in development for digital image capturing of microbial growth on media. The review focuses on the advantages and challenges of DPR. Peer-reviewed studies describing the utility and quality of these novel DPR systems are largely lacking, and professional guidelines for DPR implementation and quality management are needed. Further development and more widespread adoption of DPR are anticipated (Rhoads, D. D.; et al. J. Pathol. Inform.
Automation, Consolidation, and Integration in Autoimmune Diagnostics
Over the past two decades, we have witnessed an extraordinary change in autoimmune diagnostics, characterized by the progressive evolution of analytical technologies, the availability of new tests, and the explosive growth of molecular biology and proteomics. Aside from these huge improvements, organizational changes have also occurred, bringing about a more modern vision of the autoimmune laboratory. The introduction of automation (for harmonization of testing, reduction of human error, reduction of handling steps, increase of productivity, decrease of turnaround time, improvement of safety), consolidation (combining different analytical technologies or strategies on one instrument or on one group of connected instruments), and integration (linking analytical instruments or group of instruments with pre- and postanalytical devices) opens a new era in immunodiagnostics.
In this article, Tozzoli et al. review the most important changes that have occurred in autoimmune diagnostics and present some models related to the introduction of automation in the autoimmunology laboratory, such as automated indirect immunofluorescence and changes in the two-step strategy for detection of autoantibodies; automated monoplex immunoassays and reduction of turnaround time; and automated multiplex immunoassays for autoantibody profiling (Tozzoli, R.; et al. Auto. Immun. Highlights.
Automated Non-Stepwise Preparation of Bioanalytical Calibration Standards and Quality Controls Using an Ultra-Low Volume Digitizing Liquid Dispenser
Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. In a report by Liao et al., an alternative noncontact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of the calibration curve and QCs in bioanalysis.
Fluorescein is initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small-molecule compound, in neat solution and in dog plasma are prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples are extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 are analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions.
In the 3-day precision and accuracy assessment of dispensing volumes between 13 pL and 411.2 nL, the intraday precision and accuracy range from 1.4% to 10.3% and −12.7% to 12.8%, respectively. The interday precision and accuracy range from 3.5% to 7.8% and −6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed show a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters are physiologically comparable as well.
Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved, and tests for bioanalytical standards and QC preparations pass the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser is found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices (Liao, D.; et al. Rapid Commun Mass Spectrom.
The Evolution of MALDI-TOF Mass Spectrometry toward Ultra-High-Throughput Screening: 1536-Well Format and Beyond
Mass spectrometry (MS) offers a label-free, direct-detection method, in contrast to fluorescent or colorimetric methodologies. Over recent years, solid-phase extraction-based techniques, such as the Agilent (Santa Clara, CA) RapidFire system, have emerged and are capable of analyzing samples in
In this report, Haslam et al. describe the development and validation of assays for both small-molecule and peptide analytes using MALDI-TOF coupled with nanoliter liquid handling. Using the JMJD2c histone demethylase and acetylcholinesterase as model systems, the authors generate robust data in a 153-well format and also increase sample deposition to 6144 samples per target. Using these methods, the authors demonstrate that this technology can deliver fast sample analysis time with low sample volume and data comparable to that of current RapidFire assays (Haslam, C.; et al. J. Biomol. Screen.