Abstract
Laboratory Automation and High-Throughput Chemistry
Online Solid Phase Extraction and Liquid Chromatography–Mass Spectrometric Determination of Nucleoside Drugs in Plasma
The bioanalysis and especially the sample preparation of nucleoside drugs in complex media, such as human plasma, has been challenging due to the high polarity and high solubility of these drugs in water. Online solid phase extraction (SPE) offers significant advantages, such as automation and time saving. Thus, several types of SPE columns have been developed for compounds with different polarities.
In this study, SPE is applied to overcome the issue of sample pretreatment of nucleoside drugs in human plasma with the final aim of establishing a robust analytical platform for drugs with similar structures. A simple, easy-to-use, and efficient method is described for the simultaneous determination of lamivudine, zidovudine, didanosine, and emtricitabine in human plasma via online SPE and high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS).
Following a simple centrifugation step, a 10 μL plasma sample is injected directly onto the HPLC system. A Waters Oasis MCX cartridge is washed, and the analytes are removed by back-flushing directly onto the analytical column. The analytes are quantified using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Similarly, with the development and application of a Bond Elut phenylboronic acid (PBA) SPE cartridge, a fully automated online SPE-HPLC-MS/MS method is established for the simultaneous determination of ribavirin and taribavirin in human plasma. Linear calibration curves are obtained over the range of 0.5–2000 ng mL−1, and the limit of quantification ranged from 0.5 to 10 ng mL−1, which is sensitive enough for clinical drug monitoring. The intra- and interday precisions are in the range of 0.2%−8.9%, and the trueness ranges between 88.9% and 113.1%. Excellent recoveries from plasma are achieved with a range between 86.7% and 105.1%.
This procedure is easier to perform and requires less sample handling than methods previously described in the literature. This high-throughput method involving the direct injection of plasma samples may provide a practical solution for the analysis of multiple nucleoside drugs in clinical research. The method is tested in plasma samples from some patients and showed good performance. (Wang, M.; et al. Talanta
High-Throughput DNA Extraction of Forensic Adhesive Tapes
Tape-lifting has since its introduction in the early 2000s become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward, while direct lysis DNA extraction is more challenging due to the “stickiness,” rigidity, and size of the tape.
Forsberg et al. have developed, validated, and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labor. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provides higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences are not significant when using clothes worn in a controlled fashion as reference material (p = 0.13 and p = 0.34 for T-shirts and button-down shirts, respectively).
Through in-house validation, the authors show that the method is fit for purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01 ng/μL increases from 71% to 76%. Apart from providing higher DNA yields than the previous method, the introduction of the developed direct lysis protocol also reduces the amount of manual labor by half and doubles the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as cost-effective alternatives to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. (Forsberg, C.; et al. Forensic Sci. Int. Genet.
Feedback in Flow for Accelerated Reaction Development
The pharmaceutical industry is investing in continuous flow and high-throughput experimentation as tools for rapid process development and accelerated scale-up. Coupled with automation, these technologies offer the potential for comprehensive reaction characterization and optimization, but with the cost of conducting exhaustive multifactor screens. Automated feedback in flow offers researchers an alternative strategy for efficient characterization of reactions based on the use of continuous technology to control chemical reaction conditions and optimize in lieu of screening. Optimization with feedback allows experiments to be conducted where the most information can be gained from the chemistry, enabling product yields to be maximized and kinetic models to be generated while the total number of experiments is minimized.
This report opens by reviewing select examples of feedback optimization in flow and applications to chemical research. Systems in the literature are classified as (1) deterministic “black box” optimization systems that do not model the reaction system and are therefore limited in the utility of results for scale-up, (2) deterministic model-based optimization systems from which reaction kinetics and/or mechanisms can be automatically evaluated, and (3) stochastic systems. Although diverse in application, flow feedback systems have predominantly focused on the optimization of continuous variables, such as time, temperature, and concentration, that can be ramped from one experiment to the next. Unfortunately, this implies that the screening of discrete variables, such as catalyst, ligand, or solvents, generally does not factor into automated flow optimization, resulting in incomplete process knowledge.
Reizman and Jensen present a system and strategy for optimizing discrete and continuous variables of a chemical reaction simultaneously. The approach couples automated feedback with high-throughput reaction screening in droplet flow microfluidics. This report details the system configuration for on-demand creation of sub-20 μL droplets with interchangeable reagents and catalysts. These droplets are reacted in a fully automated microfluidic system and analyzed online by liquid chromatography–mass spectrometry. Feeding back from the online analytical results, a design of experiments–based adaptive response surface algorithm is employed that deductively removes candidate reagents from the optimization as optimal reaction conditions are refined, leading to rapid convergence. Using the automated optimization platform, case studies are presented for solvent selection in a competitive alkylation chemistry and for catalyst–ligand selection in heteroaromatic Suzuki–Miyaura cross-coupling chemistries.
For the monoalkylation of trans-1,2-diaminocyclohexane, polar aprotic solvents at moderate temperatures are shown to be favorable, with optimality accurately identified with DMSO as the solvent in 67 experiments. For Suzuki–Miyaura cross-couplings, the optimality of precatalysts and continuous variable conditions are observed to change in accordance with the coupling reagents, providing insights into catalyst behavior in the context of the reaction mechanism. Future opportunities in automated reaction development include the incorporation of chemoinformatics for faster analysis and machine learning algorithms to guide and optimize the synthesis.
Adoption of this technology stands to reduce graduate student and postdoc time on routine tasks in the laboratory, while feeding back knowledge used to guide new research directions. Moreover, the application of this technology in industry promises to lessen the cost and time associated with advancing pharmaceutical molecules through development and scale-up. (Reizman, B. J.; Jensen, K. F. Acc. Chem. Res.
A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity
Aldehyde dehydrogenase (ALDH) enzymes have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization, and structure–activity relationship (SAR) studies of small-molecule ALDH1A1 inhibitors with cellular activity, Yasgar et al. show the miniaturization to a 1536-well format and automation of a high-content cell-based ALDEFLUOR assay.
The authors demonstrate the utility of this assay by generating dose–response curves on a comprehensive set of prior art inhibitors, as well as hundreds of ALDH1A1 inhibitors synthesized in-house. Finally, the authors establish a screening paradigm using a pair of cell lines with low and high ALDH1A1 expressions, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of more than 1000 small molecules. (Yasgar, A.; et al. PLoS One
Microfluidics and Microbioreactors
Microfluidic Tools toward Industrial Biotechnology
Microfluidics is a technology that operates with small amounts of fluids and makes possible the investigation of cells, enzymes, and biomolecules, and the encapsulation of biocatalysts in a greater variety of conditions than permitted using conventional methods. This review discusses technological possibilities that can be applied in the field of industrial biotechnology, presenting the principal definitions and fundamental aspects of microfluidic parameters to better understand advanced approaches. Specifically, concentration gradient generators, droplet-based microfluidics, and microbioreactors are explored as useful tools that can contribute to industrial biotechnology. These tools present potential applications, inclusive as commercial platforms for optimization in bioprocess development as screening cells and encapsulating biocatalysts, and for determining critical kinetic parameters. (Oliveira, A. F.; et al. Biotechnol. Prog.
Hydrogel Droplet Microfluidics for High-Throughput Single-Molecule/Cell Analysis
Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single-molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single-molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways.
Various powerful techniques have been developed for single-molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, and single-molecule fluorescence spectroscopy. However, some of them require analysis of single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single-cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single-molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversifies applications in single-molecule/cell analysis.
The hydrogel can act as a three-dimensional cell culture matrix to mimic the extracellular environment for long-term single-cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype–genotype linkages in the hydrogel matrix for high-throughput molecular evolution.
In this report, Zhu and Yang review the hydrogel droplet generation on microfluidics, single-molecule/cell encapsulation in hydrogel droplets, and the progress made by their group and others in the application of hydrogel droplet microfluidics for single-molecule/cell analysis, including single-cell culture, single-molecule/cell detection, single-cell sequencing, and molecular evolution. (Zhu, Z.; Yang, C. J. Acc. Chem. Res.
Molecular Profiling of Single Circulating Tumor Cells from Lung Cancer Patients
Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic.
Park et al. report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, the authors demonstrate multigene expression profiling of individual CTCs from non-small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, the authors report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring. (Park, S. M.; et al. Proc. Natl. Acad. Sci. U.S.A.
In Vitro Microfluidic Models of Tumor Microenvironment to Screen Transport of Drugs and Nanoparticles
Advances in nanotechnology have enabled numerous types of nanoparticles (NPs) to improve drug delivery to tumors. While many NP systems have been proposed, their clinical translation has been less than anticipated primarily due to failure of current preclinical evaluation techniques to adequately model the complex interactions between the NP and physiological barriers of the tumor microenvironment.
This review focuses on microfluidic tumor models for characterization of delivery efficacy and toxicity of cancer nanomedicine. Microfluidics offer significant advantages over traditional macroscale cell cultures by enabling recapitulation of tumor microenvironments through precise control of physiological cues, such as hydrostatic pressure, shear stress, oxygen, and nutrient gradients. Microfluidic systems have recently started to be adapted for screening of drugs and NPs under physiologically relevant settings. So far, the two primary applications of microfluidics in this area have been high-throughput screening using traditional culture settings, such as single cells or multicellular tumor spheroids, and mimicry of the tumor microenvironment for study of cancer-related cell–cell and cell–matrix interactions. These microfluidic technologies are also useful in modeling specific steps in NP delivery to the tumor and characterize NP transport properties and outcomes by systematic variation of physiological conditions. Ultimately, it will be possible to design drug screening platforms uniquely tailored for individual patient physiology using microfluidics. These in vitro models can contribute to the development of precision medicine by enabling rapid and patient-specific evaluation of cancer nanomedicine. (Ozcelikkale, A.; et al. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. Epub ahead of print. 2017 Feb 14. doi: 10.1002/wnan.1460)
Analytical Chemistry
Multianalyte Profiling of Per- and Polyfluoroalkyl Substances in Liquid Commercial Products
The chemical properties of poly- and perfluoroalkyl substances (PFASs) make them widespread for use in a number of industrial and commercial products to confer water and oil repellency characteristics and to reduce surface tension, for example, in aqueous film-forming foams (AFFFs). Some PFASs, especially perfluoroctane sulfonate, and several perfluoroalkyl carboxylic acids, are known to cause significant human and environmental negative impact. Knowledge of the content of PFASs in products remains scarce, impeding any precise assessment of human exposure and environmental release upon use.
This study aims to analyze a wide variety of liquid products (n = 194) likely to contain PFASs, including impregnating agents, lubricants, cleansers, polishes, AFFFs, and other industrial products. By means of liquid chromatography (LC)– and gas chromatography–tandem mass spectrometry (GC-MS/MS) analytical techniques, 24 PFASs (from 41 targeted PFASs) are detected and quantified in 55% of samples. PFAS quantification and profiling is found to be consumer product specific. PFASs are mostly detected in AFFF (90%) and impregnating agents (60%) with mainly ionic and neutral species, respectively. In particular, the 6:2, 8:2, and 10:2 fluorotelomer alcohols are detected in 40%–50% of impregnating agents.
Further investigation by fast atom bombardment MS (FAB-MS) on a set of AFFF samples allows the characterization of eight different PFAS classes as major components in these formulations. Results demonstrate that numerous and diversified PFASs are currently used in specific commercial products, implying significant human exposure and environmental release that necessitate further research concerning their toxicological impact. (Favreau, P.; et al. Chemosphere
Proteomics Analysis of O-GalNAc Glycosylation in Human Serum by an Integrated Strategy
The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods.
In this study, an integrated strategy is developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision-induced decomposition (beam-CID) detection, and in silico deglycosylation for spectra interpretation. The intact O-GalNAc glycopeptides are selectively enriched, and the original spectra obtained by time of flight–CID are preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease search space. This strategy is applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contain at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation.
Up to now, this is the largest dataset of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems. (Qin, H.; et al. Anal. Chem.
Advances in Emerging Technologies
Altered Gut Microbiome Composition and Tryptic Activity of the 5xFAD Alzheimer’s Mouse Model
The regulation of physiological gut functions such as peristalsis or secretion of digestive enzymes by the central nervous system via the Nervus vagus is well known. Recent investigations highlight that pathological conditions of neurological or psychiatric disorders might directly interfere with the autonomous neuronal network of the gut, the enteric nervous system, or even derive from there.
Using a murine Alzheimer’s disease model, Brandscheid et al. investigate a potential influence of disease-associated changes on gastrointestinal properties. 5xFAD mice at three different ages are compared to wild-type littermates in regard to metabolic parameters and enzymes of the gut by fluorimetric enzyme assay and Western blotting. Overexpression of human amyloid-β protein precursor (AβPP) within the gut is assessed by qPCR and immunohistochemistry; fecal microbiome analysis is conducted by 16S rRNA quantitation of selected phyla and species. While the general composition of fecal samples, locomotion, and food consumption of male 5xFAD animals are not changed, the authors observed reduced body weights at early pathological stages. Human AβPP is expressed not only within the brain of these mice but also in gut tissue. Analysis of fecal proteins reveals a reduced trypsin amount in the 5xFAD model mice compared with the wild type. In addition, the authors observe changes in fecal microbiota composition along with age.
Brandscheid et al. therefore suggest that the presence of the mutated transgenes (AβPP and PS1), which are per se the basis for the genetic form of Alzheimer’s disease in humans, directly interferes with gut function, as shown here for the disease model mice. (Brandscheid, C.; et al. J. Alzheimers Dis.
Pooled CRISPR Screening with Single-Cell Transcriptome Readout
CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts, including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts, such as transcriptome profiling, but at much lower throughput.
Datlinger et al. combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells. Their method for CRISPR droplet sequencing (CROP-seq) enables pooled CRISPR screens with single-cell transcriptome resolution, which facilitates high-throughput functional dissection of complex regulatory mechanisms and heterogeneous cell populations. (Datlinger, P.; et al. Nat. Methods
Automated Microraft Platform to Identify and Collect Nonadherent Cells Successfully Gene Edited with CRISPR-Cas9
Microraft arrays have been used to screen and then isolate adherent and nonadherent cells with very high efficiency and excellent viability, but manual screening and isolation limit the throughput and utility of the technology. In this report, novel hardware and software automate the microraft array platform.
The analysis software identifies microrafts on the array with greater than 99% sensitivity and cells on the microrafts with 100% sensitivity. The software enables time-lapse imaging and the use of temporally varying characteristics as sort criteria. The automated hardware releases microrafts with 98% efficiency and collects released microrafts with 100% efficiency. The automated system is used to examine the temporal variation in enhanced green fluorescent protein (EGFP) expression in cells transfected with CRISPR-Cas9 components for gene editing.
Of 11,499 microrafts possessing a single cell, 220 microrafts are identified as possessing temporally varying EGFP expression. Candidate cells (n = 172) are released and collected from the microraft array and screened for the targeted gene mutation. Two cell colonies are successfully gene edited, demonstrating the desired mutation. (Attayek, P. J.; et al. Biosens. Bioelectron.