Abstract
Laboratory Automation and High-Throughput Chemistry
Comparison of Two Highly Automated Nucleic Acid Extraction Systems for Quantitation of Human Cytomegalovirus in Whole Blood
In this study, Mengelle et al., from the Federative Institute of Biology, compare and discuss the automated extraction processes for nucleic acids from blood. Studies including such comparisons help users choose the appropriate method for specific research and can use the methods to optimize protocols. The authors evaluate analytical performances of the COBAS Ampliprep and compare extraction from whole blood on the COBAS Ampliprep and on the MagNA Pure instruments (Roche Diagnostics, Mannheim, Germany) for quantifying human cytomegalovirus (HCMV) DNA with real-time PCR. The limit of detection using the COBAS Ampliprep is 10 copies/run (150 copies/mL, i.e., 2.20 log(10) copies/mL). Quantitation of HCMV-DNA is linear from 3.0 to 6.0 log(10) copies/mL. The intra-assay variation ranges from 11.1% to 0.4%, and interassay variation is 11.3%. A total of 107 samples are tested using both extraction systems. Only 3 samples give discrepant results. Correlation between HCMV virus loads is good (r = 0.73; p < 0.001). Mean virus load is lower (–0.49 log(10) copies/mL) with COBAS Ampliprep than with the MagNA Pure extraction system. Both MagNA Pure and COBAS Ampliprep provide reliable and high-throughput platforms for real-time PCR HCMV quantitation of DNA extracted from whole blood. (Mengelle, C., et al. Diagn. Microbiol. Infect. Dis.
From Screen to Structure With a Harvestable Microfluidic Device
Macromolecular crystallization screening has remained a tedious task for several decades. Minor variations in screening conditions will determine the presence or absence of crystals in crystallization trials. In this study, the authors demonstrate that microfluidic crystal former devices identify more crystallization conditions than vapor diffusion methods. These are encouraging results for researchers who fail to obtain crystals in their trials, providing an alternative strategy. In this study from Brookhaven National Laboratories, the authors compare crystal former devices with vapor diffusion methods. Advances in automation have facilitated the widespread adoption of high-throughput vapor diffusion methods for initial crystallization screening. For many proteins, however, screening thousands of crystallization conditions fails to yield crystals of sufficient quality for structural characterization. Here, the rates of crystal identification for thaumatin, catalase, and myoglobin using microfluidic crystal former devices and sitting-drop vapor diffusion plates are compared. It is shown that the crystal former results in a greater number of identified initial crystallization conditions compared with vapor diffusion. Furthermore, crystals of thaumatin and lysozyme obtained in the crystal former are used directly for structure determination both in situ and upon harvesting and cryocooling. On the basis of these results, a crystallization strategy is proposed that uses multiple methods with distinct kinetic trajectories through the protein phase diagram to increase the output of crystallization pipelines. (Stoianoff, V. Acta Crystallogr Sect F. Struct Biol. Cryst. Commun.
Reliable Protein Production in a Pseudomonas Fluorescens Expression System
A bottleneck to product development can be reliable expression of active target protein. A wide array of recombinant proteins in development, including an ever growing number of nonnatural proteins, is being expressed in a variety of expression systems. A pseudomonas fluorescens expression platform has been developed specifically for recombinant protein production. The development of an integrated molecular toolbox of expression elements and host strains, along with automation of strain screening, is described by Retallack. Examples of strain screening and scale-up experiments show rapid development of expression strains, producing a wide variety of proteins in a soluble active form. (Retallack, D. M. Protein Expr. Purif.
Microfluidic Chip Technology and Micro Reactor Technology
Multianalyte On-Chip Native Western Blotting
In this report, Tia et al. introduce and characterize multiplexed native Western blotting in an automated and unified microfluidic format. Although slab gel Western blotting is slow and laborious, conventional multiplexed blotting (“reblotting”: probing one sample with multiple antibodies) requires even more resources. The authors detail three key advances that enable an automated and rapid microfluidic alternative to slab gel reblotting. First, they introduce both assay and microdevice designs that integrate protein blotting against multiple antibody blotting regions with native polyacrylamide gel electrophoresis. This microfluidic integration strategy overcomes nonspecific material losses inherent to harsh antibody stripping steps typically needed for conventional reblotting; said conditions can severely limit analyte quantitation. Second, to inform rational design of the multiplexed microfluidic device, they develop an analytical model for analyte capture on the blotting regions. Comparison to empirical observations is reported with capture efficiencies of >85%. Third, they introduce label-free detection that makes simultaneous and quantitative multiplexed measurements possible without the need for prelabeling of samples. Assay linear dynamic range spans 8 to 800 nM with assay completion in 5 min. Owing to the speed, automation, enhanced quantitation capability, and the difficulty of conventional slab gel Western reblotting, microfluidic multiplexed native Western blotting should find use in systems biology, particularly in analyses of protein isoforms and multimeric protein complexes. (Tia, S. Q., et al. Anal. Chem.
Parallel Single-Cell Analysis Microfluidic Platform
In this study from the Institute of Nanotechnology, University of Twente, the Netherlands, the authors report a PDMS (polydimethyl siloxane) microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or noninvasive analysis schemes are performed. First, they report single-cell trapping in a fast (2–5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol that is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using a nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible (lithium dodecyl sulfate) way. This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electro-osmotic flow (EOF)–based extraction of the cell content. It is validated using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content. (Van Den Brink, F. T., et al. Electrophoresis
Simultaneous Removal and Evaluation of Organic Substrates and NH3-N by a Novel Combined Process in Treating Chemical Synthesis-Based Pharmaceutical Wastewater
Chen et al., from Harbin, China, report a novel process in the treatment of wastewater. A full-scale novel combined anaerobic/microaerobic and two-stage aerobic biological process is used for the treatment of actual chemical synthesis-based pharmaceutical wastewater containing amoxicillin. The anaerobic system is an up-flow anaerobic sludge blanket (UASB); the microaerobic system is a novel microaerobic hydrolysis acidification reactor (NHAR), and the two-stage aerobic process comprises a cyclic activated sludge system (CASS) and biological contact oxidation tank (BCOT). The influent wastewater was high in COD and NH3-N, varying daily from 4016 to 13 093 mg COD L–1 and 156.4 to 650.2 mg NH3-N L–1, with amoxicillin varying weekly between 69.1 and 105.4 mg amoxicillin L–1, respectively. Almost all the COD, NH3-N, and amoxicillin are removed by the biological combined system, with removal percentages of 97%, 93.4%, and 97.2%, respectively, leaving around 104 mg COD L–1, 9.4 mg NH3-N L–1, and 2.6 ± 0.8 mg amoxicillin L–1 in the final clarifier effluent. The performance evaluation of the wastewater treatment plant (WWTP) by mathematical statistic methods shows that most of the time, effluent can meet the higher treatment discharge standard. In addition, the fate of amoxicillin in the full-scale WWTP and the amoxicillin removal rate of each different removal route in UASB, NHAR, CASS, BCOT, and final clarifier processes are investigated in this study. The results show that biodegradation, adsorption, and hydrolysis are the major mechanisms for amoxicillin removal. (Chen, Z., et al. J. Hazard Mater.
Automated Cellular Sample Preparation Using a Centrifuge-on-a-Chip
The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Mach et al. present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allow enrichment of cancer cells from spiked blood samples at the mL min–1 scale, followed by fluorescent labeling of intra- and extracellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge analogue may open opportunities in automated, low-cost, and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource-poor settings. (Mach, A. J., et al. LabChip
High-Throughput Analytics
A Label-Free Methodology for Selective Protein Quantification by Means of Absorption Measurements
Label-free methods have gained wide importance in the past several years. In this study, Hansen et al. from Karlsruhe Institute of Technology, Germany, discuss a label-free method for analyzing protein mixtures and point to the benefits of this application in a commercial production environment. The application of high-throughput experimentation (HTE) in the protein purification process development has created an analytical bottleneck. Using a new label-free and noninvasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements, the authors show that the analytical throughput for selective protein quantification can be increased significantly. An analytical assay based on this new methodology generates very precise results. Furthermore, the assay is successfully applied as analytics for a resin screening performed in HTE mode. The increase in analytical throughput is obtained without decreasing the level of information when compared with analytical chromatography. This proves its potential as a valuable analytical tool in conjugation with high-throughout process development (HTPD). Furthermore, fast selective protein quantification can enhance process control in a commercial production environment and, hence, minimize the need for offline release analysis. (Hansen, S. K., et al. Biotechnol. Bioeng.
IPM: An Integrated Protein Model for False Discovery Rate Estimation and Identification in High-Throughput Proteomics
In high-throughput mass spectrometry proteomics, peptides and proteins are not simply identified as present or not present in a sample; rather, the identifications are associated with differing levels of confidence. The false discovery rate (FDR) has emerged as an accepted means for measuring the confidence associated with identifications. In this study, the authors develop the Systematic Protein Investigative Research Environment (SPIRE) for the purpose of integrating the best available proteomics methods. Two successful approaches to estimating the FDR for mass spectrometry (MS) protein identifications are the MAYU (a strategy with no acronym) and the current SPIRE methods. Higdon et al. present a method to combine these two approaches to estimating the FDR for MS protein identifications into an integrated protein model (IPM). They further illustrate the high-quality performance of this IPM approach through testing on two large publicly available proteomics data sets. MAYU and SPIRE show remarkable consistency in identifying proteins in these data sets. Still, IPM results in a more robust FDR estimation approach and additional identifications, particularly among low-abundance proteins. IPM is now implemented as a part of the SPIRE system. (Higdon, R., et al. J. Proteomics
High-Throughput Analysis of Testosterone in Serum Samples by Online Solid-Phase Extraction Liquid Chromatography–Tandem Mass Spectrometry
In this study, Salvolainen et al. present an alternate process for analysis of testosterone in serum. They compare the new method with the previous method and demonstrate that the new method is superior to the existing method in terms of specifications. Furthermore, the new process reduces the workload on lab personnel. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques are more and more common in the measurement of testosterone concentrations in biological samples. LC/MS/MS methods are more laborious than streamlined automated immunochemistry methods because of the need for tedious prepurification of the sample before the mass spectrometric analysis. Salvolainen et al. therefore develop a robust and rapid sample cleanup method to improve the throughput of the whole LC/MS/MS analysis procedure by applying an automated online solid-phase extraction (SPE) technique instead of the widely used conventional liquid-liquid extraction. The authors adopt a method to purify testosterone by the online SPE column-switching technique after rapid precipitation of serum samples by zinc sulfate/internal standard solution before LC/MS/MS analysis. The results are compared with those of the previous routine LC/MS/MS method using liquid-liquid extraction with tert-butyl methyl ether for the prepurification of the samples.
The tested online SPE-LC/MS/MS method reaches the specifications of the previous method with liquid-liquid extraction. The precision of the new method is notably better, especially in the lower concentration range, than with the former method; the total variation is below 10% in the whole quantitation range of 0.25 to 35 nmol/L. The new method liberates more than 50% of hands-on time of laboratory technicians as well as expensive instrument time for other applications compared with the older method. The online SPE-prepurification technique tested in long-term use offers a rapid and reliable technique in the LC/MS/MS analysis of serum testosterone and is a valuable tool in the improvement of efficiency in laborious steroid analytics. (Savolainen, K., et al. Clin. Chem. Lab. Med.
Fully Automated Solid-Phase Microextraction–Fast Gas Chromatography–Mass Spectrometry Method Using a New Ionic Liquid Column for High-Throughput Analysis of Sarcosine and N-Ethylglycine in Human Urine and Urinary Sediments
A fully automated, noninvasive, rapid, and high-throughput method for the direct determination of sarcosine and N-ethylglycine in urine and urinary sediments using hexyl chloroformate derivatization followed by direct-immersion solid-phase microextraction and fast gas chromatography (GC)–mass spectrometric analysis is developed and validated in this report by Bianchi et al. The use of a new ionic liquid narrow-bore column, as well as the automation and miniaturization of the preparation procedure by a customized configuration of the used XYZ robotic system, allows friendly use of the GC apparatus to achieve a quantitation limit of 0.06 µg L–1 for sarcosine, as well as good repeatability, with a coefficient of variance (CV) always lower than 7% and reduced analysis times useful for point-of-care testing. The method is then applied for the analysis of 56 samples of urine and urinary sediments in healthy subjects, in those with benign prostatic hypertrophy, and in patients with clinically localized prostate cancer. The results show that the medians of sarcosine/creatinine in urine are 103, 137, and 267 µg/g–1, respectively, thus assessing the potential use of sarcosine as a urinary biomarker for prostate cancer detection. The highest values of sensitivity (79%) and specificity (87%) are obtained in correspondence of a cut-off value of 179 µg sarcosine/g creatinine–1; thus, by using this cutoff threshold, sarcosine is significantly associated with the presence of cancer (p < 0.0001). Finally, receiver operating characteristic (ROC) analyses prove that the discrimination between clinically localized prostate cancer and patients without evidence of tumor is significantly correlated with sarcosine. (Bianchi, F., et al. Anal. Chim. Acta
Automation Systems
Use of Robotics in High-Throughput DNA Sequencing
Until relatively recently, full sequencing of genes consisting of more than several exons was not considered practicable within a routine diagnostic context. As a result, many approaches to unknown mutation detection in a specific gene involve a mutation prescreening step to limit the amount of DNA sequencing required. Protocols to prescreen for mutations and limit the amount of DNA sequencing may not localize every base change present and/or require considerable levels of manual intervention. Advances in technology, allied with careful protocol design, now permit direct DNA sequencing to be applied to larger areas of the gene sequence, allowing unequivocal mutation identification in the area of a gene being analyzed. The protocol described in this report uses robotic systems, allied to custom-designed PCR primers, to facilitate rapid DNA sequencing of multiple gene targets. The general approach is amenable to adaptation for use with multichannel pipettes. (Keeney, S. Methods Mol. Biol.
A Vision-Guided Hybrid Robotic Prototype System for Stereotactic Surgery
Robot-assisted surgery systems help surgeons perform accurate operations, but a number of drawbacks render them not yet suitable for clinical theaters and procedures. In this study, the authors present a novel vision-guided robotic system proposed to facilitate navigation procedures. A vision-guided hybrid robotic system is designed, consisting of a passive serial arm and an active parallel frame. Navigation is accomplished in three steps: approaching, aiming, and insertion. First, the target is safely approached with the passive arm. Second, the trajectory is automatically aligned using the parallel frame, and then the target is reached by manual insertion. A stereo camera is used to position fiducials, the robot, and the surgical tool. It also provides working area images for professional surgeons at a remote site.
The prototype system accomplished phantom and animal trials with satisfactory accuracy. The robot can be adjusted easily to avoid obstacles and quickly set up on an optimal “approaching” place. The surgical tool is automatically aligned with the trajectory. The system can withdraw from the working area and restore the aiming posture freely. With the help of the working area images, some important navigation steps can be handled remotely. The vision-guided robotic system enables surgeons to fit the system to the clinical theater. System safety and feasibility are enhanced by multistep navigation procedures and remote image monitoring. The system can be operated easily by general clinical staff. (Wei, J., et al. Int. J. Med. Robot
An Automated Point-of-Care System for Immunodetection of Staphylococcal Enterotoxin B
An automated point-of-care (POC) immunodetection system for immunological detection of staphylococcal enterotoxin B (SEB) is designed, fabricated, and tested in a report by Yang et al. The system combines several elements: (1) enzyme-linked immunosorbent assay–lab-on-a-chip (ELISA-LOC) with fluidics, (2) a charge-coupled device (CCD) camera detector, (3) pumps and valves for fluid delivery to the ELISA-LOC, (4) a computer interface board, and (5) a computer for controlling the fluidics, logging, and data analysis of the CCD data.
The ELISA-LOC integrates a simple microfluidic system into a miniature 96-well sample plate, allowing the user to carry out immunological assays without a laboratory. The analyte is measured in a sandwich ELISA assay format combined with a sensitive electrochemiluminescence (ECL) detection method. Using the POC system, SEB, a major food-borne toxin, is detected at concentrations as low as 0.1 ng/mL. This is similar to the reported sensitivity of conventional ELISA. The open platform with simple modular fluid delivery automation design is interchangeable between detection systems, and because of its versatility, it can also be used to automate many other LOC systems, simplifying LOC development. This new POC system is useful for carrying out various immunological and other complex medical assays without a laboratory and can easily be adapted for high-throughput biological screening in remote and resource-poor areas. (Yang, M., et al. Anal. Biochem.