Limitations In Terms Of Throughput Research Articles

Open microfluidics: droplet microarrays as next generation multiwell plates for high throughput screening.

Multiwell plates are prominent in the biological and chemical sciences; however, they face limitations in terms of throughput and deployment in emerging bioengineering fields. Droplet microarrays, as an open microfluidic technology, organise tiny droplets typically in the order of thousands, on an accessible plate. In this perspective, we summarise current approaches for generating droplets, fluid handling on them, and analysis within droplet microarrays. By enabling unique plate engineering opportunities, demonstrating the necessary experimental procedures required for manipulating and interacting with biological cells, and integrating with label-free analytical techniques, droplet microarrays can be deployed across a more extensive experimental domain than what is currently covered by multiwell plates. Droplet microarrays thus offer a solution to the bottlenecks associated with multiwell plates, particularly in the areas of biological cultivation and high-throughput compound screening.

From celebration to contamination: Analysing microplastics released by burst balloons

Air balloons are a ubiquitous presence in our daily lives, and their rupture may release a substantial quantity of debris, as investigated herein. We employ Raman imaging to capture the fragments resulting from balloon explosions, enabling the identification and direct visualisation of minute microplastic particles / fragments with an improved signal-to-noise ratio for precise quantification. To circumvent the generation of misleading confocal Raman images, we recommend employing terrain mapping to scan the three-dimensional surface of the sample. It is important to acknowledge that the analysis of microplastics at the micro-scale inherently poses limitations in terms of throughput, as it necessitates a trade-off between low and high magnifications. We conduct explosive experiments on ten-to-hundred balloons, collecting debris from various angles and positions. Our investigation involves the random testing of multiple samples / sample positions at the micro-scale, with subsequent extrapolation to estimate the total amount of microplastics. The amalgamation of these results through statistical analysis indicates that each balloon explosion can potentially release tens-to-thousands of microplastics, highlighting a concern that has hitherto received insufficient attention. The characterisation approach, particularly the random Raman scanning method in combination with SEM and the statistical analysis on accumulated samples employed in this report, has the potential to serve as a useful tool in future research on microplastics and even nanoplastics.

Rapid LC-MS/MS detection of 25-hydroxyvitamin D in dried blood spots

BackgroundThe detection of 25-hydroxyvitamin D (25OHD) from dried blood spots (DBS) has been widely studied. However, the existing pretreatment methods suffer from limitations in terms of throughput (usually exceeding 2 h), complexity (involving liquid-liquid extraction or solid-phase extraction), and contamination (including multiple steps of organic solvent evaporation). ResultsWe first released 25OHD from DBS samples by 50% acetonitrile solution through ultrasonication. Subsequently, the cold-induced phase separation technique was introduced for in-situ concentration and purification. Afterward, the PTAD derivatization of 25OHD was performed directly, profiting from the high acetonitrile content in the concentrated solution. In the end, the resulting solution was submitted to LC-MS/MS for quantification. The established LC-MS/MS methodology possessed favorable analytical performance, possessing lower limit of quantification of 1 ng/mL pointing to plasma, accuracy of 86.8–110.1% and imprecision of 5.4–16.8%. Method comparison with plasma samples demonstrated that over 93% of the detections met the acceptance limit for cross-validation of ±20%. Significance and noveltyThe novel sample preparation can be finished within 15 min and eliminated the traditional steps of extraction and organic solvent evaporation. Based on this high-throughput, reliable and applicable LC-MS/MS method, the detection of 25OHD in DBS samples can be better achieved for clinical patients and researchers with relevant demands.

Development of a Macrophage-Based ADCC Assay.

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

Improving caching resource management: A pricing economic approach using Cournot, Bertrand, and Stackelberg game models

SummaryOver‐The‐Top broadcasts a huge number of medias that mobile network operators have to manage efficiently before to deliver it to their subscribers. We propose an economic pricing approach to address caching resource management issues in the 5G wireless networks and to overcome limitations in terms of throughput, latency, and reliability. Moreover, we consider this approach based on an oligopolistic multi‐market deducted from Cournot, Stackelberg, and Bertrand models. For simulation purpose, we consider the routing protocol (Ad‐hoc On‐Demand Distance Vector [AODV]) commonly used for the wireless network. We use the NS‐2 package, and we analyze results in terms of End‐to‐End delay representing latency, throughput, packet delivery ratio, and normalized network load.

Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip

High-throughput DNA sequencing techniques haveenabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3× 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56× 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interactionnetworks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.

High-Throughput Manipulation of Circulating Tumor Cells Using a Multiple Single-Cell Encapsulation System with a Digital Micromirror Device.

Circulating tumor cells (CTCs) are potential precursors of metastatic cancer, and genomic information obtained from CTCs have the potential to provide new insights into the biology of cancer metastasis. We previously developed a technique for single-cell manipulation based on the encapsulation of a single cell in a photopolymerized hydrogel that can be used for subsequent genetic analysis. However, this technique has limitations in terms of throughput because light irradiation must be performed on each individual cell from the confocal laser-scanning microscopy. Here, we present a high-throughput cell manipulation technique using a multiple single-cell encapsulation system with a digital micromirror device. This system enables rapid cell imaging within a microcavity array, a microfilter for the recovery of CTCs from blood samples, as well as the simultaneous encapsulation of several CTCs with hydrogels photopolymerized using a multiple light-irradiation system. Furthermore, single-cell labeling using two differently shaped hydrogels was examined to distinguish between NCI-H1975 cells and A549 cells, demonstrating the utility of the system for single-cell gene mutation analysis. In addition to CTCs, our system can be widely applied for analyses of mammalian cells and microorganisms.

AN ALTERNATIVE TREATMENT TO ELECTROPOLISHING: CHEMICAL POLISHING

Anodic alumina template containing hexagonally ordered nanopores has been widely used over the last decade for the development of numerous functional nanostructures such as nanoscale sensors, computing networks, optoelectronic devices, thin film micro circuits, ultra filtration membranes, keying layers for organic adhesives to hard, wear and corrosion resistant layers and memories. The long range pore order requires the starting aluminum surface to be extremely smooth. Electropolishing is the most commonly used method for surface planarization prior to anodization. While prevalent, this method has several limitations in terms of throughput, polishing area and requirement of special experimental setups. In this work, we report a new treatment called-chemical polishing which offers a viable, simpler, safer and faster alternative to electropolishing

Fabrication of hexagonally ordered nanopores in anodic alumina: An alternative pretreatment

Anodic aluminum oxide (AAO) or anodic alumina template containing hexagonally ordered nanopores has been widely used over the last decade for the development of numerous functional nanostructures such as nanoscale sensors, computing networks and memories. The long range pore order requires the starting aluminum surface to be extremely smooth. Electropolishing is the most commonly used method for surface planarization prior to anodization. While prevalent, this method has several limitations in terms of throughput, polishing area and requirement of special experimental setups, which introduce additional speed bottlenecks in the intrinsically slow AAO-based nanofabrication process. In this work we report a new generation of the so-called “chemical polishing” approach which circumvents these stumbling blocks in the pretreatment phase and offers a viable, simpler, safer and faster alternative to electropolishing. These benefits are obtained without sacrificing the quality of the final AAO template. In this work we have ( a) identified the optimum parameter regime for chemical polishing and ( b) determined process conditions for which a novel parallel nanoridge configuration self-assembles and extends over a distance of several microns. Such patterns can be used as a mask for fabricating nanocrossbars, which are the main structural components in myriad nanoscale memories and crosspoint architectures.

High-speed, Thermo-chemical Nanolithography for Biological Applications

Scanning probe-based chemical nanolithography has been recognized as an essential part of future nanofabrication processes. However, most of the present strategies still have significant limitations in terms of throughput, resolution and substrate variety. Recently, we have developed a new chemical nanolithography technique called thermo-chemical nanolithography (TCNL). TCNL utilizes a resistively-heated atomic force microscope tip to thermally activate a chemical reaction on an arbitrary organic or inorganic substrate surface. TCNL can write well-defined chemical features at a rate of mm/s, with sub-15 nm resolution [1]. In particular, we have demonstrated that carboxylic ester groups on a polymer surface can be deprotected by TCNL to give carboxylic acids and further modified to form anhydrides [2]. Therefore, TCNL can tune surface wettability with in situ write-read-overwrite capability. In addition, nanoarrays of TCNL-activated amine groups have been used as anchor sites to immobilize a variety of biological ligands [3]. This proves TCNL to be a powerful tool to control the physical placement of biomolecules and cells. It can be applied to a range of molecular cell biology studies such as ligand-receptor recognition and cell signaling.1. R. Szoszkiewicz, T. Okada, S. C. Jones, T.-D. Li, W. P. King, S. R. Marder, and E. Riedo, Nano Lett. 7, 1064 (2007).2. D. B. Wang, R. Szoszkiewicz, T. Okada, S. C. Jones, M. Lucas, J. Lee, W. P. King, S. R. Marder, and E. Riedo, Appl. Phys. Lett. 91, 243104 (2007).3. D. Wang, V. Kodali, W. D. Underwood, J. E. Jarvholm, T. Odaka, S. J. Jones, C. Rumi, W. P. King, S. R. Marder, J.E. Curtis and E. Riedo, (2008) in preparation.