Relative Quantitation of Neuropeptides over a Thousand-fold Concentration Range

Neuropeptides are essential cell-to-cell signaling molecules that influence diverse regulatory and behavioral functions within biological systems. Differing in their amino acid sequences and post-translational modifications, hundreds of neuropeptides are produced via a series of enzymatic processing steps, and their levels vary with location, time, and physiological condition. Due to their wide range of endogenous concentrations and inherent chemical complexity, using mass spectrometry (MS) to accurately quantify changes in peptide levels can be challenging. Here we evaluate three different MS systems for their ability to accurately measure neuropeptide levels: capillary liquid chromatography-electrospray ionization-ion trap (CapLC-ESI-IT) MS, ultraperformance liquid chromatography-electrospray ionization-quadrupole-time-of-flight (UPLC-LC-ESI-Q-TOF) MS, and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS. Specifically, eight sample mixtures composed of five neuropeptide standards, with four technical replicates of each, were labeled with H₄/D₄-succinic anhydride, followed by relative peptide quantitation using the three MS platforms. For these samples, the CapLC-ESI-IT MS platform offered the most robust ability to accurately quantify peptides over a concentration range of 1200-fold, although it required larger sample sizes than the other two platforms. Both the UPLC-ESI-Q-TOF MS and the MALDI-TOF MS systems had lower limits of quantification, with the MALDI-TOF having the lowest. By implementing several data acquisition schemes and optimizing the data analysis approaches, we were able to accurately quantify peptides over a three orders of magnitude concentration range using either the UPLC or MALDI-TOF platforms. Overall these results increase our understanding of both the capabilities and limits of using MS-based approaches to measure peptides.     

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.     

A review of Cloud computing technologies for comprehensive microRNA analyses

Cloud computing revolutionized many fields that require ample computational power. Cloud platforms may also provide huge support for microRNA analysis mainly through disclosing scalable resources of different types. In Clouds, these resources are available as services, which simplifies their allocation and releasing. This feature is especially useful during the analysis of large volumes of data, like the one produced by next generation sequencing experiments, which require not only extended storage space but also a distributed computing environment. In this paper, we show which of the Cloud properties and service models can be especially beneficial for microRNA analysis. We also explain the most useful services of the Cloud (including storage space, computational power, web application hosting, machine learning models, and Big Data frameworks) that can be used for microRNA analysis. At the same time, we review several solutions for microRNA and show that the utilization of the Cloud in this field is still weak, but can increase in the future when the awareness of their applicability grows.     

Computational binding study of cardiac troponin I antibody towards cardiac versus skeletal troponin I

A computational study of the interaction of cardiac troponin I (cTnI) with its specific antibody and of that antibody with skeletal troponin I (sTnI), the principal interferon of cTnI, is carried out. Computational and simulation tools such as FTSite, FTMap, FTDock and pyDock are used to determine the binding sites of these molecules and to study their interactions and molecular docking performance, allowing us to obtain relevant information related with the antigen-antibody interaction for each of the targets. In the context of the development of immunosensing platforms, this type of computational analysis allows performing a preliminary in-silico affinity study of the available bioreceptors for a better selection when moving to the experimental stage, with the subsequent saving in cost and time.     

Capillary electrochromatography of peptides and proteins

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure-assisted CEC or voltage-assisted micro- high-performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.     

Centrifugally-driven sample extraction, preconcentration and purification in microfluidic compact discs

Centrifugally-driven microfluidic compact discs (μ-CDs) have attracted significant interest within the analytical science community in the past decade, with the primary focus on the potential of such platforms for performing parallel and/or multiplex biological assays and further application in biomedical diagnostics. More recently, μ-CD-based devices were also applied to environmental analysis as platforms for multi-sample extraction and transportation, prior to off-disc analysis in the laboratory. This review critically summarizes recent developments in μ-CD platforms for sample extraction, preconcentration, fractionation and purification in bioanalytical and environmental applications. We also summarize the common methods employed in the fabrication of μ-CD platforms. Further, we discuss preparation of stationary phases in microfluidic channels embedded in μ-CDs, as applications of μ-CDs in sample extraction are generally based on enclosed series of extraction phases and microcolumns.     

Activity study of self-assembled proteins on nanoscale diblock copolymer templates

Novel methods for affixing functional proteins on surfaces with high areal density have the potential to promote basic biological research as well as various bioarray applications. The use of polymeric templates under carefully balanced thermodynamic conditions enables spontaneous, self-assembled protein immobilization on surfaces with spatial control on the nanometer scale. To assess the full potential of such nanometer-scale protein platforms in biosensing applications, we report for the first time the biological activity of proteins on diblock copolymer platforms. We utilized horseradish peroxidase, mushroom tyrosinase, enhanced green fluorescent protein, bovine immunoglobulin G, fluorescein isothiocyanate conjugated anti-bovine IgG, and protein G as model systems in our protein activity studies. When specific catalytic functions of HRP and MT, immobilized on selective domains of microphase-separated PS-b-PMMA, are evaluated over a long period of time, these enzymes retain their catalytic activity and stability for well over 3 months. By performing confocal fluorescence measurements of self-fluorescing proteins and interacting protein/protein systems, we have also demonstrated that the binding behavior of these proteins is unaffected by surface immobilization onto PS-b-PMMA diblock copolymer microdomains. Our polymer platforms provide highly periodic, high-density, functional, stable surface-bound proteins with spatial control on the nanometer scale. Therefore, our diblock copolymer-guided protein assembly method can be extremely beneficial for high-throughput proteomic applications.     

Electrochemical Nanobiosensors as Point-of-Care Testing Solution to Cytokines Measurement Limitations

Most cytokines are present at reduced amounts in body fluids due to their biological features of production, release, and action mechanisms. The required time between sampling and their measurement is critical for diagnosis and treatment. Electrochemical nanobiosensors offer the possibility to be tailor-made and cost affordable, producing direct and rapid readouts with low sample volume, explaining their feasibility in timely measurements and potential in designing unique and multiplexed Point-Of-Care (POC) testing platforms. This review summarizes and discusses the measurement limitations of the standard methods and the recent progress on electrochemical nanobiosensors as a plausible alternative to measuring them.     

Superparamagnetic nanoparticle-polystyrene bead conjugates as pathogen capture mimics: a parametric study of factors affecting capture efficiency and specificity

There is currently significant interest in the miniaturization of disease detection platforms. As detection platforms decrease in size there is a need for the development of sample preparation protocols by which cells or biomarkers of interest can be concentrated from large volumes down to volumes more amenable to analysis within microfluidic devices. To address this issue, we present a series of magnetic confinement assays for polystyrene (PS) beads mediated through their covalent modification with a series of superparamagnetic nanoparticles, where the PS beads have many properties similar to bacteria, but are not pathogenic. The magnetic confinement of the PS beads is investigated as a function of (1) the overall nanoparticle size, (2) the loading of superparamagnetic content within the nanoparticle matrix, and (3) the viscosity and volume of the dispersion medium. We demonstrate that the time required for the magnetic capture of the PS beads by the superparamagnetic nanoparticles (1) decreases as the loading of superparamagnetic material into the nanoparticles increases and (2) increases as the viscosity and volume of the dispersion medium are increased. However, limitations in the magnetic confinement efficiency for the PS beads labeled with nanoparticles comprised of low loadings of superparamagnetic material can be overcome through the use of magnetic columns. These magnetic columns provide a practical and fast mode of sample preparation that should facilitate the magnetic concentration of cells and biomarkers from large volumes to volumes more amenable to incorporation into a microfluidic-based analysis system, where they can be analyzed/detected.     

New tools and resources in metabolomics: 2016–2017

Rapid advances in mass spectrometry (MS) and nuclear magnetic resonance (NMR)‐based platforms for metabolomics have led to an upsurge of data every single year. Newer high‐throughput platforms, hyphenated technologies, miniaturization, and tool kits in data acquisition efforts in metabolomics have led to additional challenges in metabolomics data pre‐processing, analysis, interpretation, and integration. Thanks to the informatics, statistics, and computational community, new resources continue to develop for metabolomics researchers. The purpose of this review is to provide a summary of the metabolomics tools, software, and databases that were developed or improved during 2016–2017, thus, enabling readers, developers, and researchers access to a succinct but thorough list of resources for further improvisation, implementation, and application in due course of time.     

Implantable Nanotube Sensor Platform for Rapid Analyte Detection

The use of nanoparticles within living systems is a growing field, but the long‐term effects of introducing nanoparticles to a biological system are unknown. If nanoparticles remain localized after in vivo implantation unanticipated side effects due to unknown biodistribution can be avoided. Unfortunately, stabilization and retention of nanoparticles frequently alters their function.[1] In this work multiple hydrogel platforms are developed to look at long‐term localization of nanoparticle sensors with the goal of developing a sensor platform that will stabilize and localize the nanoparticles without altering their function. Two different hydrogel platforms are presented, one with a liquid core of sensors and another with sensors decorating the hydrogel's exterior, that are capable of localizing the nanoparticles without inhibiting their function. With the use of these new hydrogel platforms nanoparticle sensors can be easily implanted in vivo and utilized without concerns of nanoparticle impact on the animal.     

Manufacturing electrochemical platforms: Direct-write dispensing versus screen printing

We report the fabrication of electrochemical platforms using recently developed direct-write dispensing technology, which are compared directly with those produced with screen printing. The manufactured electrodes were examined with the outer-sphere electron transfer probe potassium ferrocyanide, and their structure and morphology were examined with SEM. The electrochemical platforms produced via the direct-write technology performed in a similar fashion with those from screen printing and exhibited excellent voltammetric reproducibility and electrode morphology, suggesting that the direct-write methodology can be used for the routine production of electrochemical platforms. This technology offers an alternative production method for the maskless production of electrochemical platforms which will find diverse applications in the field of electrochemistry.     

Multiparametric microsensor chips for screening applications

The identification of drug targets for pharmaceutical screening can be greatly accelerated by gene databases and expression studies. The identification of leading compounds from growing libraries is realized by high throughput screening platforms. Subsequently, for optimization and validation of identified leading compounds studies of their functionality have to be carried out, and just these functionality tests are a limiting factor. A rigorous preselection of identified compounds by in vitro cellular screening is necessary prior to using the drug candidates for the further time consuming and expensive stage, e.g. in animal models. Our efforts are focused to the parallel development, adaptation and integration of different microelectronic sensors into miniaturized biochips for a multiparametric, functional on-line analysis of living cells in physiologically environments. Parallel and on-line acquisition of data related to different cellular targets is required for advanced stages of drug screening and for economizing animal tests.     

DNA microarrays: is there a role for analytical chemistry?

DNA microarrays, or "DNA chips", represent a relatively new technology that is having a profound impact on biology and medicine, yet analytical research into this area is somewhat sparse. This article presents an overview of DNA microarrays and their application to gene expression analysis from the perspective of analytical chemistry, treating aspects of array platforms, measurement, image analysis, experimental design, normalization, and data analysis. Typical approaches are described and unresolved issues are discussed, with a view to identifying some of the contributions that might be made by analytical chemists.     

1001 Ways to run AutoDock Vina for virtual screening

Large-scale computing technologies have enabled high-throughput virtual screening involving thousands to millions of drug candidates. It is not trivial, however, for biochemical scientists to evaluate the technical alternatives and their implications for running such large experiments. Besides experience with the molecular docking tool itself, the scientist needs to learn how to run it on high-performance computing (HPC) infrastructures, and understand the impact of the choices made. Here, we review such considerations for a specific tool, AutoDock Vina, and use experimental data to illustrate the following points: (1) an additional level of parallelization increases virtual screening throughput on a multi-core machine; (2) capturing of the random seed is not enough (though necessary) for reproducibility on heterogeneous distributed computing systems; (3) the overall time spent on the screening of a ligand library can be improved by analysis of factors affecting execution time per ligand, including number of active torsions, heavy atoms and exhaustiveness. We also illustrate differences among four common HPC infrastructures: grid, Hadoop, small cluster and multi-core (virtual machine on the cloud). Our analysis shows that these platforms are suitable for screening experiments of different sizes. These considerations can guide scientists when choosing the best computing platform and set-up for their future large virtual screening experiments.     

Alginate Self-Crosslinking Ink for 3D Extrusion-Based Cryoprinting and Application for Epirubicin-HCl Delivery on MCF-7 Cells

3D-printed hydrogels are particularly advantageous as drug-delivery platforms but their loading with water-soluble active compounds remains a challenge requiring the development of innovative inks. Here, we propose a new 3D extrusion-based approach that, by exploiting the internal gelation of the alginate, avoids the post-printing crosslinking process and allows the loading of epirubicin-HCl (EPI). The critical combinations of alginate, calcium carbonate and d-glucono-δ-lactone (GDL) combined with the scaffold production parameters (extrusion time, temperature, and curing time) were evaluated and discussed. The internal gelation in tandem with 3D extrusion allowed the preparation of alginate hydrogels with a complex shape and good handling properties. The dispersion of epirubicin-HCl in the hydrogel matrix confirmed the potential of this self-crosslinking alginate-based ink for the preparation of 3D-printed drug-delivery platforms. Drug release from 3D-printed hydrogels was monitored, and the cytotoxic activity was tested against MCF-7 cells. Finally, the change in the expression pattern of anti-apoptotic, pro-apoptotic, and autophagy protein markers was monitored by liquid-chromatography tandem-mass-spectrometry after exposure of MCF-7 to the EPI-loaded hydrogels.     

Advances in Biohybridized Planar Polymer Membranes and Membrane-Like Matrices

The past few decades have seen increasing growth in the field of biomimetic membranes and thus also a rapid expansion of their biomedical and technological applications. Versatility, stability and scalability have moved biohybrid polymer membranes into the limelight. This review focuses on planar, soft polymer membranes and polymer-based matrices and their role as a host for different types of biomolecules. Because biomimetic polymer platforms present an extensive, ever-growing field, we limit ourselves mostly to the discussion of producing planar polymer membranes on solid supports that lend themselves to functionalization by biomolecules. We present an overview of the major highlights and challenges associated with the biohybridization of such polymer platforms. In particular, we elaborate on procedures developed to maintain optimal peptide and membrane protein performance in a customized polymer membrane or membrane-like environment. Finally, we discuss a number of applications of such biohybridized polymer platforms and contemplate future developments to further exploit their potential.     

微流控芯片-质谱联用细胞分析方法研究进展

细胞分析和代谢物分析在生物系统中起着重要的作用。微流控技术已成为细胞生物学研究的一个重要工具。该文总结了最近微流控芯片在细胞和代谢物的分析,尤其是微流控芯片与质谱联用技术的应用。同时对微流控芯片上细胞的生物学研究提出了见解和看法,希望能对感兴趣者提供一些启发。     

Inductively coupled plasma mass spectrometry based platforms for studies involving nanoparticle effects in biological samples

The widespread application of nanoparticles (NPs) in recent times has caused concern because of their effects in biological systems. Although NPs can be produced naturally, industrially synthesized NPs affect the metabolism of a given organism because of their high reactivity. The biotransformation of NPs involves different processes, including aggregation/agglomeration, and reactions with biomolecules that will be reflected in their toxicity. Several analytical techniques, including inductively coupled plasma mass spectrometry (ICP‐MS), have been used for characterizing and quantifying NPs in biological samples. In fact, in addition to providing information regarding the morphology and concentration of NPs, ICP‐MS‐based platforms, such as liquid chromatography/ICP‐MS, single‐particle ICP‐MS, field‐flow fractionation (asymmetrical flow field‐flow fractionation)‐ICP‐MS, and laser ablation‐ICP‐MS, yield elemental information about molecules. Furthermore, such information together with speciation analysis enlarges our understanding of the interaction between NPs and biological organisms. This study reports the contribution of ICP‐MS‐based platforms as a tool for evaluating NPs in distinct biological samples by providing an additional understanding of the behavior of NPs and their toxicity in these organisms.     

Analysis of inorganic and small organic ions with the capillary electrophoresis microchip

Capillary electrophoresis microchip devices are receiving considerable attention due to their versatility, portability, and sample handling capabilities. This article is a comprehensive review of the analysis of inorganic and small, charged organic species on microchip platforms. The application of conductivity, amperometry, laser-induced fluorescence, absorbance, and chemiluminescence detection methods are discussed. The potential utilization of these devices for miniaturized analytical systems is described.