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Colorimetric methods are convenient for the determination of inorganic phosphate. However, the acidic conditions required can complicate measurement of ATPase through non-enzymatic ATP hydrolysis. Here we present an optimized antimony-phosphomolybdate microassay for the simple and rapid detection of ATPase activity, with micromolar sensitivity. The low acidity of the color reagent results in no interference for samples containing up to 0.5-5 mM ATP, dependent on the sample volume. The assay is compatible with common assay conditions and was similar in accuracy to an established continuous method. The simplicity of this method makes it ideal for medium to high throughput applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Flavonoid compounds are known for their antibacterial, anti-inflammatory, and anticancer properties. Therefore, they can influence membrane properties that interest us, modifying both their structure and functions. We used kaempferol (K) and myricetin (M) as representatives of this group. We investigated the influence of the abovementioned compounds on model cell membranes' properties (i.e., Langmuir monolayers and liposomes). The basic research methods used in these studies were the Langmuir method with Brewster angle microscopy and microelectrophoresis. The π-A isotherms were registered for the pure components and mixtures of these compounds with phosphatidylcholine (PC) in appropriate volume ratios. Using mathematical equations, we established that kaempferol, myricetin, and the lipids formed complexes at 1:1 ratios. We derived the parameters characterizing the formed complexes, i.e., the surfaces occupied by the complexes and the stability constants of the formed complexes. Using the microelectrophoretic method, we determined the dependence of the lipid membranes' surface charge density as a function of the pH (in the range of 2 to 10) of the electrolyte solution. The presented results indicate that the PC membrane's modification with kaempferol or myricetin affected changes in the surface charge density and isoelectric point values., Competing Interests: The authors declare no conflict of interest.

Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 μl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 μl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 μl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 μl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl2. We found 4 or 3 μl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

O/W nanoemulsions are isotropic colloidal systems constituted of oil droplets dispersed in continuous aqueous media and stabilised by surfactant molecules. Nanoemulsions hold applications in more widespread technological domains, more crucially in the pharmaceutical industry. Innovative nanoemulsion-based drug delivery system has been suggested as a powerful alternative strategy through the useful means of encapsulating, protecting, and delivering the poorly water-soluble bioactive components. Consequently, there is a need to generate an emulsion with small and consistent droplets. Diverse studies acknowledged that ultrasonic cavitation is a feasible and energy-efficient method in making pharmaceutical-grade nanoemulsions. This method offers more notable improvements in terms of stability with a lower Ostwald ripening rate. Meanwhile, a microstructured reactor, for instance, microchannel, has further been realised as an innovative technology that facilitates combinatorial approaches with the acceleration of reaction, analysis, and measurement. The recent breakthrough that has been achieved is the controlled generation of fine and monodispersed multiple emulsions through microstructured reactors. The small inner dimensions of microchannel display properties such as short diffusion paths and high specific interfacial areas, which increase the mass and heat transfer rates. Hence, the combination of ultrasonic cavitation with microstructures (microchannel) provides process intensification of creating a smaller monodispersed nanoemulsion system. This investigation is vital as it will then facilitate the creation of new nanoemulsion based drug delivery system continuously. Following this, the fabrication of microchannel and setup of its combination with ultrasound was conducted in the generation of O/W nanoemulsion, as well as optimisation to analyse the effect of varied operating parameters on the mean droplet diameter and dispersity of the nanoemulsion generated, besides monitoring the stability of the nanoemulsion. Scanning transmission electron microscopy (STEM) images were also carried out for the droplet size measurements. In short, the outcomes of this study are encouraging, which necessitates further investigations to be carried out to advance a better understanding of coupling microchannel with ultrasound to produce pharmaceutical-grade nanoemulsions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Measuring mitochondrial respiration in brain tissue is very critical in understanding the physiology and pathology of the central nervous system. Particularly, measurement of respiration in isolated mitochondria provides the advantage over the whole cells or tissues as the changes in respiratory function are intrinsic to mitochondrial structures rather than the cellular signaling that regulates mitochondria. Moreover, a high-throughput technique for measuring mitochondrial respiration minimizes the experimental time and the sample-to-sample variation. Here, we provide a detailed protocol for measuring respiration in isolated brain non-synaptosomal mitochondria using Agilent Seahorse XFe24 Analyzer. We optimized the protocol for the amount of mitochondria and concentrations of ADP, oligomycin, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) for measuring respiratory parameters for complex I-mediated respiration. In addition, we measured complex II-mediated respiratory parameters. We observed that 10 µg of mitochondrial protein per well, ADP concentrations ranging between 2.5 and 10 mmol/L along with 5 µmol/L of oligomycin, and 5 µmol/L of FCCP are ideal for measuring the complex I-mediated respiration in isolated mouse brain mitochondria. Furthermore, we determined that 2.5 µg of mitochondrial protein per well is ideal for measuring complex II-mediated respiration. Notably, we provide a discussion of logical analysis of data and how the assay could be utilized to design mechanistic studies for experimental stroke. In conclusion, we provide detailed experimental design for measurement of various respiratory parameters in isolated brain mitochondria utilizing a novel high-throughput technique along with interpretation and analysis of data.

Background: Δ 9 -Tetrahydrocannabinol (THC) is already considered one of the most addictive substances since an increasing number of consumers/abusers of THC and THC based products are observed worldwide. In this work, the capabilities of a novel miniaturized and portable MicroNIR spectrometer were investigated in order to propose a practical and intelligible test allowing the rapid and easy screening of Δ 9 -Tetrahydrocannabinol (THC) oral fluids without any pretreatment., Methods: Specimens from volunteers were collected in order to consider any sources of variability in the spectral response and spiked with increasing amount of THC in order to realize predictive models to be used in real cases. Partial Least Square-Discriminant Analysis (PLS-DA) and Partial Least Square regression (PLSr) for the simultaneously detection and quantification of THC, were applied to baseline corrected spectra pre-treated by first derivative transform., Results: Results demonstrated that MicroNIR/Chemometric platform is statistically able to identify THC abuse in simulated oral fluid samples containing THC from 10 to 100 ng/ml, with a precision and a sensitivity of about 1.51% and 0.1% respectively., Conclusions: The coupling MicroNIR/Chemometrics permits to simplify THC abuse monitoring for roadside drug testing or workplace surveillance and provides the rapid interpretation of results, as once the model is assessed, it can be used to process real samples in a "click-on" device., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. In the present work, we explore the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. N-alkylation reactions were used as the test case due to their ease of reactant and product detection by electrospray ionization mass spectrometry (ESI-MS) and their great importance in API synthesis. While DESI-MS narrowed the scope of possibilities for reaction selection among some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. DESI-MS high throughput experimentation (HTE) reaction evaluation significantly reduced the search space for flow chemistry optimization, thus representing a significant savings in time and materials to achieve a desired transformation with high efficiency.

As an important natural polysaccharide biomaterial from marine organisms, alginate and its derivatives have shown great potential in the fabrication of biomedical materials such as tissue engineering, cell biology, drug delivery, and pharmaceuticals due to their excellent biological activity and controllable physicochemical properties. Ionic crosslinking is the most common method used in the preparation of alginate-based biomaterials, but ionic crosslinked alginate hydrogels are prone to decompose in physiological solution, which hinders their applications in biomedical fields. In this study, dual crosslinked alginate hydrogel microfibers were prepared for the first time. The ionic crosslinked methacrylated alginate (Alg-MA) hydrogel microfibers fabricated by Microfluidic Fabrication (MFF) system were exposed to ultraviolet (UV) light and covalent crosslink between methacrylate groups avoided the fracture of dual crosslinked macromolecular chains in organizational environment. The chemical structures, swelling ratio, mechanical performance, and stability were investigated. Cell-encapsulated dual crosslinked Alg-MA hydrogel microfibers were fabricated to explore the application in tissue engineering for the first time. The hydrogel microfibers provided an excellent 3D distribution and growth conditions for cells. Cell-encapsulated Alg-MA microfibers scaffolds with functional 3D tissue structures were developed which possessed great potential in the production of next-generation scaffolds for tissue engineering and regenerative medicine.

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library., Competing Interests: The authors have declared that no competing interests exist.

This review describes briefly the high rate of counterfeiting of antimicrobial drugs with focus upon its immediate health consequences. The major part of this review encompasses accounts of the improvements achieved in the domain of miniaturization of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C 4 D). The application of this principle into the development of portable devices as well as its application to counter the health-system-crippling phenomenon of counterfeit antibiotic formulations, are discussed in the context of developing countries., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Protein-polymer microcapsules have attracted much attention, due to their special features and potential in biological use. How to make the most of this type of bio-abiotic hybrid material is an intriguing question. Nevertheless, several unsatisfactory technical issues significantly limited the application of these materials. For instance, introducing various biomolecules and crosslinking for the capsules remains challenging and problematic. In this report, recombinant mCherry protein was covalently linked with poly(N-isopropylacrylamide) (PNIPAAm) to form amphiphilic protein-polymer conjugates, which assembled into microcapsules. These microcapsules are thermoresistant and can be used in the polymerase chain reaction (PCR). In this setting, the reactant molecules can be readily and easily introduced into the microcapsules, and crosslinking and water-oil phase transition are not necessary. This protein-polymer microcapsule PCR system has potential in various biological applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This review summarizes recent developments, applications, and innovations of capillary electrophoretically mediated microanalysis methods. As a follow up of an earlier review, it covers the literature from early 2015 to early 2017. This article is divided into three parts. In the first part, different types of mixing procedures and applications of enzyme mediated microanalysis are discussed; the second part summarizes immobilized enzyme reactors (IMERs), while the third part deals with recent advances in on-line derivatization reactions., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report peptide vesicles with chiral membranes as enantioselective nanoreactors. The peptide vesicles are able to selectively encapsulate only a single enantiomer from a racemic mixture solution through preferential diffusion across the membranes. Notably, the confined space of the vesicle acts as an enantioselective nanoreactor for the encapsulated enantiomer which undergoes further chemical transformations to yield a highly enantiopure product.

Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris are the standard platforms for biopharmaceutical production with 40% of all between 2010 to 2014 approved protein drugs produced in those microbial hosts. Typically, products overexpressed E. coli and S. cerevisiae remain in the cytosol or are secreted into the periplasm. Consequently, efficient cell disruption is essential for high product recovery during microbial production. Process development platforms at microscale are essential to shorten time to market. While high-pressure homogenization is the industry standard for cell disruption at large scale this method is not practicable for experiments in microscale. This review describes microscale methods for cell disruption at scales as low as 200 µL. Strategies for automation, parallelization and miniaturization, as well as comparability of the results at this scale to high pressure homogenization are considered as those criteria decide which methods are most suited for scale down. Those aspects are discussed in detail for protein overexpression in E. coli and yeast but also the relevance for alternative products and host such as microalgae are taken into account. The authors conclude that bead milling is the best comparable microscale method to large scale high-pressure homogenization and therefore the most suitable technique for automated process development of microbial hosts with the exception of pDNA production., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

There has been an intense research for developing techniques that can produce filaments with helical shapes, given the widespread of potential applications. In this work, how helices with different curvatures can be precisely imprinted in microfilaments is shown. It is also shown that using this technique, it is possible to produce, in a single fiber, helices with different curvatures. This striking and innovative behavior is observed when one side of the stretched filaments is irradiated with UV light, modifying the mechanical properties at surface. Upon release, the regions with higher curvature start to curl first, while regions with lower intrinsic curvature remain stretched until start to curl later. The results presented here can be important to understand why structures adopt a helical shape in general, which can be of interest in nanotechnology, biomolecular science, or even to understand why plant filaments curl., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bipolar disorder is characterized as a manic-depressive psychiatric syndrome with life-threatening risks to the patient. Diagnosed individuals undergo long-term lithium therapy which has proven to be effective for mood stabilization. Maintaining blood lithium concentration levels within a narrow therapeutic window between 0.6 and 1.5 mM is vital for the patient as slightly elevated concentrations of the order of 0.1 mM can be toxic. This paper aims to evaluate the merits of tetrapolar electrical impedance spectroscopy as an alternative method in monitoring blood lithium levels. Measurements were performed using a custom-made tetrapolar probe in human blood plasma with lithium concentrations covering the therapeutic range. The results indicate a limit of detection less than 0.1 mM and a response time of less than 5 s. Prediction of lithium concentration levels using impedance values is in good agreement with conventional standard techniques to approximately 0.05 mM. This technique provides a basis for further development of instrumentation for point of care healthcare technologies.

Chemical composition and shape determine the basic properties of any object. Commonly, chemical synthesis and shaping follow each other in a sequence, although their combination into a single process would be an elegant simplification. Here, a pathway of simultaneous synthesis and shaping as applied to polysiloxanes on the micro- and nanoscale is presented. Complex structures such as stars, chalices, helices, volcanoes, rods, or combinations thereof are obtained. Varying the shape-controlling reaction parameters including temperature, water saturation, and the type of substrate allows to direct the reaction toward specific structures. A general mechanism of growth is suggested and analytical evidence and thermodynamic calculations to support it are provided. An aqueous droplet in either gaseous atmosphere or in a liquid organic solvent serves as a spatially confined polymerization volume. By substituting the starting materials, germanium-based nanostructures are also obtained. This transferability marks this approach as a major step toward a generally applicable method of chemical synthesis including in situ shaping., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pesticide residue in fruits & vegetables is one of the key issues affecting the export of rural products in India. Pesticide exposure or intake causes major nervous system problems in children. The solutions to quantitate them in field are rare and the pesticide residue detection in the parts per billion (ppb) ranges is challenging. Except ELISA, none of the existing methods can detect pesticide residues in ppb range in the field. We employed a new approach of concentrating field samples and used sodium polyacrylate (SPA) as water absorbing material. The SPA beads concentrate the field samples and obtained a sub ppb range detection using an existing FPIA system and could improve overall sensitivity by 10-100 fold. The developed assay can be done in few seconds. We have used three pesticides 2,4-D, atrazine and methyl parathion with 0.1, 0.5 and 3 ppb detection limit respectively. We developed a simple field ready FPIA device and used sodium poly acrylate (SPA) in this biochemical FPIA to enhance sensitivity. Our tests with spiked field samples offers a possibility of using SPA concentration assisted FPIA in field. This study will have far reaching applications of both qualitative & quantitative analysis chemical analytes in field samples.

Human epidermal growth factor is a small peptide consisting of 53 amino acid residues, which stimulates cell proliferation and is associated with several human carcinomas. A simple sandwich-type ultramicroELISA assay (UMELISA), based on the advantages of high affinity reaction between streptavidin and biotin has been developed for the measurement of EGF in human serum samples. Strips coated with a high affinity monoclonal antibody directed against EGF are used as solid phase, to ensure the specificity of the assay. The EGF assay was completed in 18 hr, with a measuring range of 39-2500 pg/mL. The intra- and inter-assay coefficients of variation were 4.4-7.3% and 0-5.1%, respectively, depending on the EGF concentrations evaluated. Percentage recovery ranged from 96-104%. Regression analysis showed a good correlation with the commercially available Human EGF Immunoassay Quantikine ® ELISA kit (n = 130, r = 0.92, P < 0.01). The analytical performance characteristics of our UMELISA EGF endorse its use for the quantification of EGF in human serum samples.

Glucometers present an important self-monitoring tool for diabetes patients and, therefore, must exhibit high accuracy as well as good usability features. Based on an invasive photometric measurement principle that drastically reduces the volume of the blood sample needed from the patient, we present a framework that is capable of dealing with small blood samples, while maintaining the required accuracy. The framework consists of two major parts: 1) image segmentation; and 2) convergence detection. Step 1 is based on iterative mode-seeking methods to estimate the intensity value of the region of interest. We present several variations of these methods and give theoretical proofs of their convergence. Our approach is able to deal with changes in the number and position of clusters without any prior knowledge. Furthermore, we propose a method based on sparse approximation to decrease the computational load, while maintaining accuracy. Step 2 is achieved by employing temporal tracking and prediction, herewith decreasing the measurement time, and, thus, improving usability. Our framework is tested on several real datasets with different characteristics. We show that we are able to estimate the underlying glucose concentration from much smaller blood samples than is currently state of the art with sufficient accuracy according to the most recent ISO standards and reduce measurement time significantly compared to state-of-the-art methods.

Heart failure (HF) continues to have a sufficient impact on morbidity, mortality, and disability in developed countries. Growing evidence supports the hypothesis that microparticles (MPs) might contribute to the pathogenesis of the HF development playing a pivotal role in the regulation of the endogenous repair system, thrombosis, coagulation, inflammation, immunity, and metabolic memory phenomenon. Therefore, there is a large body of data clarifying the predictive value of MP numerous in circulation among subjects with HF. Although the determination of MP signature is better than measurement of single MP circulating level, there is not yet close confirmation that immune phenotype of cells produced MPs are important for HF prediction and development. The aim of the chapter is to summarize knowledge regarding the role of various MPs in diagnosis and prognosis of HF. The role of MPs as a delivery vehicle for drugs attenuated cardiac remodeling is considered., (© 2017 Elsevier Inc. All rights reserved.)

The Fast Micromethod is a rapid and convenient microplate procedure for the determination of DNA integrity in cell suspensions and in solid tissues. The procedure is based on the ability of fluorochromes to preferentially interact with double-stranded DNA in alkaline conditions. Rapid sample lysis is followed by denaturation at high pH during 15 min. Only 30 ng of DNA from cell suspensions or tissue homogenates per single well are required for the analyses. The whole analysis is performed within 3 h or less (for one 96-well microplate).The Fast Micromethod is broadly used in biology and medicine. Its applications range from environmental pollution tests in marine invertebrates to the analysis of biopsy samples in cancer patients to detect DNA alterations caused by irradiation or chemotherapy.The procedure presented here describes the Fast Micromethod applied for the determination of DNA integrity in cell suspensions (HeLa cells) and solid tissues (mussel gills).

We developed a protocol of noninvasive prenatal testing (NIPT), employing a higher-resolution picodroplet digital PCR, to detect genetic imbalance in maternal plasma DNA (mpDNA) caused by cell-free fetal DNA (cffDNA). In the present study, this approach was applied to four families with autosomal recessive (AR) congenital sensorineural hearing loss. First, a fraction of the fetal DNA in mpDNA was calculated. Then, we made artificial DNA mixtures (positive and negative controls) to simulate mpDNA containing the fraction of cffDNA with or without mutations. Next, a fraction of mutant cluster signals over the total signals was measured from mpDNA, positive controls, and negative controls. We determined whether fetal DNA carried any paternal or maternal mutations by calculating and comparing the sum of the log-likelihood of the study samples. Of the four families, we made a successful prediction of the complete fetal genotype in two cases where a distinct cluster was identified for each genotype and the fraction of cffDNA in mpDNA was at least 6.4%. Genotyping of only paternal mutation was possible in one of the other two families. This is the first NIPT protocol potentially applicable to any AR monogenic disease with various genotypes, including point mutations.

Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.

This review shows the recent advances and state of the art in paper-based analytical devices (PADs) through the analysis of their integration with microfluidics and LOC micro- and nanotechnologies, electrochemical/optical detection and electronic devices as the convergence of various knowledge areas. The important role of the paper design/architecture in the improvement of the performance of sensor devices is discussed. The discussion is fundamentally based on μPADs as the new generation of paper-based (bio)sensors. Data about the scientific publication ranking of PADs, illustrating their increase as an experimental research topic in the past years, are supplied. In addition, an analysis of the simultaneous evolution of PADs in academic lab research and industrial commercialization highlighting the parallelism of the technological transfer from academia to industry is displayed. A general overview of the market behaviour, the leading industries in the sector and their commercialized devices is given. Finally, personal opinions of the authors about future perspectives and tendencies in the design and fabrication technology of PADs are disclosed.

From its early beginnings in characterizing aerosol particles to its recent applications for investigating natural waters and waste streams, single particle inductively coupled plasma-mass spectrometry (spICP-MS) has proven to be a powerful technique for the detection and characterization of aqueous dispersions of metal-containing nanomaterials. Combining the high-throughput of an ensemble technique with the specificity of a single particle counting technique and the elemental specificity of ICP-MS, spICP-MS is capable of rapidly providing researchers with information pertaining to size, size distribution, particle number concentration, and major elemental composition with minimal sample perturbation. Recently, advances in data acquisition, signal processing, and the implementation of alternative mass analyzers (e.g., time-of-flight) has resulted in a wider breadth of particle analyses and made significant progress toward overcoming many of the challenges in the quantitative analysis of nanoparticles. This review provides an overview of spICP-MS development from a niche technique to application for routine analysis, a discussion of the key issues for quantitative analysis, and examples of its further advancement for analysis of increasingly complex environmental and biological samples. Graphical Abstract Single particle ICP-MS workflow for the analysis of suspended nanoparticles.

Highly sensitive, specific, and selective immunoassays are of great significance for not only clinical diagnostics but also food safety, environmental monitoring, and so on. Enzyme-linked immunosorbent assays and fluorescence-based and electrochemical immunoassays are important intensively investigated immunoassay techniques. However, they might suffer from low sensitivity or false-positive results. In this work, a simple, reliable, and ultrasensitive magnetic-bead-based immunoassay was performed using biofunctionalized ZnS semiconductor nanocrystals as resonant Raman probes. The resonant Raman scattering of ZnS nanocrystals displays evenly spaced multi-phonon resonant Raman lines with narrow bandwidths and has strong resistance to environmental variation due to the nature of the electron-phonon interaction, thus rendering reliable signal readout in the immunoassays. The superparamagnetic Fe3O4 nanoparticles facilitated greatly the separation, purification, and concentration processes. It is beneficial for both reducing the labor intensity and amplifying the detection signals. The immobilization of antibodies on the surface of magnetic beads, the preparation of resonant Raman probes, and the immunological recognition between the antibody and analyte all occurred in the liquid phase, which minimized the diffusion barriers and boundary layer constraints. All these factors contributed to the ultralow detection limit of human IgG, which was determined to be about 0.5 fM (∼0.08 pg/ml). It is nearly the highest sensitivity obtained for IgG detection. This work shall facilitate the design of nanoplatforms for ultrasensitive detections of proteins, DNAs, bacteria, explosives, and so on. Graphical abstract An ultrasensitive magnetic-bead-based immunoassay was performed using multi-phonon resonant Raman lines of ZnS nanoparticles as detection signals.

Interlayer spray is proposed as a convenient ionization source for direct analysis by mass spectrometry. Two slices of non-absorbent substrate hold the liquid sample to form a sandwich structure. By applying a high voltage to the sample, spray is generated at the tip of the substrate. The sampling procedure can be operated easily in an open condition and the spray is processed in a semi-enclosed condition, which leads to a relatively stable process. An ultralow amount (<2 μL) of the liquid sample can be analyzed without dilution, which ensures that the natural concentration and properties of the target are maintained. Less influence from the substrate is achieved compared with the spray methods based on porous absorbent materials, which results in a sensitivity enhancement of large molecule samples. It is demonstrated that the interlayer spray is applicable for the analysis of various compounds, including therapeutic drugs, peptides, and proteins. Good linearity can be obtained at a concentration as low as 50 ng/mL in the quantitative analysis for imatinib. We also show the ability to identify the chemical residuals on surfaces with high sensitivity by the "wipe-spray" method, which is useful for the fast screening of illicit substances. Interlayer spray working with mass spectrometry provides a promising method for direct analysis in an ambient environment. Graphical Abstract The schematic of the interlayer spray ionization source.

We have investigated and compared a number of sample conditions on different NMR platforms in the search of maximum SNR and optimal experiment time efficiency for structure elucidation and quantitation of natural products. Using restricted volume 3 mm Shigemi microcell assembly in conjunction with a 900 MHz NMR spectrometer equipped with a 5 mm carbon-sensitive inverse cryoprobe, it was possible to achieve a substantial increase in SNR (46-fold) as compared with a conventional room temperature 400 MHz instrument. Switching from standard 5 mm NMR tube to 3 mm Shigemi microcell assembly typically improved SNR by threefold on either 600 or 900 MHz cryoplatform. A quantitation method that relies on a calibrated residual protonated NMR solvent signal as internal standard was developed using the same hardware setup and restricted sample volume tubes. Linearity of the method spans over 3 orders of magnitude, from low microgram to milligram quantities. We successfully applied this method to quantify a low micrgram sample of paclitaxel, verified by a UV/VIS quantitation measurement., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objectives: Circulating plasma DNA is being increasingly used for biomedical and clinical research as a substrate for genetic testing. However, cell lysis can occur hours after venipuncture when using standard tubes for blood collection, leading to an increase in contaminating cellular DNA that may hinder analysis of circulating plasma DNA. Cell stabilization agents can prevent cellular lysis for several days, reducing the need for immediate plasma preparation after venipuncture, thereby facilitating the ease of blood collection and sample preparation for clinical research. However, the majority of cell stabilizing reagents have not been formally tested for their ability to preserve circulating plasma tumor DNA., Design & Methods: In this study, we compared the properties of two cell stabilizing reagents, the cell-free DNA BCT tube and the PAXgene tube, by collecting blood samples from metastatic breast cancer patients and measuring genome equivalents of plasma DNA by droplet digital PCR. We compared wild type PIK3CA genome equivalents and also assayed for two PIK3CA hotspot mutations, E545K and H1047R., Results: Our results demonstrate that blood stored for 7 days in BCT tubes did not show evidence of cell lysis, whereas PAXgene tubes showed an order of magnitude increase in genome equivalents, indicative of considerable cellular lysis., Conclusions: We conclude that BCT tubes can prevent lysis and cellular release of genomic DNA of blood samples from cancer patients when stored at room temperature, and could therefore be of benefit for blood specimen collections in clinical trials., (Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objectives: Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations., Design and Methods: Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs., Results: WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations., Conclusions: Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions., (Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Capillary electrophoresis and UV-visible absorbance detection are used with sample stacking to achieve detection limits ranging from 0.2 to 2 ng/mL (0.8 to 6 nM) for steroids. Stacking is accomplished using negatively charged cyclodextrin steroid-carrier molecules at a discrete pH interface between the reconstituted sample and the separation electrolyte. Steroids are then separated in under 5 min using capillary electrophoresis that incorporates secondary equilibria via sodium dodecyl sulfate and cyclodextrin. The effectiveness of the method for measurements of multiple steroids in limited sample volumes is demonstrated in individual female fish with total circulating blood volumes of 5 μL or less. Steroid recoveries from plasma following a sample processing method developed with commercial extraction cartridges range from 81 to 109 % for 17α,20β-dihydroxy-pregn-4-en-3-one, testosterone, 11-ketotestosterone, estrone, 17β-estradiol, and 17α-ethinyl estradiol. When applied to reproductively active female zebrafish, changes were detected in the levels of circulating steroids as a result of exposure to different solvents and 17β-estradiol.

A simple and dual chemical reaction-based multimodal assay for dopamine with high sensitivity and selectivity using two types of functionalized gold nanoparticles (FB-AuNPs/NsNHS-AuNPs), i.e. fluorescein modified gold nanoparticles (FB-AuNPs) and Nile blue modified gold nanoparticles (NsNHS-AuNPs), was successfully fabricated. This assay for dopamine presents colorimetric visualization and double channel fluorescence enhancement at 515 and 665 nm. The absorbance and fluorescence changes were linearly proportional to the amounts of dopamine in the range of nanomolar scale (5-100 nM). The detection limits for absorbance and fluorescence were as low as 1.2 nM and 2.9 nM (S/N = 3), respectively. Furthermore, the extent application of this multimodal assay has been successfully demonstrated in human urine samples with high reliability and applicability, showing remarkable promise in diagnostic purposes.

A sensitive label-free method was presented for the determination of silver ion (Ag+) in this paper. Cytosine-rich DNA (C-DNA) was used as Ag+ specific DNA. Without Ag+ in the solution, fluorescence of fluorescein (FAM) is quenched by C-DNA stabilized gold nanoparticles (AuNPs) in high salt environment. When Ag+ is present in the solution, however, Ag+-mediated cytosine-Ag+-cytosine (C-Ag+-C) base pairs induced the C-DNA folding into a hairpin structure, which can not stabilize AuNPs in high salt environment, thus causing AuNPs aggregation. After centrifugation to remove the aggregated AuNPs, the quenching ability of the supernatant for FAM is decreased and the fluorescence intensity of solution increases with increasing the Ag+ concentration. Due to the highly specific interaction of the C-DNA towards Ag+ and the strong fluorescent quenching ability of AuNPs for FAM, the method has high selectivity and sensitivity for Ag+. Under the optimal conditions, the fluorescence intensity at 515 nm increased linearly with the concentration of Ag+ ranging from 15 nM to 700 nM, and the detection limit was determined as 6 nM based on 3 σ/slope. This method is simple, sensitive, and may be applied to other detection systems by selecting the appropriate DNA sequences.

Because of its widespread consumption and its persistence during wastewater treatment, the artificial sweetener sucralose has gained considerable interest as a proxy to detect wastewater intrusion into usable water resources. The molecular resilience of this compound dictates that coastal and oceanic waters are the final recipient of this compound with unknown effects on ecosystems. Furthermore, no suitable methodologies have been reported for routine, ultra-trace detection of sucralose in seawater as the sensitivity of traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is limited by a low yield of product ions upon collision-induced dissociation (CID). In this work, we report the development and field test of an alternative analysis tool for sucralose in environmental waters, with enough sensitivity for the proper quantitation and confirmation of this analyte in seawater. The methodology is based on automated online solid-phase extraction (SPE) and high-resolving-power orbitrap MS detection. Operating in full scan (no CID), detection of the unique isotopic pattern (100:96:31 for [M-H](-), [M-H+2](-), and [M-H+4](-), respectively) was used for ultra-trace quantitation and analyte identification. The method offers fast analysis (14 min per run) and low sample consumption (10 mL per sample) with method detection and confirmation limits (MDLs and MCLs) of 1.4 and 5.7 ng/L in seawater, respectively. The methodology involves low operating costs due to virtually no sample preparation steps or consumables. As an application example, samples were collected from 17 oceanic and estuarine sites in Broward County, FL, with varying salinity (6-40 PSU). Samples included the ocean outfall of the Southern Regional Wastewater Treatment Plant (WWTP) that serves Hollywood, FL. Sucralose was detected above MCL in 78% of the samples at concentrations ranging from 8 to 148 ng/L, with the exception of the WWTP ocean outfall (at pipe end, 28 m below the surface) where the measured concentration was 8418 ± 3813 ng/L. These results demonstrate the applicability of this monitoring tool for the trace-level detection of this wastewater marker in very dilute environmental waters.

This study describes the development and validation of a new liquid chromatography-tandem mass spectrometry (MS/MS) method capable of simultaneous quantitation of seven ophthalmic drugs-pilocarpine, lidocaine, atropine, proparacaine, timolol, prednisolone, and triamcinolone acetonide-within regions of the rabbit eye. The complete validation of the method was performed using an Agilent 1100 series high-performance liquid chromatography system coupled to a 4000 QTRAP MS/MS detector in positive TurboIonSpray mode with pooled drug solutions. The method sensitivity, evaluated by the lower limit of quantitation in two simulated matrices, yielded lower limits of quantitation of 0.25 nmol L(-1) for most of the drugs. The precision in the low, medium, and high ranges of the calibration curves, the freeze-thaw stability over 1 month, the intraday precision, and the interday precision were all within a 15% limit. The method was used to quantitate the different drugs in the cornea, aqueous humor, vitreous humor, and remaining eye tissues of the rabbit eye. It was validated to a concentration of up to 1.36 ng/g in humors and 5.43 ng/g in tissues. The unprecedented low detection limit of the present method and its ease of implementation allow easy, robust, and reliable quantitation of multiple drugs for rapid in vitro and in vivo evaluation of the local pharmacokinetics of these compounds.

Glycochips can be used to map the carbohydrate-protein interactions in high-throughput manner. Carbohydrates are immobilized on the support surface by covalent or noncovalent binding. The immobilization strategies for glycochips were summarized herein. In addition, some recently developed techniques for detection of carbohydrate-protein interactions were also involved.

Probe electrospray ionization (PESI) is a recently developed ionization technique based on electrospray ionization (ESI) that generates electrospray from the tip of a solid needle. High tolerance to salts, requirements of a trace amount of sample and direct ambient sampling- are major advantages of PESI compared with conventional ESI. In this report, three pairs of isomeric lignans bearing tetra-hydrofuran with variable conformations from Gymnotheca chinensis were investigated by probe electrospray tandem mass spectrometry (PESI-MS/MS) in the positive ion mode. The diagnostic characteristics of these compounds were obtained and the isomers could be successfully distinguished by comparison with their breakdown curves, even though the isomers differed only in the conformation of some groups of the isomer pairs. This report provides a rapid and reliable method for the identification of trace amounts of isomeric lignans by PESI-MS/MS. Furthermore, application of PESI and breakdown curves should have value in mass spectrometry studies of isomeric natural products compounds.

The coordinating nature of the hydroxy carboxylic acids, such as tartaric and citric acids, has been utilized for the in-situ formation of anions representing the trivalent elements boron and aluminum and two dianions of the hydroxy acid selected under negative electrospray ionization mass spectral conditions. The abundance of these ions could be used for the quantification of boron and aluminum in water at concentrations ranging from 4.0 ppb to 535.0 ppb. For a period of six months, the validity of this method was tested with citric acid as the coordinating agent. Thus, the developed method offers a simple means for the quantification of boron and aluminum in water by negative electrospray ionization mass spectrometry with a single quadrupole mass spectrometer.

Optical detection of structures with dimensions smaller than an optical wavelength requires devices that work on scales beyond the diffraction limit. Here we present the possibility of using a tapered optical nanofiber as a detector to resolve individual atoms trapped in an optical lattice in the Mott insulator phase. We show that the small size of the fiber combined with an enhanced photon collection rate can allow for the attainment of large and reliable measurement signals.

The metabolome is the set of small molecular mass compounds found in biological media, and metabolomics, which refers to as the analysis of metabolome in a given biological condition, deals with the large scale detection and quantification of metabolites in biological media. It is a data driven and multidisciplinary approach combining analytical chemistry for data acquisition, and biostatistics, informatics and biochemistry for mining and interpretation of these data. Since the middle of the 2000s, high resolution mass spectrometry is widely used in metabolomics, mainly because the detection and identification of metabolites are improved compared to low resolution instruments. As the field of HRMS is quickly and permanently evolving, the aim of this work is to review its use in different aspects of metabolomics, including data acquisition, metabolite annotation, identification and quantification. At last, we would like to show that, thanks to their versatility, HRMS instruments are the most appropriate to achieve optimal metabolome coverage, at the border of other omics fields such as lipidomics and glycomics., (© 2013 Wiley Periodicals, Inc.)

This short review considers the range of modern techniques for the hydrodynamic characterisation of macromolecules - particularly large glycosylated systems used in the food, biopharma and healthcare industries. The range or polydispersity of molecular weights and conformations presents special challenges compared to proteins. The review is aimed, without going into any great theoretical or methodological depth, to help the Industrial Biotechnologist choose the appropriate methodology or combination of methodologies for providing the detail he/she needs for particular applications.

Voltammetric techniques have emerged as powerful methods for the determination and speciation of trace and ultratrace elements without any preconcentration in several research fields. Nevertheless, large sample volumes are typically required (10 mL), which strongly limits their application and/or the precision of the results. In this work, we report a 20-fold reduction in sample size for trace and ultratrace elemental determination and speciation by conventional voltammetric instrumentation, introducing the lowest amount of sample (0.5 mL) in which ultratrace detection has been performed up to now. This goal was achieved by a careful design of a new sample holder. Reliable, validated results were obtained for the determination of trace/ultratrace elements in rainwater (Cd, Co, Cu, Ni, Pb) and seawater (Cu). Moreover, copper speciation in seawater samples was consistently determined by competitive ligand equilibration-cathodic stripping voltammetry (CLE-CSV). The proposed apparatus showed several advantages: (1) 20-fold reduction in sample volume (the sample size is lowered from 120 to 6 mL for the CLE-CSV procedure); (2) decrease in analysis time due to the reduction in purging time up to 2.5 fold; (3) 20-fold drop in reagent consumption. Moreover, the analytical performances were not affected: similar detection capabilities, precision and accuracy were obtained. Application to sample of limited availability (e.g. porewaters, snow, rainwater, open ocean water, biological samples) and to the description of high resolution temporal trends may be easily foreseen., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This paper gives an overview of the advantages and associated caveats of the most common sample handling methods in surface-sensitive chemical and biological sensing. We summarize the basic theoretical and practical considerations one faces when designing and assembling the fluidic part of the sensor devices. The influence of analyte size, the use of closed and flow-through cuvettes, the importance of flow rate, tubing length and diameter, bubble traps, pressure-driven pumping, cuvette dead volumes, and sample injection systems are all discussed. Typical application areas of particular arrangements are also highlighted, such as the monitoring of cellular adhesion, biomolecule adsorption-desorption and ligand-receptor affinity binding. Our work is a practical review in the sense that for every sample handling arrangement considered we present our own experimental data and critically review our experience with the given arrangement. In the experimental part we focus on sample handling in optical waveguide lightmode spectroscopy (OWLS) measurements, but the present study is equally applicable for other biosensing technologies in which an analyte in solution is captured at a surface and its presence is monitored. Explicit attention is given to features that are expected to play an increasingly decisive role in determining the reliability of (bio)chemical sensing measurements, such as analyte transport to the sensor surface; the distorting influence of dead volumes in the fluidic system; and the appropriate sample handling of cell suspensions (e.g. their quasi-simultaneous deposition). At the appropriate places, biological aspects closely related to fluidics (e.g. cellular mechanotransduction, competitive adsorption, blood flow in veins) are also discussed, particularly with regard to their models used in biosensing., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The widespread use of wormlike micellar solutions is commonly found in household items such as cosmetic products, industrial fluids used in enhanced oil recovery and as drag reducing agents, and in biological applications such as drug delivery and biosensors. Despite their extensive use, there are still many details about the microscopic micellar structure and the mechanisms by which wormlike micelles form under flow that are not clearly understood. Microfluidic devices provide a versatile platform to study wormlike micellar solutions under various flow conditions and confined geometries. A review of recent investigations using microfluidics to study the flow of wormlike micelles is presented here with an emphasis on three different flow types: shear, elongation, and complex flow fields. In particular, we focus on the use of shear flows to study shear banding, elastic instabilities of wormlike micellar solutions in extensional flow (including stagnation and contraction flow field), and the use of contraction geometries to measure the elongational viscosity of wormlike micellar solutions. Finally, we showcase the use of complex flow fields in microfluidics to generate a stable and nanoporous flow-induced structured phase (FISP) from wormlike micellar solutions. This review shows that the influence of spatial confinement and moderate hydrodynamic forces present in the microfluidic device can give rise to a host of possibilities of microstructural rearrangements and interesting flow phenomena., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Rationale: Carbonate clumped isotope thermometry is a powerful new technique increasingly used in many fields in earth science. Recently, it has been shown that clumped isotope measurements can be performed with a Kiel carbonate preparation device and micro-volume analyses, allowing measurements of small (1.5-2 mg) carbonate samples. However, common data correction schemes rely on measurements of gases prepared offline, potentially leading to unrecognized biases in the results., Methods: We propose a new correction scheme for the Kiel device method including: (1) A pressure-sensitive baseline correction (PBL) of the raw beam signals; (2) Transfer of data to the absolute reference frame; (3) Correction for acid fractionation; (4) Correction for average standard offsets; (5) When necessary, correction for Δ47 scale compression based on offsets among standards with different ordering state. The long-term performance of the new scheme was tested with a large set of standard measurements (N = 432) obtained over the course of 15 months., Results: The PBL correction reliably removes composition-dependent artifacts, which are commonly corrected for with gas measurements, and offsets observed in micro-volume measurements when ion beams are imbalanced. We show that the shape of the PBL can vary strongly and needs to be properly characterized. Combined PBL and standard correction resulted in long-term stability with standard deviations in Δ47 of 0.012-0.016 ‰ for the five standards over the whole period, close to the average error of 0.011 ‰ observed for individual measurements consisting of 10 replicate analyses., Conclusions: Our correction scheme eliminates the need for routine gas measurements, allowing for equal treatment of samples and standards with the Kiel device setup. While the PBL and standard data obtained over 15 months reveal variable mass spectrometer behavior, they provide a robust means of correction, yielding reproducible results from small carbonate samples in the long term., (Copyright © 2014 John Wiley & Sons, Ltd.)

A method for structural elucidation of biomolecules dating to the 1980s utilized high-energy collisions (~10 keV, laboratory frame) that induced charge-remote fragmentations (CRF), a class of fragmentations particularly informative for lipids, steroids, surfactants, and peptides. Unfortunately, the capability for high-energy activation has largely disappeared with the demise of magnetic sector instruments. With the latest designs of tandem time-of-flight mass spectrometers (TOF/TOF), however, this capability is now being restored to coincide with the renewed interest in metabolites and lipids, including steroid-sulfates and other steroid metabolites. For these metabolites, structure determinations are required at concentration levels below that appropriate for NMR. To meet this need, we explored CRF with TOF/TOF mass spectrometry for two groups of steroid sulfates, 3-sulfates and 21-sulfates. We demonstrated that the current generation of MALDI TOF/TOF instruments can generate charge-remote fragmentations for these materials. The resulting collision-induced dissociation (CID) spectra are useful for positional isomer differentiation and very often allow the complete structure determination of the steroid. We also propose a new nomenclature that directly indicates the cleavage sites on the steroid ring with carbon numbers.

When the heart is stretched there is an abrupt increase in force that coincides with the stretch that is then followed by a second phase that develops slowly over a period of minutes, known as the "slow force response", or "SFR". The SFR is accompanied by a slow increase in the Ca(2+) transients, but the underlying cellular mechanisms are still debated. We therefore devised a simple bioassay system to investigate whether stretch released autocrine/paracrine factors that could explain the stretch-dependent Ca(2+) influx during the SFR. Our analysis was based around a metabolomics approach using tandem mass spectrometry whereby secreted compounds were analysed by liquid chromatography mass spectrometry (LC-MS/MS) at high resolution and accurate mass. Our bioassay consisted of two isolated cardiac preparations in series. A sustained stretch was applied to an "upstream" Langendorff-perfused heart, whilst monitoring the performance of an un-stretched, "downstream" trabecula perfused with coronary effluent collected from the "upstream" heart. Switching from "un-stretched" to "stretched" coronary effluent increased the amplitude of the Ca(2+) transients (340/380) fura-2 ratio) in the "downstream" trabeculae from 0.87 ± 0.04 to 0.94 ± 0.05 (p < 0.05, n = 4), and active stress (mN mm(-2)) from 15.4 ± 1.5 to 19.2 ± 1.9 (p < 0.01, n = 13), confirming the stretch-dependent release of one or more positively inotropic factor(s). To identify the factor, coronary effluent samples were concentrated by Solid Phase Extraction (SPE) treatment, lyophilised, then analysed using a Thermo Finnigan LTQ FT-ICR hybrid mass spectrometer. Differential profile data was obtained initially followed by targeting specific compounds at elevated levels in the stretched coronary effluent samples. Quantitative and statistical analyses of the differential LC-MS/MS results were then carried out using SIEVE 2.1, statistical software capable of performing differential analysis of peptides and small molecules. A number of substances were identified that were different between stretched and unstretched effluents (p < 0.01)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

U.S. food imports have been increasing steadily for decades, intensifying the need for a rapid and sensitive screening technique. A method has been developed that uses foam disks to sample the surface of incoming produce. This work provides complimentary information to the extensive amount of published pesticide fragmentation data collected using LCMS systems (Sack et al. Journal of Agricultural and Food Chemistry, 59, 6383-6411, 2011; Mol et al. Analytical and Bioanalytical Chemistry, 403, 2891-2908, 2012). The disks are directly analyzed using transmission-mode direct analysis in real time (DART) ambient pressure desorption ionization coupled to a high resolution accurate mass-mass spectrometer (HRAM-MS). In order to provide more certainty in the identification of the pesticides detected, a library of accurate mass fragments and isotopes of the protonated parent molecular ion (the [M+H]⁺) has been developed. The HRAM-MS is equipped with a quadrupole mass filter, providing the capability of "data-dependent" fragmentation, as opposed to "all -ion" fragmentation (where all of the ions enter a collision chamber and are fragmented at once). A temperature gradient for the DART helium stream and multiple collision energies were employed to detect and fragment 164 pesticides of varying chemical classes, sizes, and polarities. The accurate mass information of precursor ([M+H]⁺ ion) and fragment ions is essential in correctly identifying chemical contaminants on the surface of imported produce. Additionally, the inclusion of isotopes of the [M+H]⁺ in the database adds another metric to the confirmation process. The fragmentation data were collected using a Q-Exactive mass spectrometer and were added to a database used to process data collected with an Exactive mass spectrometer, an instrument that is more readily available for this screening application. The commodities investigated range from smooth-skinned produce such as apples to rougher surfaces like broccoli. The minimal sample preparation and absence of chromatography has shortened the analysis time to about 15 min per sample, and the simplicity and robustness of the technique make it ideal for rapid screening.