Minute Samples Research Articles

Development of MoSe₂ Nano-Urchins as a Sensing Platform for a Selective Bio-Capturing of Escherichia. coli Shiga Toxin DNA.

The present study was aimed to develop “fluorine doped” tin oxide glass electrode with a MoSe2 nano-urchin based electrochemical biosensor for detection of Escherichia coli Shiga toxin DNA. The study comprises two conductive electrodes, and the working electrodes were drop deposited using MoSe2 nano-urchin, and DNA sequences specific to Shiga toxin Escherichia coli. Morphological characterizations were performed using Fourier transforms infrared spectrophotometer; X-ray diffraction technique and scanning electron microscopy. All measurements were done using methylene blue as an electrochemical indicator. The proposed electrochemical geno-sensor showed good linear detection range of 1 fM–100 µM with a low detection limit of 1 fM where the current response increased linearly with Escherichia coli Shiga toxin dsDNA concentration with R2 = 0.99. Additionally, the real sample was spiked with the dsDNA that shows insignificant interference. The results revealed that the developed sensing platform significantly improved the sensitivity and can provide a promising platform for effective detection of biomolecules using minute samples due to its stability and sensitivity.

A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow‐up personalized cancer therapy. Fine‐needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n = 25) or benign lesions (n = 33), we demonstrate that these FNA‐based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11‐protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

Optimization of a capillary zone electrophoresis-contactless conductivity detection method for the determination of nisin.

Determination of natural preservatives using electrophoretic or chromatographic techniques in fermented milk products is a complex task due to the following reasons: (i) the concentrations of the analytes can be below the detection limits, (ii) complex matrix and comigrating/coeluting compounds in the sample can interfere with the analytes of the interest, (iii) low recovery of the analytes, and (iv) the necessity of complex sample preparation. The aim of this study was to apply capillary zone electrophoresis coupled with contactless conductivity detection for the separation and determination of nisin in fermented milk products. In this work, separation and determination of natural preservative-nisin in fermented milk products is described. Optimized conditions using capillary zone electrophoresis coupled with capacitance-to-digital technology based contactless conductivity detector and data conditioning, which filter the noise of the electropherogram adaptively to the peak migration time, allowed precise, accurate, sensitive (limit of quantification: 0.02μg/mL), and most importantly requiring very minute sample preparation, determination of nisin. Sample preparation includes following steps: (i) extraction/dilution and (ii) centrifugation. This method was applied for the determination of nisin in real samples, i.e. fermented milk products. The values of different nisin forms were ranging from 0.056±0.003μg/mL to 9.307±0.437μg/g.

PreDicta chip-based high resolution diagnosis of rhinovirus-induced wheeze

Rhinovirus (RV) infections are major triggers of acute exacerbations of severe respiratory diseases such as pre-school wheeze, asthma and chronic obstructive pulmonary disease (COPD). The occurrence of numerous RV types is a major challenge for the identification of the culprit virus types and for the improvement of virus type-specific treatment strategies. Here, we develop a chip containing 130 different micro-arrayed RV proteins and peptides and demonstrate in a cohort of 120 pre-school children, most of whom had been hospitalized due to acute wheeze, that it is possible to determine the culprit RV species with a minute blood sample by serology. Importantly, we identify RV-A and RV-C species as giving rise to most severe respiratory symptoms. Thus, we have generated a chip for the serological identification of RV-induced respiratory illness which should be useful for the rational development of preventive and therapeutic strategies targeting the most important RV types.

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis.

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d-1) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

Sound-mapping a coniferous forest-Perspectives for biodiversity monitoring and noise mitigation.

Acoustic diversity indices have been proposed as low-cost biodiversity monitoring tools. The acoustic diversity of a soundscape can be indicative of the richness of an acoustic community and the structural/vegetation characteristics of a habitat. There is a need to apply these methods to landscapes that are ecologically and/or economically important. We investigate the relationship between the acoustic properties of a coniferous forest with stand-age and structure. We sampled a 73 point grid in part of the UK’s largest man-made lowland coniferous plantation forest, covering a 320ha mosaic of different aged stands. Forest stands ranged from 0–85 years old providing an age-gradient. Short soundscape recordings were collected from each grid point on multiple mornings (between 6am-11am) to capture the dawn chorus. We repeated the study during July/August in 2014 and again in 2015. Five acoustic indices were calculated for a total of 889 two minute samples. Moderate relationships between acoustic diversity with forest stand-age and vegetation characteristics (canopy height; canopy cover) were observed. Ordinations suggest that as structural complexity and forest age increases, the higher frequency bands (4-10KHz) become more represented in the soundscape. A strong linear relationship was observed between distance to the nearest road and the ratio of anthropogenic noise to biological sounds within the soundscape. Similar acoustic patterns were observed in both years, though acoustic diversity was generally lower in 2014, which was likely due to differences in wind conditions between years. Our results suggest that developing these relatively low-cost acoustic monitoring methods to inform adaptive management of production landscapes, may lead to improved biodiversity monitoring. The methods may also prove useful for modelling road noise, landscape planning and noise mitigation.

A hook effect-free immunochromatographic assay (HEF-ICA) for measuring the C-reactive protein concentration in one drop of human serum

The immunochromatographic (ICA) assay is a highly promising platform for rapid and simple detection of C-reactive protein (CRP) which is an indicator of the different phases of various diseases, as well as of inflammation and infection. However, the hook effect in the ICA assay limits the quantification of CRP levels at high CRP concentrations.Methods: In this study, we developed a hook effect-free immunochromatographic assay (HEF-ICA) to detect CRP over a wide concentration range. The hook effect results from the simultaneous reaction of an excess target antigens with both immobilized and labeled antibodies respectively. To reduce the potential occurrence of this simultaneous reaction, we separated the migration of the target antigen and gold nanoparticle (AuNP)-labeled antibodies on a nitrocellulose membrane and analyzed the time profiles by modifying the ICA structure.Results: The signal intensity of HEF-ICA was saturated at high CRP concentrations, without decreasing. The titration curve of HEF-ICA was adjusted with the Hill equation, and HEF-ICA was performed with the following parameters: limit of detection, 43 ng mL-1; dynamic range, 119 ng mL-1 to 100 µg mL-1. The accuracy of the newly developed assay was evaluated using 33 clinical samples via comparison with a clinical chemistry analyzer.Conclusion: HEF-ICA enabled the measurement of a wide range of CRP concentrations without the hook effect, and was suitable for point-of-care testing with fingertip blood sampling, as only a minute sample volume (2.5 µL) was required.

Issues in data expansion in understanding criticality in biological systems.

At the point of a second-order phase transition also termed as a critical point, systems display long-range order and their macroscopic behaviors are independent of the microscopic details making up the system. Due to these properties, it has long been speculated that biological systems that show similar behavior despite having very different microscopics, may be operating near a critical point. Recent methods in neuroscience are making it possible to explore whether criticality exists in neural networks. Despite being large in size, many datasets are only a minute sample of the neural system and methods have to be developed to expand these datasets to study criticality. In this work we develop an analytical method of expanding a dataset to the large N limit to make statements about the critical nature of the dataset. We show that different ways of expanding the dataset while keeping its variance and mean fixed yield different results regarding criticality. This hence casts doubts on the established procedures for deducing criticality of biological systems through expansion of finite-sized datasets.

Fast and complete electrochemical conversion of solutes contained in micro-volume water droplets

An elegant hanging-droplet or meniscus-based setup is proposed to carry out quantitative electrolyses from either an organic (hydroquinone) or an inorganic (permanganate) substrate. These examples validate the concept of using such easily accessible, fast (1–3min) and low-cost operating conditions not only for preparative applications (electrosynthesis), but also for pedagogical purposes in minute samples.

Basin‐scale thermal and fluid flow histories revealed by carbonate clumped isotopes (Δ47) – Middle Jurassic carbonates of the Paris Basin depocentre

AbstractThe reconstruction of past diagenetic conditions in sedimentary basins is often under‐constrained. This results from both the analytical challenge of performing the required analyses on the minute sample amounts available from diagenetic mineral phases and the lack of tracers for some of the diagenetic parameters. The carbonate clumped isotope thermometry (Δ47) opens new perspectives for unravelling the temperatures of diagenetic phases together with the source of their parent fluids, two parameters that are otherwise impossible to constrain in the absence of exploitable fluid inclusions. Here is reported the study of a large number of sedimentary and diagenetic carbonate phases (from Middle Jurassic reservoirs of the Paris Basin depocentre) by combining detailed petrographic observations with a large number of Δ47 data (n &gt; 45) on a well‐documented paragenetic sequence, including calcite and dolomite burial cements. The data reveal carbonate crystallization at temperatures between 29°C and 98°C from fluids with δ18Owater values between −7‰ and +2‰, in response to the progressive burial and uplift of the Paris Basin, throughout 165 Myr of basin evolution. Coupled with the time–temperature evolution previously estimated from thermal maturity modelling, these temperatures allow determining the timing of four successive cementation episodes. The overall data set indicates a history of complex water mixing with a significant contribution of hypersaline waters from the Triassic aquifers migrated upward along faults during the Cretaceous subsidence of the basin. Subsequent large‐scale infiltrations of meteoric waters induced a dilution of these pre‐existing brines in response to the Paris Basin uplift in the Tertiary. Overall, the data presented here allow proposing an integrated approach to characterize the cementation events affecting the studied carbonate reservoir units, based on temperature, oxygen isotope composition and salinity of the parent fluids as well as on petrographic grounds.

Testosterone-like immunoreactivity in hair measured in minute sample amounts - a competitive radioimmunoassay with an adequate limit of detection

The concentrations of testosterone deposited in hair during hair growth may provide a retrospective reflection of the concentrations of bioactive testosterone in plasma. The objective of this study was to develop a radioimmunoassay with a sufficiently low limit of detection to measure the testosterone-like immunoreactivity in smaller hair samples (5 mg) than used in earlier studies, and to compare three different extraction procedures. The competitive radioimmunoassay consisted of a polyclonal antiserum (immunogen testosterone-7α-BSA) and a radioligand synthesised from testosterone-3-CMO-histamine. The within-assay and total coefficients of variation in the working range was 3% and 4.5%, respectively. The limit of detection was 0.87 pg/mL, which is equivalent to 0.12 pg/mg testosterone in 5 mg of hair. The concentration of testosterone-like immunoreactivity in hair samples was 1.23 (SD 0.47) pg/mg in women and 2.67 (SD 0.58) pg/mg in men (pulverised hair). Significantly improved precision was found when pulverised hair was used compared to non-pulverised hair. Our data indicate that pulverisation of the hair prior to hormone extraction is crucial. Detection limits fit for the intended purpose are achievable with 5 mg samples of hair.

Immobilized-enzyme reactors integrated with capillary electrophoresis forpharmaceutical research.

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized-enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis-integrated immobilized-enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug-enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.

Sensitive and selective quantification of free and total malondialdehyde in plasma using UHPLC-HRMS

Quantification of malondialdehyde (MDA) as a marker of lipid peroxidation is relevant for many research fields. We describe a new sensitive and selective method to measure free and total plasmatic MDA using derivatization with 2,4-dinitrophenylhydrazine (DNPH) and ultra-HPLC-high-resolution MS. Free and total MDA were extracted from minute sample amounts (10 μl) using acidic precipitation and alkaline hydrolysis followed by acidic precipitation, respectively. Derivatization was completed within 10 min at room temperature, and the excess DNPH discarded by liquid-liquid extraction. Quantification was achieved by internal standardization using dideuterated MDA as internal standard. The method's lowest limit of quantification was 100 nM and linearity spanned greater than three orders of magnitude. Intra- and inter-day precisions for total MDA were 2.9% and 3.0%, respectively, and those for free MDA were 12.8% and 24.9%, respectively. Accuracy was 101% and 107% at low and high concentrations, respectively. In human plasma, free MDA levels were 120 nM (SD 36.26) and total MDA levels were 6.7 μM (SD 0.46). In addition, we show the applicability of this method to measure MDA plasma levels from a variety of animal species, making it invaluable to scientists in various fields.

A point-of-care microfluidic biochip for quantification of CD64 expression from whole blood for sepsis stratification

Sepsis, a potentially life-threatening complication of an infection, has the highest burden of death and medical expenses in hospitals worldwide. Leukocyte count and CD64 expression on neutrophils (nCD64) are known to correlate strongly with improved sensitivity and specificity of sepsis diagnosis at its onset. A major challenge is the lack of a rapid and accurate point-of-care (PoC) device that can perform these measurements from a minute blood sample. Here, we report a PoC microfluidic biochip to enumerate leukocytes and quantify nCD64 levels from 10 μl of whole blood without any manual processing. Biochip measurements have shown excellent correlation with the results from flow cytometer. In clinical studies, we have used PoC biochip to monitor leukocyte counts and nCD64 levels from patients’ blood at different times of their stay in the hospital. Furthermore, we have shown the biochip’s utility for improved sepsis diagnosis by combining these measurements with electronic medical record (EMR).

Fluorination technique to identify the type of resin in aged vanishes and lacquers using infrared spectroscopy

Infrared spectroscopy (FTIR) is a standard technique for the characterisation of organic materials in conservation science. This analytical technique is particularly interesting to analyse historical varnishes from cultural artefacts, as only minute samples are required. Some limitations apply, however, when analysing such chemically complex multi-component materials with interfering absorption bands. A previously introduced sample pre-treatment method using gaseous sulfur tetrafluoride SF4 has shown to widen the application fields of the IR-technique by improving its specificity. One of these new fields is its successful application to the characterisation of complex aged natural varnish and lacquer systems. The fluorination treatment leads to characteristic band shifts of four individual saturated and unsaturated acid groups attached at varying positions of the terpene backbone. This allows the separation of otherwise overlapping carbonyl bands of different ketones and ester groups. Based on this technique, inert and unique functional groups can be used as material specific markers to identify drying oil, shellac and different terpene resins in varnishes and lacquers. The proposed markers proved to be stable for several hundred years. These findings are particularly interesting, as it is now possible to trace such markers even in very old varnishes dating back to the 16th century – with a likewise simple technique.

Surface Acoustic Wave Nebulisation Mass Spectrometry for the Fast and Highly Sensitive Characterisation of Synthetic Dyes in Textile Samples

Surface acoustic wave nebulisation (SAWN) mass spectrometry (MS) is a method to generate gaseous ions compatible with direct MS of minute samples at femtomole sensitivity. To perform SAWN, acoustic waves are propagated through a LiNbO3 sampling chip, and are conducted to the liquid sample, which ultimately leads to the generation of a fine mist containing droplets of nanometre to micrometre diameter. Through fission and evaporation, the droplets undergo a phase change from liquid to gaseous analyte ions in a non-destructive manner. We have developed SAWN technology for the characterisation of organic colourants in textiles. It generates electrospray-ionisation-like ions in a non-destructive manner during ionisation, as can be observed by the unmodified chemical structure. The sample size is decreased by tenfold to 1000-fold when compared with currently used liquid chromatography–MS methods, with equal or better sensitivity. This work underscores SAWN-MS as an ideal tool for molecular analysis of art objects as it is non-destructive, is rapid, involves minimally invasive sampling and is more sensitive than current MS-based methods.Graphical ᅟ

Early prediction of non-response to anti-depressive treatment with an easy-to-use electrophysiological index dynamics

IntroductionThe evaluation of response to pharmacological treatment in MDD requires 6–8 weeks. Therefore, the ability to predict response, and especially lack of response to treatment, as early as possible after treatment onset or change, is of major significance. Many studies demonstrated significant results regarding the ability to use EEG and ERP markers. However, these markers are derived from long EEG/ERP samples, often from multiple channels, which render them impractical for frequent sampling.MethodsWe developed a new electrophysiological attention-related marker from a single channel (2 electrodes) and 1 minute samples. This work presents an initial evaluation of the ability to harness this marker, for early differentiation between responders and non-responders to anti-depressive treatment, in 26 patients with various levels of depression and heterogeneous treatment interventions and 10 healthy controls. Subjects who initiated treatment for depression were followed clinically through their Hamilton depression scores as well as their EEG activity twice a week for a period of 8 weeks. Any acceptable anti-depressive treatment been included. The improvement in Hamilton scores at the end of 8 weeks used to discriminate responders and non-responders.ResultsWithin two weeks, we could differentiate between non-responders and responders to anti-depressive treatment, with absolute discrimination between subjects with moderate to severe depression, and with 0.71 sensitivity and 0.96 specificity within the whole depressed subjects.ConclusionsThis is a proof of concept for an easy to use, cheap and quick marker for the lack of respond to anti-depressive treatment within two weeks of anti-depressive treatment.

Peptidomic and transcriptomic profiling of four distinct spider venoms.

Venom based research is exploited to find novel candidates for the development of innovative pharmacological tools, drug candidates and new ingredients for cosmetic and agrochemical industries. Moreover, venomics, as a well-established approach in systems biology, helps to elucidate the genetic mechanisms of the production of such a great molecular biodiversity. Today the advances made in the proteomics, transcriptomics and bioinformatics fields, favor venomics, allowing the in depth study of complex matrices and the elucidation even of minor compounds present in minute biological samples. The present study illustrates a rapid and efficient method developed for the elucidation of venom composition based on NextGen mRNA sequencing of venom glands and LC-MS/MS venom proteome profiling. The analysis of the comprehensive data obtained was focused on cysteine rich peptide toxins from four spider species originating from phylogenetically distant families for comparison purposes. The studied species were Heteropoda davidbowie (Sparassidae), Poecilotheria formosa (Theraphosidae), Viridasius fasciatus (Viridasiidae) and Latrodectus mactans (Theridiidae). This led to a high resolution profiling of 284 characterized cysteine rich peptides, 111 of which belong to the Inhibitor Cysteine Knot (ICK) structural motif. The analysis of H. davidbowie venom revealed a high richness in term of venom diversity: 95 peptide sequences were identified; out of these, 32 peptides presented the ICK structural motif and could be classified in six distinct families. The profiling of P. formosa venom highlighted the presence of 126 peptide sequences, with 52 ICK toxins belonging to three structural distinct families. V. fasciatus venom was shown to contain 49 peptide sequences, out of which 22 presented the ICK structural motif and were attributed to five families. The venom of L. mactans, until now studied for its large neurotoxins (Latrotoxins), revealed the presence of 14 cysteine rich peptides, out of which five were ICK toxins belonging to the CSTX superfamily. This in depth profiling of distinct ICK peptide families identified across the four spider species highlighted the high conservation of these neurotoxins among spider families.

Efficient, high-resolution resonance laser ionization spectroscopy using weak transitions to long-lived excited states

Laser spectroscopic studies on minute samples of exotic radioactive nuclei require very efficient experimental techniques. In addition, high resolving powers are required to allow extraction of nu- clear structure information. Here we demonstrate that by using weak atomic transitions, resonance laser ionization spectroscopy is achieved with the required high efficiency (1-10%) and precision (linewidths of tens of MHz). We illustrate experimentally and through the use of simulations how the narrow experimental linewidths are achieved and how distorted resonance ionization spec- troscopy lineshapes can be avoided. The role of the delay of the ionization laser pulse with respect to the excitation laser pulse is crucial: the use of a delayed ionization step permits the best resolving powers and lineshapes. A high efficiency is maintained if the intermediate level has a lifetime that is at least of the order of the excitation laser pulse width. A model that describes this process re- produces well the observed features and will help to optimize the conditions for future experiments.

Label-free Detection of Influenza Viruses using a Reduced Graphene Oxide-based Electrochemical Immunosensor Integrated with a Microfluidic Platform

Reduced graphene oxide (RGO) has recently gained considerable attention for use in electrochemical biosensing applications due to its outstanding conducting properties and large surface area. This report presents a novel microfluidic chip integrated with an RGO-based electrochemical immunosensor for label-free detection of an influenza virus, H1N1. Three microelectrodes were fabricated on a glass substrate using the photolithographic technique, and the working electrode was functionalized using RGO and monoclonal antibodies specific to the virus. These chips were integrated with polydimethylsiloxane microchannels. Structural and morphological characterizations were performed using X-ray photoelectron spectroscopy and scanning electron microscopy. Electrochemical studies revealed good selectivity and an enhanced detection limit of 0.5 PFU mL−1, where the chronoamperometric current increased linearly with H1N1 virus concentration within the range of 1 to 104 PFU mL−1 (R2 = 0.99). This microfluidic immunosensor can provide a promising platform for effective detection of biomolecules using minute samples.