Showing papers in "Analytical and Bioanalytical Chemistry in 2015"

Review of plasmonic fiber optic biochemical sensors: improving the limit of detection.

Abstract: This paper presents a brief overview of the technologies used to implement surface plasmon resonance (SPR) effects into fiber-optic sensors for chemical and biochemical applications and a survey of results reported over the last ten years. The performance indicators that are relevant for such systems, such as refractometric sensitivity, operating wavelength, and figure of merit (FOM), are discussed and listed in table form. A list of experimental results with reported limits of detection (LOD) for proteins, toxins, viruses, DNA, bacteria, glucose, and various chemicals is also provided for the same time period. Configurations discussed include fiber-optic analogues of the Kretschmann–Raether prism SPR platforms, made from geometry-modified multimode and single-mode optical fibers (unclad, side-polished, tapered, and U-shaped), long period fiber gratings (LPFG), tilted fiber Bragg gratings (TFBG), and specialty fibers (plastic or polymer, microstructured, and photonic crystal fibers). Configurations involving the excitation of surface plasmon polaritons (SPP) on continuous thin metal layers as well as those involving localized SPR (LSPR) phenomena in nanoparticle metal coatings of gold, silver, and other metals at visible and near-infrared wavelengths are described and compared quantitatively.

Non-target screening with high-resolution mass spectrometry: critical review using a collaborative trial on water analysis

Abstract: In this article, a dataset from a collaborative non-target screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target screening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches and methods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a “fully-automated identification workflow” remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution of more spectra from standard substances into (openly accessible) databases.

Recent advances in quantitative LA-ICP-MS analysis: challenges and solutions in the life sciences and environmental chemistry

Abstract: Laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) is a widely accepted method for direct sampling of solid materials for trace elemental analysis. The number of reported applications is high and the application range is broad; besides geochemistry, LA-ICP-MS is mostly used in environmental chemistry and the life sciences. This review focuses on the application of LA-ICP-MS for quantification of trace elements in environmental, biological, and medical samples. The fundamental problems of LA-ICP-MS, such as sample-dependent ablation behavior and elemental fractionation, can be even more pronounced in environmental and life science applications as a result of the large variety of sample types and conditions. Besides variations in composition, the range of available sample states is highly diverse, including powders (e.g., soil samples, fly ash), hard tissues (e.g., bones, teeth), soft tissues (e.g., plants, tissue thin-cuts), or liquid samples (e.g., whole blood). Within this article, quantification approaches that have been proposed in the past are critically discussed and compared regarding the results obtained in the applications described. Although a large variety of sample types is discussed within this article, the quantification approaches used are similar for many analytical questions and have only been adapted to the specific questions. Nevertheless, none of them has proven to be a universally applicable method.

Effects of pre-analytical processes on blood samples used in metabolomics studies

Abstract: Every day, analytical and bio-analytical chemists make sustained efforts to improve the sensitivity, specificity, robustness, and reproducibility of their methods Especially in targeted and non-targeted profiling approaches, including metabolomics analysis, these objectives are not easy to achieve; however, robust and reproducible measurements and low coefficients of variation (CV) are crucial for successful metabolomics approaches Nevertheless, all efforts from the analysts are in vain if the sample quality is poor, ie if preanalytical errors are made by the partner during sample collection Preanalytical risks and errors are more common than expected, even when standard operating procedures (SOP) are used This risk is particularly high in clinical studies, and poor sample quality may heavily bias the CV of the final analytical results, leading to disappointing outcomes of the study and consequently, although unjustified, to critical questions about the analytical performance of the approach from the partner who provided the samples This review focuses on the preanalytical phase of liquid chromatography–mass spectrometry-driven metabolomics analysis of body fluids Several important preanalytical factors that may seriously affect the profile of the investigated metabolome in body fluids, including factors before sample collection, blood drawing, subsequent handling of the whole blood (transportation), processing of plasma and serum, and inadequate conditions for sample storage, will be discussed In addition, a detailed description of latent effects on the stability of the blood metabolome and a suggestion for a practical procedure to circumvent risks in the preanalytical phase will be given

A novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor for sensitive detection of prostate specific antigen.

Abstract: Prostate specific antigen (PSA) is a significant and the most widely used biomarker for the early diagnosis of prostate cancer and its subsequent treatment. A MoS2 nanosheet is a two-dimensional (2D) layered nanomaterial analogous to graphene. However, a MoS2 nanosheet has a higher fluorescence-quenching ability than graphene when applied to a dye-labeled single-stranded DNA probe. In this work, we propose a novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor that detects PSA. The binding of the aptamer to the target PSA induces a rigid aptamer structure which makes the integration with the MoS2 nanosheet very weak. This results in the release of the aptamer probe from the nanosheet surface and restores the quenched fluorescence. This approach has the advantage of simple design and rapid detection of PSA. The biosensor has the merits of high sensitivity and high selectivity with a detection limit for the PSA of 0.2 ng/mL. The biosensor was also successfully applied to the detection of PSA in human serum samples with satisfactory results. The foregoing indicates its promising application to real-life biological samples.

Identification of microplastics by FTIR and Raman microscopy: a novel silicon filter substrate opens the important spectral range below 1300 cm−1 for FTIR transmission measurements

Abstract: The presence of microplastics in aquatic ecosystems is a topical problem and leads to the need of appropriate and reliable analytical methods to distinctly identify and to quantify these particles in environmental samples. As an example transmission, Fourier transform infrared (FTIR) imaging can be used to analyze samples directly on filters without any visual presorting, when the environmental sample was afore extracted, purified, and filtered. However, this analytical approach is strongly restricted by the limited IR transparency of conventional filter materials. Within this study, we describe a novel silicon (Si) filter substrate produced by photolithographic microstructuring, which guarantees sufficient transparency for the broad mid-infrared region of 4000–600 cm-1. This filter type features holes with a diameter of 10 μm and exhibits adequate mechanical stability. Furthermore, it will be shown that our Si filter substrate allows a distinct identification of the most common microplastics, polyethylene (PE), and polypropylene (PP), in the characteristic fingerprint region (1400–600 cm-1). Moreover, using the Si filter substrate, a differentiation of microparticles of polyesters having quite similar chemical structure, like polyethylene terephthalate (PET) and polybutylene terephthalate (PBT), is now possible, which facilitates a visualization of their distribution within a microplastic sample by FTIR imaging. Finally, this Si filter can also be used as substrate for Raman microscopy—a second complementary spectroscopic technique—to identify microplastic samples.

Label-free surface-enhanced Raman spectroscopy of biofluids: fundamental aspects and diagnostic applications.

Abstract: In clinical practice, one objective is to obtain diagnostic information while minimizing the invasiveness of the tests and the pain for the patients. To this end, tests based on the interaction of light with readily available biofluids including blood, urine, or saliva are highly desirable. In this review we examine the state of the art regarding the use of surface-enhanced Raman spectroscopy (SERS) to investigate biofluids, focusing on diagnostic applications. First, a critical evaluation of the experimental aspects involved in the collection of SERS spectra is presented; different substrate types are introduced, with a clear distinction between colloidal and non-colloidal metal nanostructures. Then the effect of the excitation wavelength is discussed, along with anomalous bands and artifacts which might affect SERS spectra of biofluids. The central part of the review examines the literature available on the SERS spectra of blood, plasma, serum, urine, saliva, tears, and semen. Finally, diagnostic applications are critically discussed in the context of the published evidence; this section clearly reveals that SERS of biofluids is most promising as a rapid, cheap, and non-invasive tool for mass screening for cancer.

Tissue protein imaging at 1 μm laser spot diameter for high spatial resolution and high imaging speed using transmission geometry MALDI TOF MS

Abstract: We have achieved protein imaging mass spectrometry capabilities at sub-cellular spatial resolution and at high acquisition speed by integrating a transmission geometry ion source with time of flight mass spectrometry. The transmission geometry principle allowed us to achieve a 1-μm laser spot diameter on target. A minimal raster step size of the instrument was 2.5 μm. Use of 2,5-dihydroxyacetophenone robotically sprayed on top of a tissue sample as a matrix together with additional sample preparation steps resulted in single pixel mass spectra from mouse cerebellum tissue sections having more than 20 peaks in a range 3–22 kDa. Mass spectrometry images were acquired in a standard step raster microprobe mode at 5 pixels/s and in a continuous raster mode at 40 pixels/s.

Size and shape characterization of hydrated and desiccated exosomes

Abstract: Exosomes are stable nanovesicles secreted by cells into the circulation. Their reported sizes differ substantially, which likely reflects the difference in the isolation techniques used, the cells that secreted them, and the methods used in their characterization. We analyzed the influence of the last factor on the measured sizes and shapes of hydrated and desiccated exosomes isolated from the serum of a pancreatic cancer patient and a healthy control. We found that hydrated exosomes are close-to-spherical nanoparticles with a hydrodynamic radius that is substantially larger than the geometric size. For desiccated exosomes, we found that the desiccated shape and sizing are influenced by the manner in which drying occurred. Isotropic desiccation in aerosol preserves the near-spherical shape of the exosomes, whereas drying on a surface likely distorts their shapes and influences the sizing results obtained by techniques that require surface fixation prior to analysis.

The many facets of Raman spectroscopy for biomedical analysis.

Abstract: A critical review is presented on the use of linear and nonlinear Raman microspectroscopy in biomedical diagnostics of bacteria, cells, and tissues. This contribution is combined with an overview of the achievements of our research group. Linear Raman spectroscopy offers a wealth of chemical and molecular information. Its routine clinical application poses a challenge due to relatively weak signal intensities and confounding overlapping effects. Nonlinear variants of Raman spectroscopy such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) have been recognized as tools for rapid image acquisition. Imaging applications benefit from the fact that contrast is based on the chemical composition and molecular structures in a label-free and nondestructive manner. Although not label-free, surface enhanced Raman scattering (SERS) has also been recognized as a complementary biomedical tool to increase sensitivity. The current state of the art is evaluated, illustrative examples are given, future developments are pointed out, and important reviews and references from the current literature are selected. The topics are identification of bacteria and single cells, imaging of single cells, Raman activated cell sorting, diagnosis of tissue sections, fiber optic Raman spectroscopy, and progress in coherent Raman scattering in tissue diagnosis. The roles of networks-such as Raman4clinics and CLIRSPEC on a European level-and early adopters in the translation, dissemination, and validation of new methods are discussed.

Applications of ion-mobility mass spectrometry for lipid analysis

Abstract: The high chemical complexity of the lipidome is one of the major challenges in lipidomics research. Ion-mobility spectrometry (IMS), a gas-phase electrophoretic technique, makes possible the separation of ions in the gas phase according to their charge, shape, and size. IMS can be combined with mass spectrometry (MS), adding three major benefits to traditional lipidomic approaches. First, IMS–MS allows the determination of the collision cross section (CCS), a physicochemical measure related to the conformational structure of lipid ions. The CCS is used to improve the confidence of lipid identification. Second, IMS–MS provides a new set of hybrid fragmentation experiments. These experiments, which combine collision-induced dissociation with ion-mobility separation, improve the specificity of MS/MS-based approaches. Third, IMS–MS improves the peak capacity and signal-to-noise ratio of traditional analytical approaches. In doing so, it allows the separation of complex lipid extracts from interfering isobaric species. Developing in parallel with advances in instrumentation, informatics solutions enable analysts to process and exploit IMS–MS data for qualitative and quantitative applications. Here we review the current approaches for lipidomics research based on IMS–MS, including liquid chromatography–MS and direct-MS analyses of “shotgun” lipidomics and MS imaging.

A critical review of recent progress in analytical laser-induced breakdown spectroscopy

Abstract: Laser-induced breakdown spectroscopy (LIBS) has become an established analytical atomic spectrometry technique and is valued for its very compelling set of advantageous analytical and technical characteristics. It is a rapid, versatile, non-contact technique, which is capable of providing qualitative and quantitative analytical information for practically any sample, in a virtually non-destructive way, without any substantial sample preparation. The instrumentation is simple, robust, compact, and even enables remote analysis. This review attempts to give a critical overview of the diverse progress of the field, focusing on the results of the last five years. The advancement of LIBS instrumentation and data evaluation is discussed in detail and selected results of some prominent applications are also described.

Improving surface and defect center chemistry of fluorescent nanodiamonds for imaging purposes-a review

Abstract: Diamonds are widely used for jewelry owing to their superior optical properties accounting for their fascinating beauty. Beyond the sparkle, diamond is highly investigated in materials science for its remarkable properties. Recently, fluorescent defects in diamond, particularly the negatively charged nitrogen-vacancy (NV-) center, have gained much attention: The NV- center emits stable, nonbleaching fluorescence, and thus could be utilized in biolabeling, as a light source, or as a Forster resonance energy transfer donor. Even more remarkable are its spin properties: with the fluorescence intensity of the NV- center reacting to the presence of small magnetic fields, it can be utilized as a sensor for magnetic fields as small as the field of a single electron spin. However, a reproducible defect and surface and defect chemistry are crucial to all applications. In this article we review methods for using nanodiamonds for different imaging purposes. The article covers (1) dispersion of particles, (2) surface cleaning, (3) particle size selection and reduction, (4) defect properties, and (5) functionalization and attachment to nanostructures, e.g., scanning probe microscopy tips.

Simultaneous determination of major type A and B trichothecenes, zearalenone and certain modified metabolites in Finnish cereal grains with a novel liquid chromatography-tandem mass spectrometric method

Abstract: A reliable and sensitive liquid chromatography-tandem mass spectrometric method was developed for the simultaneous quantitative determination in cereals of the Fusarium mycotoxins HT-2 toxin, T-2 toxin, deoxynivalenol, nivalenol and zearalenone, as well as the modified metabolites 3-acetyl-deoxynivalenol, α-zearalenol, β-zearalenol, deoxynivalenol-3-glucoside, HT-2-3-glucoside, nivalenol-3-glucoside, zearalenone-14-glucoside, zearalenone-14-sulphate, zearalenone-16-glucoside, α-zearalenol-14-glucoside and β-zearalenol-14-glucoside. The ‘dilute and shoot’ approach was used for sample preparation after extraction with acetonitrile:water:acetic acid (79:20:1, v/v/v). Separation was carried out using reversed-phase liquid chromatography, and detection was performed using tandem mass spectrometry in the selected reaction monitoring mode. The method was in-house validated according to performance characteristics, established in Commission Regulation EC No 401/2006 and Commission Decision EC No 657/2002, prior to its application in a nationwide survey for the analysis of barley, oat and wheat samples (n = 95) harvested in Finland during 2013. Deoxynivalenol and its glucosylated form were the most abundant of the analytes, being detected in 93 and 81 % of the samples, respectively. Concentrations of deoxynivalenol were unusually high in 2013, especially in oats, with some cases exceeding the maximum legislative limits for unprocessed oats placed on the market for first-stage processing. All modified mycotoxins analysed were detected, and the natural occurrence of some of these compounds (e.g. zearalenone-16-glucoside and nivalenol-3-glucoside) in barley, oats and/or wheat was documented for the first time.

Functionalizable low-fouling coatings for label-free biosensing in complex biological media: advances and applications

Abstract: This review focuses on recent advances in the development of functionalizable antifouling coatings and their applications in label-free optical biosensors. Approaches to the development of antifouling coatings, ranging from self-assembled monolayers and PEG derivatives to ultra-low-fouling polymer brushes, are reviewed. Methods of preparation and characterization of antifouling coatings and the functionalization of antifouling coatings with bioreceptors are reviewed, and the effect of functionalization on the fouling properties of biofunctional coating is discussed. Special attention is given to biofunctional coatings for label-free bioanalysis of blood plasma and serum for medical diagnostics.

Determination of bisphenol A and related substitutes/analogues in human breast milk using gas chromatography-tandem mass spectrometry

Abstract: Bisphenol A (BPA) is an industrial chemical widely used in the production of polycarbonate and epoxy resins. Identified as an endocrine-disrupting chemical (EDC), BPA is a matter of existing or ongoing restrictive regulations and then is increasingly being replaced by other analogues used as BPA’s substitutes. Human biomonitoring studies focusing on both BPA and emerging related analogues consequently appear as a requirement either for documenting the efficiency of regulatory actions toward BPA and for fuelling incoming risk assessment studies toward BPA’s substitutes. In particular, the increasing concern about the late effects consecutive to early exposures naturally identify human breast milk as a target biological matrix of interest for priority exposure assessment focused on critical sub-populations such as pregnant women, fetuses, and/or newborns. In this context, an accurate and sensitive analytical method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed for the quantification of 18 “BPA-like” compounds in breast milk samples at trace levels (<0.05 μg kg−1). The method includes a preliminary protein precipitation step followed by two successive solid-phase extraction (SPE) stages. Quantification of the targeted compounds was achieved according to the isotopic dilution method using 13C12-BPA as internal standard. The method was validated according to current EU guidelines and criteria. Linearity (R 2) was better than 0.99 for each molecule within the concentration range 0–5 μg kg−1. The detection and quantification limits ranged from 0.001 to 0.030 μg kg−1 and from 0.002 to 0.050 μg kg−1, respectively. The analytical method was successfully applied to the first set of human breast milk samples (n = 30) originating from French women in the Region Pays-de-la-Loire. The measured levels of BPA were found in the

Nontargeted SWATH acquisition for identifying 47 synthetic cannabinoid metabolites in human urine by liquid chromatography-high-resolution tandem mass spectrometry.

Abstract: Clandestine laboratories constantly produce new synthetic cannabinoids to circumvent legislative scheduling efforts, challenging and complicating toxicological analysis. Sundstrom et al. (Anal Bioanal Chem 405(26):8463-8474, [9]) and Kronstrand et al. (Anal Bioanal Chem 406(15):3599-3609, [10]) published nontargeted liquid chromatography, high-resolution, quadrupole/time-of-flight mass spectrometric (LC-QTOF) assays with validated detection of 18 and 38 urinary synthetic cannabinoid metabolites, respectively. We developed and validated a LC-QTOF urine method for simultaneously identifying the most current 47 synthetic cannabinoid metabolites from 21 synthetic cannabinoid families (5-fluoro AB-PINACA, 5-fluoro-AKB48, 5-fluoro PB-22, AB-PINACA, ADB-PINACA, AKB48, AM2201, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, JWH-398, MAM2201, PB-22, RCS-4, UR-144, and XLR11). β-Glucuronidase-hydrolyzed urine was extracted with 1-mL Biotage SLE+ columns. Specimens were reconstituted in 150-μL mobile phase consisting of 80% A (0.1% formic acid in water) and 20% B (0.1% formic acid in acetonitrile). Fifty microliters was injected, and SWATH™ MS data were acquired in positive electrospray mode. The LC-QTOF instrument consisted of a Shimadzu UFLCxr system and an ABSciex 5600+ TripleTOF® mass spectrometer. Gradient chromatographic separation was achieved with a Restek Ultra Biphenyl column with a 0.5-mL/min flow rate and an overall run time of 15 min. Identification criteria included molecular ion mass error, isotopic profiles, retention time, and library fit criteria. Limits of detection were 0.25-5 μg/L (N = 10 unique fortified urine samples), except for two PB-22 metabolites with limits of 10 and 20 μg/L. Extraction efficiencies and matrix effects (N = 10) were 55-104 and -65-107%, respectively. We present a highly useful novel LC-QTOF method for simultaneously confirming 47 synthetic cannabinoid metabolites in human urine.

Identification of lipopeptide isoforms by MALDI-TOF-MS/MS based on the simultaneous purification of iturin, fengycin, and surfactin by RP-HPLC

Abstract: A three-stage linear gradient strategy using reverse-phase high-performance liquid chromatography (HPLC) was optimized for rapid, high-quality, and simultaneous purification of the lipopeptide isoforms of iturin, fengycin, and surfactin, which may differ in composition by only a single amino acid and/or the fatty acid residue. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was applied to detect the lipopeptides harvested from each reversed-phase HPLC peak. Amino acid analysis based on phenyl isothiocyanate derivatization was further used for confirmation of the amino acid species and molar ratio in a certain HPLC fraction. By this MALDI-TOF-MS/MS coupled with amino acid analysis, it was revealed that iturin at m/z 1,043 consists of a circular Asn-Tyr-Asn-Gln-Pro-Asn-Ser peptide and C14 β-OH fatty acid. Surfactin homologs from Bacillus subtilis THY-7 at m/z 1,030, 1,044, 1,058, and 1,072 possess a circular Glu-Leu-Leu-Val-Asp-Leu-Leu peptide and the β-OH fatty acid with a different length (C13–C16). Fengycin species at m/z 1,463 and 1,477 are homologs possessing the circular peptide Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile linked to a C16 or C17 γ-OH fatty acid, whereas fengycin at m/z 1,505 contains a Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile sequence with a Val instead of Ala at position 6. The method developed in this work provided an efficient approach for characterization of diverse lipopeptide isoforms from the iturin, fengycin, and surfactin families.

Simultaneous determination of 148 pharmaceuticals and illicit drugs in sewage sludge based on ultrasound-assisted extraction and liquid chromatography-tandem mass spectrometry.

Abstract: This paper describes the development and validation of a new method for the simultaneous determination of 148 substances in sewage sludge. The selected compounds belong to different classes of pharmaceuticals, including antibiotics, analgesic and/or anti-inflammatory drugs, antiepileptics, benzodiazepines, antipsychotics, and antidepressants, among others, and illicit drugs, including opiates, opioids, cocaine, amphetamines, cannabinoids, and their metabolites. As far as the authors are aware, this is the first method in the peer-reviewed literature covering such a large number of target drugs for determination in a complex matrix like sewage sludge. The method presented herein combines ultrasound-assisted extraction (USE) and liquid chromatography coupled to tandem mass spectrometry. Good analytical performance was achieved, with limit-of-detection values below 10 ng g−1 d.w. for 91 % of the analytes and absolute recovery in the range 50–110 % for more than 77 % of the studied compounds. A combination of methanol and acidified water, also containing EDTA, proved to be the optimum solvent mixture to perform the extractions. An extra solid-phase-extraction clean-up step was not required, substantially reducing sample-preparation time and solvent consumption. Finally, the developed method was applied to the analysis of different sewage-sludge samples from five wastewater treatment plants of Santorini Island (Greece). Out of the 148 target compounds, 36 were detected. Several compounds, including acetylsalicylic acid, citalopram, and ciprofloxacin among others, had maximum concentrations above 100 ng g−1 d.w.

Ion selective optodes: from the bulk to the nanoscale

Abstract: This review describes recent advances in the miniaturization of ion selective optodes into microscale and nanoscale sensors. The topics include a comparison between film-based and miniaturized ion optodes, equilibrium and exhaustive detection modes, recent preparation methodologies and applications of microscale and nanoscale ion optodes, criteria for the design of optode sensors, and other future perspectives.

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials

Abstract: The value assignment for properties of six certified reference materials (ERM-AD623a–f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per μL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad’s new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.

Subcellular-level resolution MALDI-MS imaging of maize leaf metabolites by MALDI-linear ion trap-Orbitrap mass spectrometer.

Abstract: A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 μm for the commercial configuration down to ~9 μm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-μm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed.

Applying 'Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra' (SWATH) for systematic toxicological analysis with liquid chromatography-high-resolution tandem mass spectrometry.

Abstract: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an indispensable analytical technique in clinical and forensic toxicology for detection and identification of potentially toxic or harmful compounds. Particularly, non-target LC-MS/MS assays enable extensive and universal screening requested in systematic toxicological analysis. An integral part of the identification process is the generation of information-rich product ion spectra which can be searched against libraries of reference mass spectra. Usually, ‘data-dependent acquisition’ (DDA) strategies are applied for automated data acquisition. In this study, the ‘data-independent acquisition’ (DIA) method ‘Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra’ (SWATH) was combined with LC-MS/MS on a quadrupole-quadrupole-time-of-flight (QqTOF) instrument for acquiring informative high-resolution tandem mass spectra. SWATH performs data-independent fragmentation of all precursor ions entering the mass spectrometer in 21m/z isolation windows. The whole m/z range of interest is covered by continuous stepping of the isolation window. This allows numerous repeat analyses of each window during the elution of a single chromatographic peak and results in a complete fragment ion map of the sample. Compounds and samples typically encountered in forensic casework were used to assess performance characteristics of LC-MS/MS with SWATH. Our experiments clearly revealed that SWATH is a sensitive and specific identification technique. SWATH is capable of identifying more compounds at lower concentration levels than DDA does. The dynamic range of SWATH was estimated to be three orders of magnitude. Furthermore, the >600,000 SWATH spectra matched led to only 408 incorrect calls (false positive rate = 0.06 %). Deconvolution of generated ion maps was found to be essential for unravelling the full identification power of LC-MS/MS with SWATH. With the available software, however, only semi-automated deconvolution was enabled, which rendered data interpretation a laborious and time-consuming process.

ddpcRquant: threshold determination for single channel droplet digital PCR experiments

Abstract: Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy

Abstract: We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use.

Nanometer-sized materials for solid-phase extraction of trace elements.

Abstract: This review presents a comprehensive update on the state-of-the-art of nanometer-sized materials in solid-phase extraction (SPE) of trace elements followed by atomic-spectrometry detection. Zero-dimensional nanomaterials (fullerene), one-dimensional nanomaterials (carbon nanotubes, inorganic nanotubes, and nanowires), two-dimensional nanomaterials (nanofibers), and three-dimensional nanomaterials (nanoparticles, mesoporous nanoparticles, magnetic nanoparticles, and dendrimers) for SPE are discussed, with their application for trace-element analysis and their speciation in different matrices. A variety of other novel SPE sorbents, including restricted-access sorbents, ion-imprinted polymers, and metal–organic frameworks, are also discussed, although their applications in trace-element analysis are relatively scarce so far.

Tip-enhanced Raman spectroscopy: tip-related issues

Abstract: After over 15 years of development, tip-enhanced Raman spectroscopy (TERS) is now facing a very important stage in its history TERS offers high detection sensitivity down to single molecules and a high spatial resolution down to sub-nanometers, which make it an unprecedented nanoscale analytical technique offering molecular fingerprint information The tip is the core element in TERS, as it is the only source through which to support the enhancement effect and provide the high spatial resolution However, TERS suffers and will continue to suffer from the limited availability of TERS tips with a high enhancement, good stability, and high reproducibility This review focuses on the tip-related issues in TERS We first discuss the parameters that influence the enhancement and spatial resolution of TERS and the possibility to optimize the performance of a TERS system via an in-depth understanding of the enhancement mechanism We then analyze the methods that have been developed for producing TERS tips, including vacuum-based deposition, electrochemical etching, electrodeposition, electroless deposition, and microfabrication, with discussion on the advantages and weaknesses of some important methods We also tackle the issue of lifetime and protection protocols of TERS tips which are very important for the stability of a tip Last, some fundamental problems and challenges are proposed, which should be addressed before this promising nanoscale characterization tool can exert its full potential Graphical Abstract ᅟ

Quantitative evaluation of bias in PCR amplification and next-generation sequencing derived from metabarcoding samples.

Abstract: Unbiased identification of organisms by PCR reactions using universal primers followed by DNA sequencing assumes positive amplification. We used six universal loci spanning 48 plant species and quantified the bias at each step of the identification process from end point PCR to next-generation sequencing. End point amplification was significantly different for single loci and between species. Quantitative PCR revealed that Cq threshold for various loci, even within a single DNA extraction, showed 2,000-fold differences in DNA quantity after amplification. Next-generation sequencing (NGS) experiments in nine species showed significant biases towards species and specific loci using adaptor-specific primers. NGS sequencing bias may be predicted to some extent by the Cq values of qPCR amplification.

LC-QTOF MS screening of more than 1,000 licit and illicit drugs and their metabolites in wastewater and surface waters from the area of Bogotá, Colombia

Abstract: A large screening of around 1,000 emerging contaminants, focused on licit and illicit drugs and their metabolites, has been made in urban wastewaters (both influent and effluent) and surface waters from the area of Bogota, Colombia. After a simple generic solid-phase extraction (SPE) step with Oasis hydrophilic-lipophilic balanced (HLB) cartridges, analyses were made by ultra high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS) under MSE mode (sequential acquisition of mass spectra at low energy (LE) and high collision energy (HE)). Accurate mass measurements and the information provided by MSE on the presence of the (de)protonated molecule and fragment ions allowed the reliable identification of the compounds detected, even without reference standards being available in some cases (tentative identification). The compounds most frequently found were acetaminophen/paracetamol, carbamazepine and its dihydro-dihydroxylated metabolite, clarithromycin, diclofenac, ibuprofen, gemfibrozil, lincomycin, losartan, valsartan, the two metabolites of metamizole (4-acetamido-antipyrine and 4-formylamino-antipyrine), sucralose, and cocaine and its main metabolite benzoylecgonine. Caffeine, the sweetener saccharin, and two hydroxylated metabolites of losartan were tentatively identified in almost all samples analyzed. Pharmaceutical lidocaine was tentatively identified and subsequently confirmed with reference standard. For the first time, a general overview of the occurrence of drugs and their metabolites in the aquatic environment of Colombia has been reported. In the near future, target methodologies, typically based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), will need to be set up for accurate and sensitive quantification of the contaminants selected on the basis on the information provided in the present paper.

Human sweat metabolomics for lung cancer screening

Abstract: Sweat is one of the less employed biofluids for discovery of markers in spite of its increased application in medicine for detection of drugs or for diagnostic of cystic fibrosis. In this research, human sweat was used as clinical sample to develop a screening tool for lung cancer, which is the carcinogenic disease with the highest mortality rate owing to the advanced stage at which it is usually detected. In this context, a method based on the metabolite analysis of sweat to discriminate between patients with lung cancer versus smokers as control individuals is proposed. The capability of the metabolites identified in sweat to discriminate between both groups of individuals was studied and, among them, a trisaccharide phosphate presented the best independent performance in terms of the specificity/sensitivity pair (80 and 72.7%, respectively). Additionally, two panels of metabolites were configured using the PanelomiX tool as an attempt to reduce false negatives (at least 80% specificity) and false positives (at least 80% sensitivity). The first panel (80% specificity and 69% sensitivity) was composed by suberic acid, a tetrahexose, and a trihexose, while the second panel (69% specificity and 80% sensitivity) included nonanedioic acid, a trihexose, and the monoglyceride MG(22:2). Thus, the combination of the five metabolites led to a single panel providing 80% specificity and 79% sensitivity, reducing the false positive and negative rates to almost 20%. The method was validated by estimation of within-day and between-days variability of the quantitative analysis of the five metabolites.