Label-free targeted LC-ESI-MS 2 analysis of human milk oligosaccharides (HMOS) and related human milk groups with enhanced structural selectivity Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-16 Marko Mank, Philipp Welsch, Albert J. R. Heck, Bernd Stahl
Human milk (HM) supports the healthy development of neonates and exerts many of its beneficial effects via contained free human milk oligosaccharides (HMOS). These HMOS exhibit a complexity and structural diversity that pose a significant analytical challenge. A detailed characterization of HMOS is essential as every individual structure may have a different function/activity. Certain HMOS isomers may even fundamentally differ in their biological function, and especially their characterization by LC or LC-MS is often impaired by co-elution phenomena. Thus, more efficient analytical methodologies with enhanced structural selectivity are required. Therefore, we developed a negative ion mode LC-ESI-MS2 approach featuring straightforward sample preparation, environmentally friendly EtOH gradient elution, and enhanced, semiquantitative characterization of distinct native HMOS by multiple reaction monitoring (MRM). Our MRM-LC-MS setup takes advantage of highly selective, glycan configuration-dependent collision-induced dissociation (CID) fragments to identify individual neutral and acidic HMOS. Notably, many human milk oligosaccharide isomers could be distinguished in a retention time-independent manner. This contrasts with other contemporary MRM approaches relying on rather unspecific MRM transitions. Our method was used to determine the most abundant human milk tri-, tetra-, penta-, and hexaoses semiquantitatively in a single LC-MS assay. Detected HMO structures included fucosyllactoses (e.g., 2′-FL), lacto-N-difucotetraose (LDFT), lacto-N-tetraoses (LNTs), lacto-N-fucopentaoses (e.g., LNFP I, LNFP II and III), lacto-N-difucohexaoses (LNDFHs) as well as sialyllactoses (SLs) and tentatively assigned blood group A and B tetrasaccharides from which correct human milk type assignment could be also demonstrated. Correctness of milk typing was validated for milk groups I–IV by high pressure anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (HPAEC-PAD).
Correction to: Affinity capillary electrophoresis for identification of active drug candidates in myotonic dystrophy type 1 Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-15 Ioan O. Neaga, Stephanie Hambye, Ede Bodoki, Claudio Palmieri, Jean Jacques Vanden Eynde, Eugénie Ansseau, Alexandra Belayew, Radu Oprean, Bertrand Blankert
Unfortunately the name of Jean Jacques Vanden Eynde was missing as co-author of this contribution. The correct list of authors is: Ioan O. Neaga, Stephanie Hambye, Ede Bodoki, Claudio Palmieri, Jean Jacques Vanden Eynde, Eugénie Ansseau, Alexandra Belayew, Radu Oprean, Bertrand Blankert.
Ratiometric fluorescence probe of MIPs@CdTe QDs for trace malachite green detection in fish Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-14 Hui Ran, Zheng-Zhong Lin, Qiu-Hong Yao, Cheng-Yi Hong, Zhi-Yong Huang
A facile and practical ratiometric fluorescence probe based on two CdTe quantum dots (QDs) coated with molecularly imprinted polymers (MIPs) was prepared for the detection of trace malachite green (MG) in fish. Two CdTe QDs coated with MIPs were fabricated by a one-pot method using MG, (3-aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as template, functional monomer, and cross-linker, respectively. CdTe QDs with λem 530 nm (gQDs) and 630 nm (rQDs) were used as the referential fluorophore and target sensitive fluorophore, respectively. The fluorescence intensity of gQDs remained unchanged in the presence of MG, while the fluorescence of rQDs could be quantitatively quenched by MG based on the strategy of fluorescence resonance energy transfer. The ratiometric fluorescence probe (MIPs@gQDs&rQDs) was characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The linear range of MG detection was 0.1–32 μmol L−1 with a detection limit of 8.8 μg kg−1. The constructed probe has been successfully applied to the detection of MG in fish with the recoveries of 92.3–109.1%, which were validated by the method of HPLC. The result indicated that the probe possessed rapid response, wide linear range, high sensitivity, and relatively high selectivity, and was low-cost and easy in operation in the detection of MG in fish samples.
Correction to: Advanced analytical strategies for measuring free bioactive milk sugars: from composition and concentrations to human metabolic response Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-24 Aifric O’Sullivan, Jaime Salcedo, Josep Rubert
The authors should like to call your attention to the fact that Figure 5 of the article was taken from the book “Prebiotics and Probiotics in Human Milk” by Sarah S. Comstock and Sharon M. Donovan and printed with the permission from Elsevier. Unfortunately, the right referencing was missing in the original article.
Rapid screening of drug candidates against EGFR/HER2 signaling pathway using fluorescence assay Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-18 Farkhondeh Khanjani, Reza H. Sajedi, Sadegh Hasannia
Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca2+ release was investigated. The changes in the concentration of intracellular Ca2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca2+ entry into the cytoplasm and fluorescence emission decreased. The IC50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways.
NiMoO 4 nanosheet arrays anchored on carbon cloth as 3D open electrode for enzyme-free glucose sensing with improved electrocatalytic activity Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-23 Mujia Huang, Daiping He, Mingzhu Wang, Ping Jiang
High electrical conductivity and more active sites exposure are crucial for improving the performance of electrocatalyst. Binary metal oxide nanoarray grown on conductive substrate offers a 3D self-supported electrode with a great promise in boosting its performance in enzyme-free glucose sensing. Here, NiMoO4 nanosheet arrays anchored on carbon cloth (NiMoO4 NSA/CC) was prepared via a simple hydrothermal synthesis and used as 3D self-supported electrode for enzyme-free glucose sensing. The morphology and composition of NiMoO4 nanosheet have been characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The electrochemical results show that NiMoO4 NSA/CC exhibits remarkable high catalytic activity towards glucose oxidation, with a wide linear response ranging from 1 μM to 0.9 mM, a high sensitivity of 4.13 mA/mM·cm2, and a low detection limit of 1 μM (S/N = 3). The enhanced performance might be attributed to the merits of nanosheet arrays with large surface area, self-supported electrode with 3D open network, as well as bimetallic component with high conductivity. Furthermore, NiMoO4 NSA/CC also shows good selectivity and reliability for glucose detection in human serum. This work offers a new pathway for the construction of enzyme-free glucose sensor with high performance.
Automated multicomponent phospholipid analysis using 31 P NMR spectroscopy: example of vegetable lecithin and krill oil Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-22 Yulia B. Monakhova, Bernd W. K. Diehl
Nuclear magnetic resonance (NMR) spectroscopy is widely applied in the field of metabolomics due to its quantitative nature and the reproducibility of data generated. However, one of the main challenges in routine NMR analysis is to obtain valuable information from large datasets of raw data in a high-throughput, automatic, and reproducible manner. In this study, a method to automatically annotate and quantify 12 phospholipids (PLs) in vegetable lecithin (soy, sunflower, rape) and krill oil is introduced. Automated routines were written in MATLAB environment for quantification of phosphatidylcholine (PC), phosphatidylinositol (PI), lyso-phosphatidylcholine (LPC), phosphatidylserine (PS), phosphatidylethanolamine (PE), diphosphatidylglycerol or cardiolipin (DPG), phosphatidylglycerol (PG), and lyso-phosphatidylethanolamine (LPE) in lecithin and of PC, PC-ether, LPC, PE, N-acyl phosphatidylethanolamine (APE), and LPE in krill oil matrix. The routine includes NMR spectra import, extraction of data points, peaking of local minima and local maxima in the data, integration, quantitation against internal standard, reporting of results as Word file, and their importing in our internal database. Our extensive studies on a representative set of more than 1000 lecithin (soy, rape, sunflower) and krill samples showed that the routine can automatically and accurately calculate the concentrations of all PLs. No systematic or proportional differences between automated and manual evaluation were detected. The developed automated program produces accurate results and requires less than 5 s for each analysis. This tool is already used in high-throughput PL analysis of krill and lecithin and will be adjusted to other matrices (egg, milk, chocolate, etc.) as well.
Affinity profiling of monoclonal antibody and antibody-drug-conjugate preparations by coupled liquid chromatography-surface plasmon resonance biosensing Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-17 Dina Lakayan, Rob Haselberg, Rabah Gahoual, Govert W. Somsen, Jeroen Kool
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are highly potent biopharmaceuticals designed for targeted cancer therapies. mAbs and ADCs can undergo modifications during production and storage which may affect binding to target receptors, potentially altering drug efficacy. In this work, liquid chromatography was coupled online to surface plasmon resonance (LC-SPR) to allow label-free affinity evaluation of mAb and ADC sample constituents (size and charge variants), under near-native conditions. Trastuzumab and its ADC trastuzumab emtansine (T-DM1) were used as a test sample and were analyzed by aqueous size-exclusion chromatography (SEC)-SPR before and after exposure to aggregate-inducing conditions. SEC-SPR allowed separation of the formed aggregates and measurement of their affinity towards the ligand-binding domain of the human epidermal growth factor receptor 2 (HER2) receptor immobilized on the surface of the SPR sensor chip. The monomer and aggregates of the mAb and ADC were shown to have similar antigen affinity. Conjugation of drugs to trastuzumab appeared to accelerate the aggregate formation. In addition, cation-exchange chromatography (CEX) was coupled to SPR enabling monitoring the maximum ligand-analyte binding capacity (Rmax) of individual charge variants present in mAbs. Deamidated species and lysine variants in trastuzumab sample were separated but did not show different binding affinities to the immobilized HER2-binding domain. In order to allow protein variant assignment, parallel MS detection was added to the LC-SPR setup using a column effluent split. The feasibility of the LC-MS/SPR system was demonstrated by analysis of trastuzumab and T-DM1 providing information on antibody glycoforms and/or determination of the drug-to-antibody ratio (DAR), while simultaneously monitoring binding of eluting species to HER2.
Investigation of mycobacteria fatty acid profile using different ionization energies in GC–MS Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-29 Marco Beccaria, Flavio A. Franchina, Mavra Nasir, Theodore Mellors, Jane E. Hill, Giorgia Purcaro
Gas chromatography (GC) coupled with electron ionization (EI) mass spectrometry (MS) is a well-established technique for the analysis of volatile and semi-volatile compounds. The main advantage is the highly repeatable fragmentation of the compounds into the ion source, generating intense and diagnostic fragmentation when the ionization is performed at 70 eV; this is considered the standard ionization condition and has been used for creating many established databases, which are of great support in the analyte identification process. However, such an intense fragmentation often causes the loss of the molecular ion or more diagnostic ions, which can be detrimental for the identification of homologous series or isomers, as for instance fatty acids. To obtain this information chemical or soft ionization can be used, but dedicated ion sources and conditions are required. In this work, we explored different ionization voltages in GC–EI–MS to preserve the intensity of the molecular ion using a conventional quadrupole MS. Twenty, 30, 50, and 70 eV were tested using a mixture of fatty acid methyl esters standards. Intensity and repeatability of the most informative ions were compared. Twenty and 70 eV were then used to analyze the fatty acid composition of six different strains of mycobacteria. Two approaches were used for elaborating the data: (1) a single average spectrum of the entire chromatogram was derived, which can be considered (in terms of concept) as a direct EI–MS analysis; (2) the actual chromatographic separation of the compounds was considered after automatic alignment. The results obtained are discussed herein.
A non-targeted metabolomic approach based on reversed-phase liquid chromatography–mass spectrometry to evaluate coffee roasting process Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-22 Raquel Pérez-Míguez, Elena Sánchez-López, Merichel Plaza, María Castro-Puyana, María Luisa Marina
In this work, a non-targeted metabolomics approach based on the use of reversed-phase liquid chromatography coupled to a high-resolution mass spectrometer has been developed to provide the characterization of coffee beans roasted at three different levels (light, medium, and dark). In this way, it was possible to investigate how metabolites change during the roasting process in order to identify those than can be considered as relevant markers. Twenty-five percent methanol was selected as extracting solvent since it provided the highest number of molecular features. In addition, the effect of chromatographic and MS parameters was evaluated in order to obtain the most adequate separation and detection conditions. Data were analyzed using both non-supervised and supervised multivariate statistical methods to point out the most significant markers that allow group discrimination. A total of 24 and 33 compounds in positive and negative ionization modes, respectively, demonstrated to be relevant markers; most of them were from the hydroxycinnamic acids family.
Analysis of the 13 C isotope ratios of amino acids in the larvae, pupae and adult stages of Calliphora vicina blow flies and their carrion food sources Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-25 Mayara P. V. Matos, Kateryna I. Konstantynova, Rachel M. Mohr, Glen P. Jackson
Adult blow flies are one of the first necrophagous insects to colonize fresh carcasses. The eggs they lay hatch into larvae, which then feed on the decomposing body. Like all organisms, blow flies “are what they eat,” meaning that the isotopic composition of their body tissues reflects their diet. This manuscript combines ecology with a forensic application by using isotope ratio mass spectrometry (IRMS) to understand the relationship between the δ13C of amino acids in different carrion sources and the blow fly that feed on them. We also measure the amino acid-level fractionation that occurs at each major life stage of the blow flies. Adult blow flies from a commercial strain of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) oviposited on raw pork muscle, beef muscle, or chicken liver. Larvae, pupae, and adult blow flies from each carrion were selected for amino acid compound-specific isotope analysis. Canonical discriminant analysis showed that flies were correctly classified to specific carrion types in 100% (original rules) and 96.8% (leave-one-out cross-validation [LOOCV]) of cases. Regarding life stages, we obtained 100% and 71% of correct classification in original rules and LOOCV, respectively. The isotope ratios of most of the essential amino acids did not significantly change between life stages (at 95% CI). However, some non-essential amino acids (Ala, Ser, and Glu) and some conditionally essential amino acids (Gly and Pro) were isotopically depleted in the adult stage. Except for the essential amino acids, the amino acids in larvae and pupae were enriched in 13C, and adult blow flies were depleted in 13C relative to the carrion on which they fed. These results make it possible to exclude potential sources of carrion as larval food. Amino acid-specific IRMS could help inform entomologists whether a fly has just arrived from another location to feed on a corpse or has emerged from a pupa whose feedstock was the corpse. Such insight could enhance the significance of blow flies for post-mortem interval determinations. The analytical ability to link organisms from one trophic level to another through the use of compound-specific isotope analysis of amino acids could have wide-reaching consequences in a variety of disciplines.
Retention-time prediction in comprehensive two-dimensional gas chromatography to aid identification of unknown contaminants Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-25 Cathrin Veenaas, Anna Linusson, Peter Haglund
Comprehensive two-dimensional (2D) gas chromatography (GC×GC) coupled to mass spectrometry (MS, GC×GC-MS), which enhances selectivity compared to GC-MS analysis, can be used for non-directed analysis (non-target screening) of environmental samples. Additional tools that aid in identifying unknown compounds are needed to handle the large amount of data generated. These tools include retention indices for characterizing relative retention of compounds and prediction of such. In this study, two quantitative structure–retention relationship (QSRR) approaches for prediction of retention times (1tR and 2tR) and indices (linear retention indices (LRIs) and a new polyethylene glycol–based retention index (PEG-2I)) in GC × GC were explored, and their predictive power compared. In the first method, molecular descriptors combined with partial least squares (PLS) analysis were used to predict times and indices. In the second method, the commercial software package ChromGenius (ACD/Labs), based on a “federation of local models,” was employed. Overall, the PLS approach exhibited better accuracy than the ChromGenius approach. Although average errors for the LRI prediction via ChromGenius were slightly lower, PLS was superior in all other cases. The average deviations between the predicted and the experimental value were 5% and 3% for the 1tR and LRI, and 5% and 12% for the 2tR and PEG-2I, respectively. These results are comparable to or better than those reported in previous studies. Finally, the developed model was successfully applied to an independent dataset and led to the discovery of 12 wrongly assigned compounds. The results of the present work represent the first-ever prediction of the PEG-2I.
Simplifying the complex: metabolomics approaches in chemical ecology Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-12 Remington X. Poulin, Georg Pohnert
Chemical signals are important mediators of organismal interactions. These interactions significantly influence ecosystem structure and thus are crucial to understand. Ecologists and analytical chemists work closely together to identify the specific molecules regulating ecological interactions. However, limitations in the analytical techniques on the one hand and time-demanding bioassays on the other have been restraining chemical ecology research. Application of metabolomics techniques has recently led to significant advancement of the field. Here, we discuss modifications to the traditional bioassay-guided fractionation approach with metabolomics techniques. We focus on two challenging topics within chemical ecology, waterborne cues and single-cell investigations, to highlight how metabolomics techniques can succeed where traditional approaches have failed.
Spatial and molecular changes of mouse brain metabolism in response to immunomodulatory treatment with teriflunomide as visualized by MALDI-MSI Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-12 Ignacy Rzagalinski, Nadine Hainz, Carola Meier, Thomas Tschernig, Dietrich A. Volmer
Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the central nervous system (CNS). One of the most promising recent medications for MS is teriflunomide. Its primary mechanism of action is linked to effects on the peripheral immune system by inhibiting dihydroorotate dehydrogenase (DHODH)-catalyzed de novo pyrimidine synthesis and reducing the expansion of lymphocytes in the peripheral immune system. Some in vitro studies suggested, however, that it can also have a direct effect on the CNS compartment. This potential alternative mode of action depends on the drug’s capacity to traverse the blood-brain barrier (BBB) and to exert an effect on the complex network of brain biochemical pathways. In this paper, we demonstrate the application of high-resolution/high-accuracy matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry for molecular imaging of the mouse brain coronal sections from animals treated with teriflunomide. Specifically, in order to assess the effect of teriflunomide on the mouse CNS compartment, we investigated the feasibility of teriflunomide to traverse the BBB. Secondly, we systematically evaluated the spatial and semi-quantitative brain metabolic profiles of 24 different endogenous compounds after 4-day teriflunomide administration. Even though the drug was not detected in the examined cerebral sections (despite the high detection sensitivity of the developed method), in-depth study of the endogenous metabolic compartment revealed noticeable alterations as a result of teriflunomide administration compared to the control animals. The observed differences, particularly for purine and pyrimidine nucleotides as well as for glutathione and carbohydrate metabolism intermediates, shed some light on the potential impact of teriflunomide on the mouse brain metabolic networks.
Improving the reliability of the iron concentration quantification for iron oxide nanoparticle suspensions: a two-institutions study Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-12 Rocio Costo, David Heinke, Cordula Grüttner, Fritz Westphal, M. Puerto Morales, S. Veintemillas-Verdaguer, Nicole Gehrke
Most iron oxide nanoparticles applications, and in special biomedical applications, require the accurate determination of iron content as the determination of particle properties from measurements in dispersions is strongly dependent on it. Inductively coupled plasma (ICP) and spectrophotometry are two typical worldwide used analytical methods for iron concentration determination. In both techniques, precise determination of iron is not straightforward and nanoparticle digestion and dilution procedures are needed prior to analysis. The sample preparation protocol has been shown to be as important as the analytical method when accuracy is aimed as many puzzling reported results in magnetic, colloidal, and structural properties are simply attributable to inadequate dissolution procedures. Therefore, a standard sample preparation protocol is needed to ensure the adequate and complete iron oxide nanoparticle dissolution and to harmonize this procedure. In this work, an interlaboratory evaluation of an optimized iron oxide nanoparticle digestion/dilution protocol was carried out. The presented protocol is simple, inexpensive, and does not involve any special device (as microwave, ultrasound, or other high-priced digestion devices). Then, iron concentration was measured by ICP-OES (performed in ICMM/CSIC-Spain) and spectrophotometry (NanoPET-Germany) and the obtained concentration values were analyzed to determine the most probable error causes. Uncertainty values as low as 1.5% were achieved after the optimized method was applied. Moreover, this article provides a list of recommendations to significantly reduce uncertainty in both sample preparation and analysis procedures.
Comprehensive MS-based screening and identification of pharmaceutical transformation products formed during enzymatic conversion Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-12 Lara F. Stadlmair, Sylvia Grosse, Thomas Letzel, Jörg E. Drewes, Johanna Grassmann
In this study, transformation products (TPs) of diclofenac, mefenamic acid, and sotalol derived from peroxidase- and laccase-catalyzed transformations were studied with different mass spectrometry (MS)-based workflows. A straightforward pre-screening of enzymatic degradation rate was performed using a robotic nano-ESI source coupled to single quadrupole MS. Accurate mass data and information on molecular hydrophobicity were obtained from a serial coupling of reversed phase liquid chromatography (RPLC) with hydrophilic interaction liquid chromatography (HILIC) to a time-of-flight-mass spectrometer (ToF-MS). These parameters were combined with fragmentation information from product ion scan operated in enhanced mode (EPI) with precursor selection in Q3 and data from multiple reaction monitoring (MRM) modes using a hybrid triple quadrupole-linear ion trap-mass spectrometer (QqQ/LIT-MS). “Suspect” MRM modes did not provide a significant sensitivity improvement compared to EPI experiments. The complementarity of the data from different MS-based workflows allowed for an increase of identification confidence. Overall, this study demonstrated that dimerization, hydroxylation, and dehydration reactions were the predominant mechanisms found for diclofenac and mefenamic acid during enzyme-catalyzed transformation, whereas a degradation product was observed for the peroxidase-catalyzed conversion of sotalol. Results can contribute to understand enzymatic mechanisms and provide a basis for assessing risks and benefits of enzyme-based remediation.
Tunable superamphiphobic surfaces: a platform for naked-eye ATP detection Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-10 Fujian Huang, Yan Chen, Yongqian Wang, Fan Xia
A superamphiphobic surface composed of two different size ranges of TiO2 nanoparticles was simply fabricated through spraying the perfluorosilane coated TiO2 nanoparticles suspension dispersing in ethanol. The surface chemistry was finely regulated through gradient UV irradiation-induced organic compound degradation to fabricate surface with gradient solid surface energy or wettability. The fabricated surface shows good droplet sorting ability, which can successfully discriminate ethanol droplets with different concentrations. As a proof-of-concept, the biosensor application of this surface was demonstrated by using it for naked-eye ATP detection. Liquid droplets with different concentrations of ATP after ATP-dependent rolling circle amplification (RCA) can be effectively sorted by the surface. This developed biosensor methodology based on droplet sorting ability of the fabricated surface is energy-efficient and economical which is promising for biosensors, point-of-care testing, and biochemical assays.
A tridecaptin-based fluorescent probe for differential staining of Gram-negative bacteria Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-10 Wei Wang, Yingyan Wang, Liyuan Lin, Yanling Song, Chaoyong James Yang
The traditional Gram-staining method, which was invented more than a century ago for differentiating bacteria as Gram positive or Gram negative, is still widely practiced in microbiology. However, Gram staining suffers from several problems which can affect the accuracy of the diagnosis. Here, we report a new Gram-negative-specific fluorescent probe, which is based on a narrow-spectrum antibiotic, tridecaptin A1, and allows selective staining of Gram-negative bacteria in different fixed bacterial samples. Solid-phase peptide synthesis was used to prepare the tridecaptin A1–fluorophore conjugate with a single structure. Labeling selectivity of the probe toward Gram-negative bacteria was confirmed by testing against a panel of bacterial species. By combining the use of a previously reported Gram-positive-specific fluorescent probe, we then further showed the capability of the new probe in differential labeling of a number of complex bacterial samples, which included a mouse gut microbiota cultured in vitro, as well as microbiotas collected from the human oral cavity, soil, and crude oil. High labeling selectivity and coverage were observed in most samples. This method offers a new Gram-negative-specific probe with a defined structure, which allows facile fluorescence-based differentiation of Gram-positive and Gram-negative bacteria for further microbial studies.
A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aβ 1–42 , α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-09 Alejandro Mahía, María Conde-Giménez, Sandra Salillas, Irantzu Pallarés, Juan J. Galano-Frutos, Íñigo Lasa, Salvador Ventura, María D. Díaz-de-Villegas, José A. Gálvez, Javier Sancho
Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1–42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aβ1–42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them.
Nitrogen-doped carbon quantum dots as a fluorescent probe to detect copper ions, glutathione, and intracellular pH Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-29 Sen Liao, Xueqian Huang, Hua Yang, Xiaoqing Chen
A facile one-step hydrothermal method was developed to synthesize nitrogen-doped carbon quantum dots (N-CQDs) by utilizing hexamethylenetetramine as the carbon and nitrogen source. The quantum yield (QY) of 21.7% was under the excitation wavelength of 420 nm with maximum emission at 508 nm. This N-CQD fluorescent probe has been successfully applied to selectively determine the concentration of copper ion (Cu2+) with a linear range of 0.1–40 μM and a limit of detection of 0.09 μM. In addition, the fluorescence of N-CQDs could be effectively quenched by Cu2+ and specifically recovered by glutathione (GSH), which render the N-CQDs as a premium fluorescent probe for GSH detection. This fluorescence “turn-on” protocol was applied to determine GSH with a linear range of 0.1–30 μM as well as a detection limit of 0.05 μM. For pH detection, there is good linearity in the pH range of 2.87–7.24. Furthermore, N-CQD is a promising and convenient fluorescent pH, Cu2+, and glutathione sensor with brilliant biocompatibility and low cytotoxicity in environmental monitoring and bioimaging applications.
Porous Co 3 O 4 nanosheets as a high-performance non-enzymatic sensor for glucose detection Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-28 Fuyan Liu, Peng Wang, Qianqian Zhang, Zeyan Wang, Yuanyuan Liu, Zhaoke Zheng, Xiaoyan Qin, Xiaoyang Zhang, Ying Dai, Lu Li, Baibiao Huang
Novel porous Co3O4 nanosheets (NSs) were synthesized on the flexible carbon cloth (CC) substrate by a facile hydrothermal method and applied to construct a non-enzymatic sensor for glucose detection. The sensor is based on the electro-catalytic oxidation of glucose on the surface of Co3O4 NSs. Since this particular nanostructure can provide large surface area and more active sites, the Co3O4 NSs non-enzymatic sensor exhibits excellent analytical performance, such as a high sensitivity (8506 μA mM−1 cm−2), a fast response time (less than 6 s), low detection limit of 1 μM, good selectivity, and long-term stability. The results suggest that the porous Co3O4 NSs have great potential applications in the development of sensors for enzyme-free detection of glucose.
Comprehensive analysis of oxylipins in human plasma using reversed-phase liquid chromatography-triple quadrupole mass spectrometry with heatmap-assisted selection of transitions Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-08 Guan-yuan Chen, Qibin Zhang
Oxylipins, a subclass of lipid mediators, are metabolites of various polyunsaturated fatty acids with crucial functions in regulation of systemic inflammation. Elucidation of their roles in pathological conditions requires accurate quantification of their levels in biological samples. We refined an ultra-performance liquid chromatography-multiple reaction monitoring-mass spectrometry (UPLC-MRM-MS)-based workflow for comprehensive and specific quantification of 131 endogenous oxylipins in human plasma, in which we optimized LC mobile phase additives, column, and gradient conditions. We employed heatmap-assisted strategy to identify unique transitions to improve the assay selectivity and optimized solid phase extraction procedures to achieve better analyte recovery. The method was validated according to FDA guidelines. Overall, 94.4% and 95.7% of analytes at tested concentrations were within acceptable accuracy (80–120%) and precision (CV < 15%), respectively. Good linearity for most analytes was obtained with R2 > 0.99. The method was also validated using a standard reference material—SRM 1950 frozen human plasma to demonstrate inter-lab compatibility.
A MnO 2 nanosheet-based ratiometric fluorescent nanosensor with single excitation for rapid and specific detection of ascorbic acid Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-07 Yanlong Lyu, Zhanhui Tao, Xiaodong Lin, Pengcheng Qian, Yunfei Li, Shuo Wang, Yaqing Liu
Ascorbic acid (AA) detection in biological sample and food sample is critical for human health. Herein, a MnO2 nanosheet (MnO2-NS)-based ratiometric fluorescent nanosensor has been developed for high sensitive and specific detection of AA. The MnO2-NS presents peroxidase-like activity and can oxidize non-fluorescent substrate of o-phenylenediamine (OPDA) into fluorescent substrate, presenting maximum fluorescence at 568 nm (F568). If MnO2-NS is premixed with AA, the MnO2-NS is then decomposed as Mn2+ by AA, decreasing the fluorescent intensity of F568. Meantime, AA is oxidized as dehydroascorbic acid (DHAA), which can react with OPDA to generate fluorescent substrate. A new fluorescence response is found at 425 nm (F425). The dual fluorescent responses can be excited with a universal excitation wavelength, simplifying the detection procedure. With F425/F568 as readout, limit of detection for AA reaches as low as 10.0 nM. Satisfactory recoveries are found for AA detection in serum and diverse beverages. The ratiometric strategy significantly eliminates false-negative and false-positive results, providing a cost-effective, rapid, and reliable way for AA detection in real sample.
New analytical approach to determine organophosphorus insecticides in blood by dried matrix spots sampling and GC-MS/MS Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-07 Sofia Soares, Teresa Castro, Tiago Rosado, Nicolás Fernández, Mário Barroso, Eugenia Gallardo
This work describes the optimization of a new method for the determination of five organophosphorus insecticides in whole blood. The analytes were extracted from the matrix (50 μL) using the dried blood spot (DBS) approach and were analyzed by gas chromatography (GC) coupled to tandem mass spectrometry (MS/MS). The studied compounds (diazinon, chlorpyrifos, parathion-ethyl, chlorfenvinphos and quinalphos) were chosen based on the statistics of intoxications in Portugal, and ethion was used as internal standard. The method was fully validated, taking into account international guidelines for bioanalytical method validation, such as those of the Food and Drug Administration (FDA), International Conference on Harmonization (ICH) and Scientific Working Group for Forensic Toxicology (SWGTOX). A linear range of 0.1–25 μg/mL was obtained for all compounds, except for diazinon and quinalphos (0.05–25 μg/mL and 0.25–25 μg/mL, respectively), presenting determination coefficients above 0.99. Concerning precision, the coefficients of variation (CVs) were lower than 14% for all compounds. Those compounds were found to be stable in the samples. Although the values obtained for recovery were low (between 1 and 12%), the method proved to be sensitive, since detection limits between 0.05 and 0.1 μg/mL were obtained. The novelty is the use of the DBS approach in the extraction of these compounds, and this is the first paper reporting it: DBS is a recent technique of bioanalysis in the field of toxicology, and in addition to its simplicity and sensitivity, it is applicable routinely in both clinical and forensic toxicology situations.
Towards metrologically traceable and comparable results in GM quantification Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-06 Philippe Corbisier, Hendrik Emons
The GM content in a food or feed product produced from or containing genetically modified organisms (GMO) has to be expressed in Europe in the form of a GM mass fraction. However, the most widely used quantification methods, based on PCR, are basically counting PCR-amplifiable DNA fragments in a sample extract. This paper outlines the requirements for obtaining comparable measurement results which are fit for regulatory decision-making. It introduces the concept of a reference measurement system which enables GMO analysis laboratories to relate their results to a universally accessible reference, thus establishing metrological traceability to a unique reference point. The conversion factors required for transforming data from one measurement unit into the other have to carry a minimum uncertainty and are anchored to specified certified reference materials. The establishment of such conversion factors and related calibration approaches to achieve comparable GM quantification results are sketched.
A methodological inter-comparison study on the detection of surface contaminant sodium dodecyl sulfate applying ambient- and vacuum-based techniques Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-06 Andrea M. Giovannozzi, Andrea Hornemann, Beatrix Pollakowski-Herrmann, Felicia M. Green, Paul Gunning, Tara L. Salter, Rory T. Steven, Josephine Bunch, Chiara Portesi, Bonnie J. Tyler, Burkhard Beckhoff, Andrea Mario Rossi
Biomedical devices are complex products requiring numerous assembly steps along the industrial process chain, which can carry the potential of surface contamination. Cleanliness has to be analytically assessed with respect to ensuring safety and efficacy. Although several analytical techniques are routinely employed for such evaluation, a reliable analysis chain that guarantees metrological traceability and quantification capability is desirable. This calls for analytical tools that are cascaded in a sensible way to immediately identify and localize possible contamination, both qualitatively and quantitatively. In this systematic inter-comparative approach, we produced and characterized sodium dodecyl sulfate (SDS) films mimicking contamination on inorganic and organic substrates, with potential use as reference materials for ambient techniques, i.e., ambient mass spectrometry (AMS), infrared and Raman spectroscopy, to reliably determine amounts of contamination. Non-invasive and complementary vibrational spectroscopy techniques offer a priori chemical identification with integrated chemical imaging tools to follow the contaminant distribution, even on devices with complex geometry. AMS also provides fingerprint outputs for a fast qualitative identification of surface contaminations to be used at the end of the traceability chain due to its ablative effect on the sample. To absolutely determine the mass of SDS, the vacuum-based reference-free technique X-ray fluorescence was employed for calibration. Convex hip liners were deliberately contaminated with SDS to emulate real biomedical devices with an industrially relevant substance. Implementation of the aforementioned analytical techniques is discussed with respect to combining multimodal technical setups to decrease uncertainties that may arise if a single technique approach is adopted.
Silver and gold nanoparticles as multi-chromatic lateral flow assay probes for the detection of food allergens Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-06 Laura Anfossi, Fabio Di Nardo, Alida Russo, Simone Cavalera, Cristina Giovannoli, Giulia Spano, Sabine Baumgartner, Kathrin Lauter, Claudio Baggiani
In this study, we report the simultaneous use of gold and silver nanoparticles to set a multicolor multiplex lateral flow immunoassay (xLFIA). Silver nanoparticles (AgNPs), spherical in shape and characterized by a brilliant yellow color, were obtained by a new viable one-step synthetic protocol. AgNPs were stable over time and acceptably robust to conditions used for fabricating LFIA devices. These AgNPs were employed as a colorimetric probe in combination with two different kinds of gold nanoparticles (AuNPs) to set a visual xLFIA for detecting allergens. Surface plasmon resonance peaks of probes (AgNPs, spherical and desert rose-like AuNPs) were centered at 420, 525, and 620 nm, respectively. Therefore, the xLFIA output was easily interpreted through a “yellow magenta cyan (YMC)” color code. The prospect of the YMC xLFIA was demonstrated by simultaneously detecting three major allergens in bakery products. Antibodies directed towards casein, ovalbumin, and hazelnut allergenic proteins were individually adsorbed onto metal nanoparticles to produce three differently colored specific probes. These were inserted in a LFIA comprising three lines, each responsive for one allergen. The trichromatic xLFIA was able to detect allergenic proteins at levels as low as 0.1 mg/l and enabled the easy identification of the allergens in commercial biscuits based on the color of the probes.
Corona discharge electrospray ionization of formate-containing solutions enables in-source reduction of disulfide bonds Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-06 Bradley B. Stocks, Jeremy E. Melanson
Disulfide bonds are critical linkages for maintaining protein structure and enzyme activity. These linkages, however, can limit peptide sequencing efforts by mass spectrometry (MS) and often require chemical reduction and alkylation. Under such conditions, information regarding cysteine connectivity is lost. Online partial disulfide reduction within the electrospray (ESI) source has recently been established as a means to identify complex cysteine linkage patterns in a liquid chromatography-MS experiment without the need for sample pre-treatment. Corona discharge (CD) is invoked as the causative factor of this in-source reduction (ISR); however, evidence remains largely circumstantial. In this study, we demonstrate that instrumental factors—nebulizing gas, ESI capillary material, organic solvent content, ESI spray needle-to-MS distance—all modulate the degree of reduction observed for the single disulfide in oxytocin, further implicating CD in ISR. Rigorous analysis of solution conditions, however, reveals that corona discharge alone can induce only minor disulfide reduction. We establish that CD-ESI of peptide solutions containing formic acid or its conjugate base results in a dramatic increase in disulfide reduction. It is also determined that ISR is exacerbated at low pH for complex peptides containing multiple disulfide bonds and possessing higher-order structure, as well as for a small protein. Overall, our results demonstrate that ESI of formate/formic acid–containing solutions under corona discharge conditions facilitates disulfide ISR, likely by a similar reduction pathway measured in γ-radiolysis studies nearly three decades ago.
Fast and facile analysis of glycosylation and phosphorylation of fibrinogen from human plasma—correlation with liver cancer and liver cirrhosis Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-05 Tim Nagel, Florentine Klaus, Ines Gil Ibanez, Henning Wege, Ansgar Lohse, Bernd Meyer
Hepatocellular carcinoma (HCC) is one of the deadliest cancers due to its late diagnosis with the main risk factor being liver cirrhosis (LC). Glycan structures from glycoproteins are usually altered in cancer. Blood plasma from 111 healthy and sick donors was analyzed to determine the post-translational modifications (PTM) of intact Aα-, Bβ-, and γ-subunits of fibrinogen, a glycoprotein predominantly produced in liver cells. Glycosylation and phosphorylation of the protein species were quantified by liquid chromatography coupled to mass spectrometry to correlate PTMs to pathological cases. Quantities of the PTMs were used for statistical classification by principal component analysis (PCA) and multivariate analysis of variance (MANOVA). As relevant clinical finding, patients with liver disease (HCC and/or LC) were distinguished from individuals without relevant chronic liver disease with 91% sensitivity and 100% specificity. Within the group of patients with liver disease, a robust separation between LC and HCC was not possible. In more detail, the phosphorylation of Aα-subunit is decreased in HCC patients, whereas the monophosphorylated state is significantly increased in LC patients. In terms of glycosylation, the amount of O-glycans in the Aα-subunit is decreased in LC patients, while sialylation and fucosylation of N-type glycans of Bβ- and γ-subunits are increased in LC and HCC. Based on PTM of fibrinogen, starting from plasma we can assign the status of an individual as healthy or as liver disease in less than 3 h.
Nanowell-mediated multidimensional separations combining nanoLC with SLIM IM-MS for rapid, high-peak-capacity proteomic analyses Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-05 Maowei Dou, Christopher D. Chouinard, Ying Zhu, Gabe Nagy, Andrey V. Liyu, Yehia M. Ibrahim, Richard D. Smith, Ryan T. Kelly
Mass spectrometry (MS)–based analysis of complex biological samples is essential for biomedical research and clinical diagnostics. The separation prior to MS plays a key role in the overall analysis, with separations having larger peak capacities often leading to more identified species and improved confidence in those identifications. High-resolution ion mobility (IM) separations enabled by Structures for Lossless Ion Manipulation (SLIM) can provide extremely rapid, high-resolution separations and are well suited as a second dimension of separation following nanoscale liquid chromatography (nanoLC). However, existing sample handling approaches for offline coupling of separation modes require microliter-fraction volumes and are thus not well suited for analysis of trace biological samples. We have developed a novel nanowell-mediated fractionation system that enables nanoLC-separated samples to be efficiently preconcentrated and directly infused at nanoelectrospray flow rates for downstream analysis. When coupled with SLIM IM-MS, the platform enables rapid and high-peak-capacity multidimensional separations of small biological samples. In this study, peptides eluting from a 100 nL/min nanoLC separation were fractionated into ~ 60 nanowells on a microfluidic glass chip using an in-house–developed robotic system. The dried samples on the chip were individually reconstituted and ionized by nanoelectrospray for SLIM IM-MS analysis. Using model peptides for characterization of the nanowell platform, we found that at least 80% of the peptide components of the fractionated samples were recovered from the nanowells, providing up to ~tenfold preconcentration for SLIM IM-MS analysis. The combined LC-SLIM IM separation peak capacities exceeded 3600 with a measurement throughput that is similar to current one-dimensional (1D) LC-MS proteomic analyses.
An ultrasensitive sensor based on quantitatively modified upconversion particles for trace bisphenol A detection Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-03 Qiaofeng Li, Jialei Bai, Shuyue Ren, Jiang Wang, Yifei Gao, Shuang Li, Yuan Peng, Baoan Ning, Zhixian Gao
Bisphenol A (BPA) is one of the endocrine-disrupting chemicals which might cause reproductive and endocrine system diseases, and poses a serious threat to the ecosystem and human health. This paper reports an ultrasensitive sensor for trace BPA detection employing fluorescence resonance energy transfer (FRET) between modified upconversion nanoparticles (UCNPs) and tetramethylrhodamine. To circumvent the problems of low luminous efficiency of FRET and low sensitivity of sensor, the upconversion nanoparticles with very strong fluorescence efficiency were prepared and quantitatively modified. Results showed that the concentrations of amino groups and streptavidin were 43 nmol/mg and 6.12 μg/mg on the surface of the UCNPs, respectively. Under the optimal detection conditions, the peak intensity of UCNPs at 547 nm was linear with the logarithm of the BPA concentration with the detection limit of 0.05 ng/mL. Without complicated pre-processing, the recoveries were in general between 91.0 and 115.0% in tap water, river water, and disposable paper cup water. Therefore, the proposed sensor is suitable for effective sensing of trace BPA in water samples.
MicroRNA sensors based on gold nanoparticles Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-11-03 Catarina Coutinho, Álvaro Somoza
MicroRNAs (miRNAs) are small regulatory RNAs, the dysregulation of which has been associated with the progression of several human diseases, including cancer. Interestingly, these molecules can be used as biomarkers for early disease diagnosis and can be found in a variety of body fluids and tissue samples. However, their specific properties and very low concentrations make their detection rather challenging. In this regard, current detection methods are complex, cost-ineffective, and of limited application in point-of-care settings or resource-limited facilities. Recently, nanotechnology-based approaches have emerged as promising alternatives to conventional miRNA detection methods and paved the way for research towards sensitive, fast, and low-cost detection systems. In particular, due to their exceptional properties, the use of gold nanoparticles (AuNPs) has significantly improved the performance of miRNA biosensors. This review discusses the application of AuNPs in different miRNA sensor modalities, commenting on recently reported examples. A practical overview of each modality is provided, highlighting their future use in clinical diagnosis.
Imidazolium ionic liquids as mobile phase additives in reversed phase liquid chromatography for the determination of iodide and iodate Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-28 Ya-nan Zhang, Hong Yu, Ya-jie Ma, Ge Cui
An analytical method for the simultaneous determination of iodide and iodate by reversed phase liquid chromatography with ultraviolet detection using imidazolium ionic liquids as mobile phase additives was developed in this paper. Imidazolium ionic liquids, pyridinium ionic liquids, and common ion pair reagent tetrabutylammonium hydroxide as mobile phase additives were compared. The results indicated that imidazolium ionic liquids as the mobile phase additives were better than any tetrabutylammonium hydroxide or pyridinium ionic liquids. The relevant mechanisms of separation and detection for the anions were discussed. Imidazolium ionic liquids acted as ion pair reagents and ultraviolet absorption reagents in the separation and detection of the anions. The optimized mobile phase of methanol/0.3 mmol/L 1-hexyl-3-methylimidazolium tetrafluoroborate aqueous solutions (12/88, v/v), detection wavelength of 210 nm, and column temperature of 35 °C were used for the determination of iodide and iodate. Under these conditions, the detection limits of iodide and iodate were 0.02 and 0.03 mg/L, respectively. To evaluate the practicability of the method, the determination of iodide and iodate in medicines and beverages was performed by this method. The results are that the spiked recoveries were greater than 95% and RSD was less than 3.0%. The method was simple, accurate, and reliable and could provide a reference for the analysis of iodide and iodate.
A label-free MALDI TOF MS-based method for studying the kinetics and inhibitor screening of the Alzheimer’s disease drug target β-secretase Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-15 Markéta Machálková, Jan Schejbal, Zdeněk Glatz, Jan Preisler
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) is a well-established method with a unique set of qualities including sensitivity, minute sample consumption, and label-free detection, all of which are highly desired in enzyme assays. On the other hand, the application of MALDI TOF MS is usually limited by high concentrations of MS-incompatible compounds in the reaction mixture such as salts or organic solvents. Here, we introduce kinetic and inhibition studies of β-secretase (BACE1), a key enzyme of the progression of Alzheimer’s disease. Compatibility of the enzyme assay with MALDI TOF MS was achieved, providing both a complex protocol including a desalting step designed for rigorous kinetic studies and a simple mix-and-measure protocol designed for high-throughput inhibitor screening. In comparison with fluorescent or colorimetric assays, MALDI TOF MS represents a sensitive, fast, and label-free technique with minimal sample preparation. In contrast to other MS-based methodological approaches typically used in drug discovery processes, such as a direct injection MS or MS-coupled liquid chromatography or capillary electrophoresis, MALDI TOF MS enables direct analysis and is a highly suitable approach for high-throughput screening. The method’s applicability is strongly supported by the high correlation of the acquired kinetic and inhibition parameters with data from the literature as well as from our previous research.
A sequential separation strategy for facile isolation and comprehensive analysis of human urine N-glycoproteome Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-31 Bianbian Huo, Mingli Chen, Junjie Chen, Yuanyuan Li, Wanjun Zhang, Jianhua Wang, Weijie Qin, Xiaohong Qian
Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for biomarker screening in clinical research. In normal human adult urine, 48% of the total urinary protein is in the sediment, 49% is soluble and the remaining 3% is contained in urinary extracellular vesicles (EVs). The soluble proteins and EV proteins in urine have attracted particular attention in recent years as cancer diagnostics. Furthermore, considering the important role of N-glycoproteins in practically all physiological processes, including regulating receptor-ligand binding, cell-cell interactions, inflammatory response and tumour progression, N-glycoproteome in human urine is an invaluable target for monitoring the physiological status and pathological changes of the kidney and urinary tract. Given the different origins of the soluble proteins and EV proteins in the urine, different N-glycoproteome patterns exist. Therefore, isolating the soluble N-glycoproteins and EV N-glycoproteins for separate analysis will provide a more specific and comprehensive view and provide a deeper understanding of human urinary N-glycoproteome. In this work, we developed a sequential separation method that isolates urinary soluble proteins and EV proteins via stepwise ultrafiltration based on their obvious size difference. A facile and reproducible protein isolation was achieved using this strategy. Subsequent N-glycoproteome enrichment and identification revealed distinct patterns in the two sub-proteomes of urine with more than 60% differential N-glycopeptides. A more comprehensive picture of the urinary N-glycoproteome with close to 1800 identified N-glycopeptides was obtained by this new analysis strategy, therefore making it advantageous for urinary biomarker screening.
Magnetic carbon nanocomposites as a MALDI co-matrix enhancing MS-based glycomics Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-08 Alireza Banazadeh, Seth Williamson, Masoud Zabet, Ahmed Hussien, Yehia Mechref
More than 50% of all known proteins are glycosylated, which is critical for many biological processes such as protein folding and signal transduction. Glycosylation has proven to be associated with different mammalian diseases such as breast and liver cancers. Therefore, characterization of glycans is highly important to facilitate a better understanding of the development and progression of many human diseases. Although matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) offers several advantages such as ease of operation and short analysis times, however, due to the complexity of glycan structures and their low ionization efficiency, there are still challenges that need to be addressed to achieve sensitive glycan analysis. Here, magnetic carbon nanocomposites (CNPs@Fe3O4 NCs) were used as a new MALDI matrix or co-matrix for the analysis of glycans derived from different model glycoproteins and human blood serum samples. The addition of CNPs@Fe3O4 NCs to the matrix significantly enhanced glycan signal intensity by several orders of magnitude, and effectively controlled/reduced/eliminated in-source decay (ISD) fragmentation. The latter was attained by modulating CNPs@Fe3O4 NCs concentrations and allowed the simultaneous study of intact and fragmented glycans, and pseudo-MS3 analysis. Moreover, CNPs@Fe3O4 NCs was also effectively employed to desalt samples directly on MALDI plate, thus enabling direct MALDI-MS analysis of unpurified permethylated glycans derived from both model glycoproteins and biological samples. On-plate desalting enhanced sensitivity by reducing sample loss.
Portable detection of serum HER-2 in breast cancer by a pressure-based platform Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-20 Qian Tao, Xinyi Wu, Qingyuan Lin, Haiyan Zheng, Wensheng Yang, Dan Liu, Chaoyong James Yang, Tianhai Ji
A high serum HER-2 extracellular domain (sHER-2 ECD) level has a reverse association with tumor behaviors. In this study, a portable platform for the disease biomarker sHER-2 ECD detection has been established using a pressure-based bioassay. The pressure bioassay consists of a monoclonal antibody immobilized on an eight-well strip, the analyte HER-2, and another monoclonal antibody labeled with the Pt nanoparticles (PtNPs), which have the catalytic ability to decompose H2O2 into H2O and O2(g). The increased pressure due to O2(g) generation is measured by a hand-held pressure meter. A total of 34 serum samples were collected to validate the performance of the pressure bioassay. The results showed that the pressure bioassay platform of HER-2 had a dynamic range from 2 to 50 ng/mL with a limit of detection (LOD) of 2 ng/mL, which was consistent with the ELISA result. In the real serum samples, there was a significant correlation between sHER-2 ECD level and several clinicopathological parameters, especially tissue HER-2 status. Furthermore, the sHER-2 ECD level was found to decrease after targeted therapy in a patient with tHER-2 positive. Overall, this bioassay can facilitate breast cancer diagnosis and prognosis in clinical scenarios and resource-limited areas.
A fluorescence turn-on probe for rapid monitoring of hypochlorite based on coumarin Schiff base Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-24 Liqiang Yan, Cunjie Hu, Jianping Li
A simple Schiff base was prepared by mild condensation reaction between a coumarin fluorescent group and diaminomaleonitrile, and it could serve as an excellent fluorescent probe for fast detection of ClO− with high selectivity and sensitivity. Along with addition of ClO−, this probe fleetingly showed noteworthy “turn-on” phenomenon accompanied by an increase of fluorescence intensity and the change of emission color from yellow to blue. This change of fluorescence is so significant that it can be observed by the naked-eye under a handheld ultraviolet light of 365 nm. However, other common reactive oxygen species exhibited no or very little fluorescent response under the same conditions. The limit of detection of this probe toward ClO− had a sensitivity feature as low as 9.6 nM. On account of these excellent features of short response time, remarkable fluorescence and color signal changes, high sensitivity and selectivity, this probe was effectively used for the fluorescence detection of ClO− in water samples. The values of the relative standard deviation were between 1.41% and 2.91%. More importantly, this probe displays excellent imaging capability in cytoplasm as well as very low cell toxicity and was unambiguously applied to image ClO− in living cells.
Sample preparation for mass spectrometry imaging of leaf tissues: a case study on analyte delocalization Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-13 Bin Li, Ying Zhang, Junyue Ge, Kehui Liu, Ping Li
Appropriate sample preparation is pivotally important to obtain high-quality mass spectrometry imaging (MSI) data. Unlike mammalian tissues, preparation of cryosections from plant tissues for MSI measurement is quite challenging due to its intrinsic complex texture and cellular structure. This is especially true for leaf samples which are generally thin, water-rich, and fragile. In this work, a systematic study was performed, aiming to evaluate three embedding materials and five mounting approaches for matrix-assisted laser desorption ionization (MALDI) MSI of secondary metabolites in cross sections of the ginkgo leaf. Delocalization of endogenous metabolites was chosen as a major indicator for evaluation of three embedding materials including ice, carboxymethyl cellulose (CMC), and gelatin and different mounting approaches. Image distortion and analyte delocalization were observed when ice was used as an embedding medium. CMC embedding provided better results compared to the ice by using modified mounting approach. Among three embedding materials, no delocalization was observed in specimens embedded with gelatin, and gelatin embedding is the least affected by different mounting approaches. An alternative approach to mitigate analyte delocalization is the removal of embedding media embraced the tissue sections before mounting, which is particularly suitable for ice-embedded samples. Additionally, the extent of analyte delocalization was closely related to their lipophilicity/hydrophilicity properties, and less analyte diffusion was observed for hydrophobic analytes than for the water-soluble compounds.
Switchable fluorescence of MoS 2 quantum dots: a multifunctional probe for sensing of chromium(VI), ascorbic acid, and alkaline phosphatase activity Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-21 Lianzhe Hu, Qian Zhang, Xiaoyan Gan, Weiling Yin, Wensheng Fu
A simple strategy for modulating the fluorescence of MoS2 quantum dots (QDs) is described. The fluorescence of MoS2 QDs was firstly switched off by the addition of Cr(VI), and the quenched fluorescence was further switched on by introducing ascorbic acid (AA) into the mixture. The fluorescence quenching of MoS2 QDs by Cr(VI) was attributed to the fluorescence inner filter effect. After the addition of AA, Cr(VI) was reduced to Cr(III), and the fluorescence was restored. This finding has been applied for the fluorescent sensing of Cr(VI) in drinking water and AA in serum samples. In addition, the present method has been extended for turn-on sensing of an important biomarker alkaline phosphatase (ALP). There is a linear relationship between the fluorescence intensity and the concentrations of ALP in the range from 2.5 to 50 U/L, and the limit of detection is 0.34 U/L. The results showed MoS2 QDs hold great potential as a multifunctional fluorescent probe for the detection of metal ions, biological small molecules, and proteins.
Dispersive solid-phase extraction based on MoS 2 /carbon dot composite combined with HPLC to determine brominated flame retardants in water Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-10 Sheying Dong, Qi Lou, Guiqi Huang, Jingjun Guo, Xiaohong Wang, Tinglin Huang
The method based on the dispersive solid-phase extraction (DSPE) by novel molybdenum disulfide modified with carbon dot (MoS2/CD) composite combined with high-performance liquid chromatography (HPLC) was developed for the determination of three brominated flame retardants (BFRs), including tetrabromobisphenol A (TBBPA), tetrabromobisphenol A bisallylether (TBBPA-BAE), and tetrabromobisphenol A bis(2,3-dibromopropyl ether) (TBBPA-BDBPE). Owing to the stacked planar structure and large surface area of MoS2, a large number of CDs can be easily loaded on the surface of MoS2. Benefiting from good dispersing capability of MoS2, similar density with analytes, and hydrogen bonds between CDs and the target analytes, the CDs on the surface of MoS2 as sorbent for the DSPE procedure exhibited good extraction performance. Under optimal conditions, application of the developed method to analyze BFRs from real water samples resulted in good recovery values ranging from 80 to 91% with relative standard deviation (RSD) values lower than 6.5%. Limits of detection (LODs) were in the range of 0.01–0.06 μg/L. The result above showed that the method has potential for the extraction and detection of trace-level BFRs from real water sample.
Matrix solid-phase dispersion coupled with gas chromatography–tandem mass spectrometry for simultaneous determination of 13 organophosphate esters in vegetables Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-28 Qing Luo, Shiyu Wang, Yue Shan, Li-na Sun, Hui Wang
In this study, matrix solid-phase dispersion coupled with gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed for the analysis of 23 organophosphate esters (OPEs) in vegetables. Under the optimal conditions, 0.5 g vegetables was dispersed with use of 2 g Florisil, 2 g anhydrous sodium sulfate, and 0.1 g graphitized carbon black, and it was transferred to an empty solid-phase extraction cartridge. The analytes were eluted with 15 mL n-hexane/acetone (1:1, v/v) and analyzed by GC–MS/MS. The method detection limits and quantitation limits ranged from 0.05 to 0.33 ng/g and from 0.16 to 1.10 ng/g, respectively. The recoveries ranged from 65.1% to 109.1%, and the relative standard deviations were less than 15%. The analysis of eight kinds of vegetables shows that the vegetables had been contaminated by OPEs; the concentrations of the sum of the OPEs ranged from 5.89 to 26.8 ng/g. The proposed method is applicable to analyze OPEs in vegetables.
High-throughput liquid chromatography differential mobility spectrometry mass spectrometry for bioanalysis: determination of reduced and oxidized form of glutathione in human blood Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-28 Sophie Bravo-Veyrat, Gérard Hopfgartner
Currently, the measure of the oxidative stress, from oxidized and reduced glutathione (GSSG and GSH respectively), for large cohorts of samples, is generally limited to spectrometric methods. In this study, a high-throughput assay for GSH after derivatization with N-ethylmaleimide and GSSG in blood sample was developed with an analysis time of 1.5 min. The method combines protein precipitation and a short LC (10-mm length) column where compounds were trapped in front-flush mode and eluted in back-flush mode. This setup is combined with modifier-assisted differential ion mobility spectrometry (DMS, SelexIon) and detection is performed in the selected reaction monitoring mode using positive electrospray ionization. In DMS, various modifiers were investigated including N2, methanol, toluene, ethanol, acetonitrile, and isopropanol to improve assay selectivity. Using EtOH as modifier, the limit of quantification (LOQ) was found to be 0.4 μM for GSSG and 3.2 μM for GS-N-ethylmaleimide (NEM) using a blood volume of 60 μL. The method is linear over a wide dynamic concentration range of 0.4 to 400 μM for GSSG and from 3.2 to 3200 μM for GS-NEM. The inter-assay precision of QC samples were ≤ 6.7%, with accuracy values between 98.3 and 103%. The method was further cross-validated with a LC Hypercarb-DMS-MS/MS method by the analysis of human blood samples. The bias between both assays ranged from − 0.3 to 0.2%.
An assessment of retention behavior for gold nanorods in asymmetrical flow field-flow fractionation Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-07 Hind El Hadri, Julien Gigault, Jiaojie Tan, Vincent A. Hackley
Applications of asymmetrical flow field-flow fractionation (AF4) continue to expand rapidly in the fields of nanotechnology and biotechnology. In particular, AF4 has proven valuable for the separation and analysis of particles, biomolecular species (e.g., proteins, bacteria) and polymers (natural and synthetic), ranging in size from a few nanometers to several micrometers. The separation of non-spheroidal structures (e.g., rods, tubes, etc.) with primary dimensions in the nanometer regime, is a particularly challenging application deserving of greater study and consideration. The goal of the present study was to advance current understanding of the mechanism of separation of rod-like nano-objects in the AF4 channel. To achieve this, we have systematically investigated a series of commercially available cetyltrimethylammonium bromide stabilized gold nanorods (AuNRs), with aspect ratios from 1.7 to 10. Results show clearly that the retention time is principally dependent on the translational diffusion coefficient of the AuNRs. Equations used to calculate translational and rotational diffusion coefficients (cylinder and prolate ellipsoid models) yield similarly good fits to experimental data. Well characterized gold nanorods (length and diameter by transmission electron microscopy) can be used as calibrants for AF4 measurements allowing one to determine the aspect ratio of nanorod samples based on their retention times.
A hierarchically porous composite monolith polypyrrole/octadecyl silica/graphene oxide/chitosan cryogel sorbent for the extraction and pre-concentration of carbamate pesticides in fruit juices Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-31 Pattamaporn Klongklaew, Thamolwan Naksena, Proespichaya Kanatharana, Opas Bunkoed
A hierarchically porous structured composite monolith sorbent of polypyrrole-coated graphene oxide and octadecyl silica incorporated in chitosan cryogel (PPY/GOx/C18/chitosan) was synthesized and used as solid-phase extraction sorbent for the determination of carbamate pesticides. Various factors affecting the characteristics of the adsorbents (chemistry of the sorbent, polymerization time, concentrations of graphene oxide and octadecyl silica) and the extraction efficiency using the prepared sorbents, such as sample loading, desorption conditions, sample volume, sample flow rate, sample pH, and ionic strength, were investigated and optimized. Under the optimal conditions of sorbent preparation and extraction, the developed composite monolith sorbent provided wide linear responses from 1.0 to 500 μg L−1 for carbofuran and diethofencarb, from 0.5 to 500 μg L−1 for carbaryl, and from 2.0 to 500 μg L−1 for isoprocarb. The limits of detection using HPLC-UV at 203, 220, and 208 nm were in the range of 0.5–2.0 μg L−1. When the composite monolith sorbent was applied for the pre-concentration and determination of carbamate in fruit juices, good recoveries (84.1–99.5%) were achieved. The developed sorbents were porous and exhibited low back pressure enabling their use at high flow rates during sample loading. Extraction and clean-up were highly efficient, and the good physical and chemical stability of the sorbent enables reuse up to 13 times.
Multi-contrast diffraction enhanced computed laminography at Beijing Synchrotron Radiation Facility Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-31 Jian Fu, Xianhong Shi, Qingxi Yuan, Wanxia Huang, Wei Guo, Peng Peng
Synchrotron radiation X-ray computed tomography (CT) enables nondestructive visualization of 3D morphological and chemical changes inside a sample and has become a powerful analysis tool to monitor reactive parts and their chemical states. However, synchrotron radiation CT imaging of specimens with lateral extensions much larger than the acceptance window of detectors is rather problematic due to strong absorption of X-rays in the lateral directions. On the other hand, X-ray computed laminography (CL) permits 3D imaging of flat samples while X-ray diffraction enhanced imaging (DEI) can provide high-quality results with different imaging contrasts such as absorption, phase and dark-field for samples with weak absorptions. Combining CL and DEI together, we have developed a multi-contrast DEI-CL system at the 4W1A beamline of the Beijing Synchrotron Radiation Facility for this kind of sample. Here we reported its design, implementation, and preliminary experimental results of carbon fiber reinforced polymer laminates with three kinds of imaging contrasts. The results have demonstrated the validity of this DEI-CL system. It will be helpful to push the applications of the state-of-the-art synchrotron radiation methods and instruments towards cutting-edge research.
Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-15 Václav Ranc, Radovan Žižka, Zuzana Chaloupková, Juraj Ševčík, Radek Zbořil
Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results.
Capillary-based chemiluminescence immunoassay for C-reactive protein with portable imaging device Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-22 Haiying Shen, Rizwanullah Khan, Xiaoqian Wang, Zulan Li, Feng Qu
A capillary-based chemiluminescence immunoassay system using a charge-coupled device (CCD) camera as detector was established in this paper. The fused quartz capillary was easily activated in one step for immobilizing capture antibody, and the chemiluminescence immunoassay was carried out in the capillary in double-antibody sandwich format. Chemiluminescence signals were recorded by the portable imaging device which was installed with the CCD camera and the results were analyzed through gray intensity. The total cost time, which included not only the time for test but also the time for the preparation of experimental materials, was only 2 h. The immunoassay was performed without any complicated or expensive instruments. The consumption of the sample was only 0.8 μL in one test, which was significantly less than other methods. In this work, C-reactive protein (CRP), as a target, was quantitatively detected from 0.3 to 160.0 μg mL−1 with high specificity and low sample volume. The reproducibility and accuracy were tested in clinic human serum samples and shown good results. Thus, this rapid, easy preparation and using, portable immunoassay system indicated its usefulness as a novel technology platform.
Sensitive determination of aldehyde metabolites in exhaled breath condensate using capillary electrophoresis with laser-induced fluorescence detection Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-13 Tingting Wang, Dan Luo, Zheyan Chen, Yining Qu, Xiuhua Ma, Jiannong Ye, Qingcui Chu, Dongping Huang
A novel capillary electrophoresis with laser-induced fluorescence detection method has been developed for the analysis of aldehyde metabolism biomarkers for oxidative stress in exhaled breath condensate (EBC), and fluorescein 5-thiosemicarbazide was used as a derivatization reagent. In a simple capillary zone electrophoresis mode, ten low molecular weight aldehydes (LMWAs) could be well separated within 30 min. The reaction efficiency was doubled by increasing sample solution pH and magnetic stirring, and the LODs of this method reached 0.16–3.4 nM (S/N = 3). Acceptable recoveries (82.1–115%) were obtained for EBC samples, and the RSD data were within 7.9%. This developed method has been applied for the analyses of EBC samples and evaluation of the correlation between smoking and the contents of aldehyde metabolites in EBC. Due to no need of buffer additives and sample preconcentration, this proposed method may provide an appealing alternative for the trace analyses of LMWAs in noninvasive biofluids.
A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-29 Agnes Magri, Matheus F. Soler, André M. Lopes, Eduardo M. Cilli, Patrick S. Barber, Adalberto Pessoa, Jorge F. B. Pereira
L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.188.8.131.52) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations.
Ultrathin ZIF-67 nanosheets as a colorimetric biosensing platform for peroxidase-like catalysis Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-01 Shujuan Wang, Dongpo Xu, Lan Ma, Jingxuan Qiu, Xiang Wang, Qingli Dong, Qi Zhang, Jing Pan, Qing Liu
In this work, we report a zeolitic imidazolate framework (ZIF-67) which could catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) to produce a yellow chromogenic reaction. ZIF-67 showed high peroxidase-like activity compared with copper-based metal−organic framework nanoparticles (Cu-MOF), zinc-based metal−organic framework nanoparticles (ZIF-8), and horseradish peroxidase (HPR). We discovered for the first time that the cobalt-based metal−organic framework nanoparticles possess intrinsic peroxidase-like activity without H2O2, which can be employed to quantitatively monitor the H2O2.
Characterization and mapping of secondary metabolites of Streptomyces sp. from caatinga by desorption electrospray ionization mass spectrometry (DESI–MS) Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-09-08 Júlia Pereira Rodrigues, Shamina Saiyara Prova, Luiz Alberto Beraldo Moraes, Demian Rocha Ifa
The discovery of new secondary metabolites is a challenge to biotechnologists due to the emergence of superbugs and drug resistance. Knowledge about biodiversity and the discovery of new microorganisms have become major objectives; thus, new habitats like extreme ecosystems have become places of interest to research. In this context, caatinga is an unexplored biome. The ecosystem caatinga is a rich habitat for thermophilic microbes. Its high temperature and dry climate cause selective microbes to flourish and become established. Actinobacteria (Caat 1-54 genus Streptomyces sp.) isolated from the soil of caatinga was investigated to characterize and map its secondary metabolites by desorption electrospray ionization mass spectrometry imaging (DESI–MSI). With this technique, the production of bioactive metabolites was detected and associated with the different morphological differentiation stages within a typical Streptomyces sp. life cycle. High-resolution mass spectrometry, tandem mass spectrometry, UV–Vis profiling and NMR analysis were also performed to characterize the metabolite ions detected by DESI–MS. A novel compound, which is presumed to be an analogue of the antifungal agent lienomycin, along with the antimicrobial compound lysolipin I were identified in this study to be produced by the bacterium. The potency of these bioactive compounds was further studied by disc diffusion assays and their minimum inhibitory concentrations (MIC) against Bacillus and Penicillium were determined. These bioactive metabolites could be useful to the pharmaceutical industry as candidate compounds, especially given growing concern about increasing resistance to available drugs with the emergence of superbugs. Consequently, the unexplored habitat caatinga affords new possibilities for novel bioactive compound discovery.
Critical assessment of different methods for quantitative measurement of metallodrug-protein associations Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-29 Luis Galvez, Sarah Theiner, Márkó Grabarics, Christian R. Kowol, Bernhard K. Keppler, Stephan Hann, Gunda Koellensperger
Quantitative screening for potential drug–protein binding is an essential step in developing novel metal-based anticancer drugs. ICP–MS approaches are at the core of this task; however, many applications lack in the capability of large-scale high-throughput screenings and proper validation. In this work, we critically discuss the analytical figures of merit and the potential method-based quantitative differences applying four different ICP–MS strategies to ex vivo drug–serum incubations. Two candidate drugs, more specifically, two Pt(IV) complexes with known differences of binding affinity towards serum proteins were selected. The study integrated centrifugal ultrafiltration followed by flow injection analysis, turbulent flow chromatography (TFC), and size exclusion chromatography (SEC), all combined with inductively coupled plasma-mass spectrometry (ICP–MS). As a novelty, for the first time, UHPLC SEC-ICP–MS was implemented to enable rapid protein separation to be performed within a few minutes at > 90% column recovery for protein adducts and small molecules.
A fluorescent material for the detection of chlortetracycline based on molecularly imprinted silica–graphitic carbon nitride composite Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-17 Shengnan Xu, Jie Ding, Ligang Chen
A new fluorescent probe based on graphitic carbon nitride (g-C3N4) combined with molecularly imprinted silica was successfully fabricated and used to selectively recognize chlortetracycline (CTC). The g-C3N4 used in this study has the characteristics of low toxicity and high chemical stability. This synthetic composite was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, UV spectroscopy, X-ray diffraction, and fluorescence spectroscopy. The material was used to detect CTC by the fluorescence quenching technique. The fluorescence quenching was due to g-C3N4 and the benzene ring of CTC through π–π electron donor–acceptor interaction and electrostatic force. Hydrogen bonds formed between CTC and 3-aminopropyltriethoxysilane during the polymerization process. Eventually, a considerable amount of selective recognition holes were formed in the composite material and could specifically recognize the template molecule CTC. In addition, the probe strategy was successfully applied to milk analysis, and the recoveries ranged from 90.1% to 95.7%, with relative standard deviations of 1.8–2.8%; the detection limit for CTC was 8 ng mL-1. The results indicate that this method combined the sensitivity of fluorescence detection with the excellent selectivity of a molecularly imprinted polymer. The new material can be widely used in the detection of dairy products.
A sample-in-digital-answer-out system for rapid detection and quantitation of infectious pathogens in bodily fluids Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-29 Haowen Yang, Zhu Chen, Xiaobao Cao, Zhiyang Li, Stavros Stavrakis, Jaebum Choo, Andrew J. deMello, Philip D. Howes, Nongyue He
A variety of automated sample-in-answer-out systems for in vitro molecular diagnostics have been presented and even commercialized. Although efficient in operation, they are incapable of quantifying targets, since quantitation based on analog analytical methods (via standard curve analysis) is complex, expensive, and challenging. To address this issue, herein, we describe an integrated sample-in-digital-answer-out (SIDAO) diagnostic system incorporating DNA extraction and digital recombinase polymerase amplification, which enables rapid and quantitative nucleic acid analysis from bodily fluids within a disposable cartridge. Inside the cartridge, reagents are pre-stored in sterilized tubes, with an automated pipetting module allowing facile liquid transfer. For digital analysis, we fabricate a simple, single-layer polydimethylsiloxane microfluidic device and develop a novel and simple sample compartmentalization strategy. Sample solution is partitioned into an array of 40,044 fL-volume microwells by sealing the microfluidic device through the application of mechanical pressure. The entire analysis is performed in a portable, fully automated instrument. We evaluate the quantitative capabilities of the system by analyzing Mycobacterium tuberculosis genomic DNA from both spiked saliva and serum samples, and demonstrate excellent analytical accuracy and specificity. This SIDAO system provides a promising diagnostic platform for quantitative nucleic acid testing at the point-of-care.
13 C quantification in heterogeneous multiphase natural samples by CMP-NMR using stepped decoupling Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-13 Paris Ning, Ronald Soong, Wolfgang Bermel, Daniel Lane, Myrna J. Simpson, André J. Simpson
Many natural and environmental samples contain combinations of liquids, gels, and solids, yet quantification in the intact state and across multiple phases is highly challenging. Comprehensive multiphase nuclear magnetic resonance (CMP-NMR) combines all the capabilities of high-resolution magic angle spinning (HR-MAS), with the addition of full solids power handling, permitting all phases (i.e., mixtures of liquids, gels, and solids) to be studied and differentiated in intact samples without pre-treatment or extraction. Here, quantification in CMP-NMR is considered. As 1H NMR is considerably broadened in the solid-state, quantification is easier to achieve through 13C which can be observed easily in all the phases. Accurate 13C quantification requires effective 1H decoupling for all the phases, but each phase requires different decoupling conditions. To satisfy these conditions, a pulse sequence termed stepped decoupling is introduced. This sequence can be used to study all components under ideal decoupling conditions resulting in high-resolution spectra without truncation artifacts and provides accurate integrals of components in all phases. The approach is demonstrated on standards and then applied to natural samples including broccoli, soil, and Arabidopsis. The approach permits accurate quantification of chemical categories (for example total carbohydrates) as well as individual species (for example glucose). Further, as the samples are studied intact, volatile species such as methanol and ethylene which are normally hard to detect in plants can be easily quantified in Arabidopsis.
Rapid monitoring of plant growth regulators in bean sprouts via automated on-line polymeric monolith solid-phase extraction coupled with liquid chromatography tandem mass spectrometry Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-08-28 Qixun Nian, Lianfeng Ai, Dongmei Li, Xuelei Chen, Lei Zhang, Manman Wang, Xuesheng Wang
An automated on-line solid-phase extraction (SPE) following liquid chromatography tandem mass spectrometry was established for the fast determination of plant growth regulator residues in soybean sprout and mung bean sprout. The crude extracted specimens were directly purified on a poly (2-(dimethylamino) ethyl methacrylate-co-ethylene dimethacrylate) monolithic column which was well-defined as the on-line SPE adsorbent. Under the optimized conditions, the developed method gave the linear range of 0.3–50 ng/mL for gibberellin and 2,4-dichlorophenoxyacetic acid, 0.2–50 ng/mL for 4-chlorophenoxyacetic acid, and 0.5–50 ng/mL for 1-naphthaleneacetic acid (r ≥ 0.998). The detection limits (S/N = 3) ranged from 1.0 to 2.5 μg/kg and the recoveries for spiked soybean sprout samples were in the range of 75.0–93.3%. Besides, the total time for one analysis was 16 min. The reusability of the monolith was up to 600 extractions. The proposed process facilitated fully automated SPE and accurate determination in one step with rapidity, simplicity, and reliability.
A simple, highly sensitive colorimetric immunosensor for the detection of alternariol monomethyl ether in fruit by non-aggregated gold nanoparticles Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-13 Yan Man, Junan Ren, Bingru Li, Xinxin Jin, Ligang Pan
Alternariol monomethyl ether (AME) is one of the major Alternaria mycotoxins present in a wide range of fruits, vegetables, grains, and their products, and possesses the properties of mutagenicity and carcinogenicity. In this study, a simple, rapid, and highly sensitive colorimetric immunosensor based on magnetic nanoparticles (MNPs) was firstly developed for the detection of AME in fruit by nonaggregated gold nanoparticles (GNPs). AME–BSA–Fe3O4 MNP conjugates and free AME molecules in samples competitively bind with monoclonal antibody (mAb)–GNP conjugates. After magnetic separation, the UV absorbance of the nonaggregated GNP supernatant was measured directly. The absorption intensity was proportional to the concentration of AME in the sample. Carboxyl-group-modified AME, AME–bovine serum albumin (BSA) conjugates, anti-AME mAbs, AME–BSA–Fe3O4 MNP conjugates, and mAb–GNP conjugates were prepared and characterized. The effect of GNP sizes (16, 24, and 40 nm) on the colorimetric determination of AME was studied. Under optimized conditions, the limit of detection and the linear range for AME were 0.16 ng/mL and 0.08–0.48 ng/mL, respectively. Moreover, the colorimetric immunosensor developed has lower cross-reactivity with AME analogues. The recoveries of spiked fruits ranged from 80.6% to 90.7%. The colorimetric immunosensor developed provides a promising method for simple, rapid, highly sensitive, and highly specific detection of other mycotoxins in the field of food safety.
IR-MALDESI mass spectrometry imaging of underivatized neurotransmitters in brain tissue of rats exposed to tetrabromobisphenol A Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-13 M. Caleb Bagley, Måns Ekelöf, Kylie Rock, Heather Patisaul, David C. Muddiman
There is a pressing need to develop tools for assessing possible neurotoxicity, particularly for chemicals where the mode of action is poorly understood. Tetrabromobisphenol A (TBBPA), a highly abundant brominated flame retardant, has lately been targeted for neurotoxicity analysis by concerned public health entities in the EU and USA because it is a suspected thyroid disruptor and neurotoxicant. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used for the analysis of neurotransmitters in the brains of rats exposed to TBBPA in gestation and lactation through their mothers. Three neurotransmitters of interest were studied in three selected regions of the brain: caudate putamen, substantia nigra (SN), and dorsal raphe. Stable isotope labeled (SIL) standards were used as internal standards and a means to achieve relative quantification. This study serves as a demonstration of a new application of IR-MALDESI, namely that neurotransmitter distributions can be confidently and rapidly imaged without derivatization.
New structural insights into the role of TROVE2 complexes in the on-set and pathogenesis of systemic lupus erythematosus determined by a combination of QCM-D and DPI Anal. Bioanal. Chem. (IF 3.307) Pub Date : 2018-10-13 Augusto Juste-Dolz, Noelle M. do Nascimento, Isidro Monzó, Elena Grau-García, Jose A. Román-Ivorra, José Luis Lopez-Paz, Jorge Escorihuela, Rosa Puchades, Sergi Morais, David Gimenez-Romero, Ángel Maquieira
The mechanism of self-recognition of the autoantigen TROVE2, a common biomarker in autoimmune diseases, has been studied with a quartz crystal microbalance with dissipation monitoring (QCM-D) and dual polarization interferometry (DPI). The complementarity and remarkable analytical features of both techniques has allowed new insights into the onset of systemic lupus erythematosus (SLE) to be achieved at the molecular level. The in vitro study for SLE patients and healthy subjects suggests that anti-TROVE2 autoantibodies may undergo an antibody bipolar bridging. An epitope-paratope-specific binding initially occurs to activate a hidden Fc receptor in the TROVE2 tertiary structure. This bipolar mechanism may contribute to the pathogenic accumulation of anti-TROVE2 autoantibody immune complex in autoimmune disease. Furthermore, the specific calcium-dependent protein-protein bridges point out at how the TRIM21/TROVE2 association might occur, suggesting that the TROVE2 protein could stimulate the intracellular immune signaling via the TRIM21 PRY-SPRY domain. These findings may help to better understand the origins of the specificity and affinity of TROVE2 interactions, which might play a key role in the SLE pathogenesis. This manuscript gives one of the first practical applications of two novel functions (−df/dD and Δh/molec) for the analysis of the data provided by QCM-D and DPI. In addition, it is the first time that QCM-D has been used for mapping hidden Fc receptors as well as linear epitopes in a protein tertiary structure.
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