The i-motif is a biologically relevant non-canonical DNA structure formed by cytosine-rich sequences. Despite the importance of the factors affecting the formation/stability of such a structure, like pH, cation type and concentration, no systematic study that simultaneously analysed their effect on the i-motif in vitro has been carried out so far. Therefore, here we report a systematic study that aims to evaluate the effect of these factors, and their possible interaction, on the formation of an i-motif structure. Our results confirm that pH plays the main role in i-motif formation. However, we demonstrate that the effect of the cation concentration on the i-motif is strictly dependent on the pH, while no significant differences are observed among the investigated cation types.
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Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized.
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A simple method for the simultaneous quantification of meropenem and the recently approved β-lactamase inhibitor, vaborbactam, in human plasma and renal replacement therapy effluent (RRTE) was developed and validated. This antibiotic combination protects a primary β-lactam, meropenem, with a new β-lactamase inhibitor, and expands the limited options for treatment of multidrug-resistant Gram-negative infections. Meropenem, vaborbactam, and the internal standards [2H6]-meropenem and sulbactam in plasma and RRTE were processed using acetonitrile followed by a chromatographic separation on a Poroshell HPH-C18 column with a gradient elution of the mobile phases and monitored using mass spectrometry detection. The calibration range was 0.05 to 100 μg mL−1 for both meropenem and vaborbactam. The intra-day and inter-day precision and accuracy were less than 15% for both meropenem and vaborbactam and the recovery from plasma was 96% for both meropenem and vaborbactam and the recovery from RRTE was 93% and 103% for meropenem and vaborbactam, respectively. This methodology was successfully applied to an ex vivo characterisation study of the effects of renal replacement therapy modalities on the pharmacokinetics of meropenem and vaborbactam (Antimicrob Agents Chemother 62(10), 2018).
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A multichannel chip containing 16 microchambers was developed for fast and sensitive immunoassays. In each chamber, antibody-functionalized nonmagnetic beads were applied as the solid phase to capture target antigens. Four types of IgGs (human, rabbit, chicken, and mouse) could be detected simultaneously by our combining this microchip with a sandwich immunoassay technique. A three-layer chip structure was investigated for integration of multiple processes, including washing, immune reaction, and detection, in one microchip. Moreover, the proposed chip design could improve batch-to-batch repeatability and avoid interferences between different channels without the preparation of complex microvalves. The total operation time of this system was less than 30 min, with a desirable detection limit of 0.2 pg/mL. The results indicate that the microfluidic platform is promising for the immunoassay of multiple clinical biomarkers.
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G-quadruplexes have been widely researched as new targets for cancer treatment owing to their non-canonical structure and crucial role in biological processes. Although attention has been paid to the development of selective G-quadruplex ligands, few studies have focused on the binding affinity of stereoisomers towards G-quadruplex, which will be conducive to support the optimal design of G-quadruplex ligands in future studies. Here, tetrandrine and isotetrandrine were used to study the binding affinity and difference of stereoisomers towards G-quadruplex structures. The results showed that tetrandrine had a high possibility of binding to the N-myc and Bcl-2 G-quadruplexes through hydrogen bonding, whereas the possibility of binding of isotetrandrine was low and it seemed to have no possibility of forming hydrogen bonds. Our study shows that optical isomerism of ligand molecules has an important effect on G-quadruplex recognition, which is helpful for the design of G-quadruplex ligands in future studies.
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Desorption atmospheric pressure photoionization (DAPPI) is an ambient mass spectrometry (MS) technique that allows the analysis of both polar and nonpolar compounds directly from the surfaces of various sample types. Here, DAPPI was used to study the chemical profiles in different parts of birch and alder tree barks. Four distinct fractions of Betula pendula (silver birch) bark were collected from three different developmental stages of the stem, after which the chemical profiles of the different tissue types were measured. Of special interest were triterpenoids, a class of important defensive substances, which are found in the bark of the silver birch. Additionally, the chemical profiles of lenticels and the surrounding surfaces in the phellem of B. pendula (silver birch), Alnus glutinosa (black alder), and Alnus incana (gray alder) were screened with DAPPI. Another ambient MS technique, laser ablation atmospheric pressure photoionization (LAAPPI), was further used for the mass spectrometry imaging of lenticels on the B. pendula phellem. All the studied birch bark fractions showed individual chemical profiles in DAPPI. The mass spectra from the young apical stem and the transition zone resembled each other more than the mature stem. Instead, the phellem was found to contain a high amount of triterpenoids in all the developmental stages of the stem. The most intense peaks in the DAPPI mass spectra of the birch bark fractions were those of betulin and lupeol. Betulinic and betulonic acid peaks were intense as well, and these compounds were detected especially in the lenticels of the tree samples.
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The cyanate anion (CNO−), formed spontaneously within cells from urea and carbamoyl phosphate, usually functions as a biomarker of some diseases such as chronic kidney disease. Therefore, accurate determination of CNO− is highly demanded. Herein, a 3-amino-2-naphthoic acid-based “turn-on” fluorescence probe was developed for specific detection of CNO−. Upon the addition of sodium cyanate, the weak-fluorescent 3-amino-2-naphthoic acid could react with CNO−, which triggered intense emission of green fluorescence. And up to 9-fold fluorescence enhancement was observed. The fluorescence enhancement ratios displayed a good linear relationship with the concentrations of CNO− in the range of 0.5–200 μM. The high selectivity and sensitivity for CNO− detection were investigated with the detection limit as low as 260 nM. The probe was further successfully applied to determine CNO− in real samples such as tap water, human urine and serum samples, which offered a promising approach in practical applications.
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In this label-free surface-enhanced Raman scattering (SERS) study of genomic DNA, we demonstrate that the cancer-specific DNA methylation pattern translates into specific spectral differences. Thus, DNA extracted from an acute myeloid leukemia (AML) cell line presented a decreased intensity of the 1005 cm−1 band of 5-methylcytosine compared to normal DNA, in line with the well-described hypomethylation of cancer DNA. The unique methylation pattern of cancer DNA also influences the DNA adsorption geometry, resulting in higher adenine SERS intensities for cancer DNA. The possibility of detecting cancer DNA based on its SERS spectrum was validated on peripheral blood genomic DNA samples from n = 17 AML patients and n = 17 control samples, yielding an overall classification of 82% based on the 1005 cm−1 band of 5-methylcytosine. By demonstrating the potential of SERS in assessing the methylation status in the case of real-life DNA samples, the study paves the way for novel methods of diagnosing cancer.
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A newly developed molecularly imprinted photonic polymer (MIPP) film, which was prepared by colloidal crystal templating and surface molecular imprinting, was used for selective capture of S-layer protein (SLP) from a complex Lactobacillus acidophilus sample. The colloidal crystal templates were formed by a dipping process followed by chemical binding of the imprinted template SLP molecules. A sandwich structure consisting of two glass slides was formed after the SLP–silica layer had been covered with a poly(methyl methacrylate) glass slide. After polymerization of the SLP–silica layer with the preprepared polymerization solution, hydrofluoric acid and acetic phosphate buffer solutions removed the silica particles and SLP molecules, respectively. The MIPP film obtained exhibited a three-dimensional, highly ordered and interconnected macroporous structure (pore size greater than 200 nm), which is specifically accessible to SLP molecules. The adsorbed SLP molecules were simply and straightforwardly detected by a fiber-optic spectrometer. The redshift of the Bragg diffraction peak of the MIPP film was linearly related to the number of SLP molecules that had been harvested in the film. The detection limit of the SLP–MMIP–fiber-optic spectrometer method for SLP was 1 ng mL-1. The MIPP sensor was successfully applied to detect SLP molecules in a crudely extracted Lactobacillus acidophilus sample. Our results prove the applicability of the SLP–MIPP film for fast and real-time measurement of SLP.
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Folic acid (FA) is an essential vitamin in humans, and thus, rapid, accurate, and sensitive methods for its quantification in different biological samples are needed. This work describes a novel, simple, and effective dual-emission fluorescence nanoprobe for FA detection and quantification. The probe was covalently linked to amino-modified orange quantum dots (QDs) and carboxyl-modified blue graphene quantum dots (GQDs). The resulting material exhibited two emission peaks at 401 and 605 nm upon excitation at 310 nm. The probe had good selectivity and sensitivity toward FA with an exceptionally low detection limit (LOD = 0.09 nM). This probe was effectively used to quantify FA in animal serum samples. The method has potential utility for FA analysis in different types of biological samples.
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A biomass nitrogen and sulfur codoped carbon dots (NS-Cdots) was prepared by a simple and clean hydrothermal method using leek, and was employed as efficient fluorescent probes for sensitive detection of organophosphorus pesticides (OPs). The leek-derived NS-Cdots emitted blue fluorescence, but was quenched by H2O2. Due to acetylcholinesterase/choline oxidase–based cascade enzymatic reaction that produces H2O2 and the inhibition effect of OPs on acetylcholinesterase activity, a NS-Cdots-based fluorescence “off-on” method to detect OPs-dichlorvos (DDVP) was developed. More sensitivity and wider linear detection range were achieved from 1.0 × 10−9 to 1.0 × 10−3 M (limit of detection = 5.0 × 10−10 M). This developed method was applied to the detection of DDVP in Chinese cabbage successfully. The average recoveries were in the range of 96.0~104.0% with a relative standard deviation of less than 3.3%. In addition, the NS-Cdots fluorescent probes were also employed successfully in multicolor imaging of living cells, manifesting that the NS-Cdots fluorescent probes have great application potential in agricultural and biomedical fields.
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In this paper, we present a new colorimetric technique as a novel assay for the easy and direct detection of α-amylase activity. This detection system utilizes the interaction of α-amylase with starch that is supporting copper/gold (Cu/Au) nanoclusters. The Cu/Au nanoclusters are synthesized using starch as a stabilizing agent at room temperature. These nanoclusters show robust peroxidase-like activity and are able to catalyze the oxidation of TMB (3,3,5,5-tetramethylbenzidine) in the presence of hydrogen peroxide (H2O2), leading to the generation of a blue-colored solution. The α-amylase detection mechanism is based on the digestion of the starch by α-amylase, which results in nanocluster aggregation, leading to increased nanoparticle size and thus decreased peroxidase-like activity of the Cu/Au NCs. Experiments showed that the gradual addition of α-amylase causes the peroxidase activity to decrease step by step in a linear fashion. Using this method, colorimetric sensing of α-amylase was achieved with a detection limit (LOD) of 0.04 U/mL and a linear range of 0.1–10 U/mL. This method is significantly selective for α-amylase and could be affordably and conveniently applied to the detection of α-amylase in blood serum.
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A novel rhodamine–tryptamine conjugate–based fluorescent and chromogenic chemosensor (RTS) for detection of Hg2+ present in water was reported. After gradual addition of Hg2+ in aqueous methanol solution of RTS, a strong orange fluorescence and deep-pink coloration were observed. The probe showed high selectivity towards Hg2+ compared to other competitive metal ions. The 1:1 binding stoichiometry between RTS and Hg2+ was established by Job’s plot analysis and mass spectroscopy. Initial studies showed that the synthesized probe RTS possessed fair non-toxicity and effectively passed through cell walls of model cell systems, viz., human neuroblastoma (SHSY5Y) cells and cervical cells (HeLa) to detect intercellular Hg2+ ions, signifying its utility in biological system. The limit of detection (LOD) was found to be 2.1 nM or 0.42 ppb by fluorescence titration. Additionally, the potential relevance of synthesized chemosensor for detecting Hg2+ ions in environmental water samples has been demonstrated.
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The cholesterol-lowering properties of 12 lactic acid bacteria (LAB) in the absence or presence of 0.3% bile salts were assessed and compared quantitatively and qualitatively in vitro. A new, more sensitive and cost-effective high-performance thin-layer chromatography method combined with digital image evaluation of derivatised chromatographic plates was developed and validated to quantify cholesterol in LAB culture media. The performance of the method was compared with that of the o-phthalaldehyde method. For qualitative assessment, assimilated fluorescently tagged cholesterol was visualised by confocal microscopy. All LAB strains exhibited a cholesterol-lowering effect of various degrees (19–59% in the absence and 14–69% in the presence of bile salts). Lactobacillus plantarum LAB12 and Pentosaceus pentosaceus LAB6 were the two best strains of lactobacilli and pediococci. They lowered cholesterol levels by 59% and 54%, respectively, in the absence and by 69% and 58%, respectively, in the presence of bile salts. Confocal microscopy showed that cholesterol was localised at the outermost cell membranes of LAB12 and LAB6. The present findings warrant in-depth in vivo study.
(A) 3D plots based on scan at 525 nm of (B) derivatized HPTLC plate of separated cholesterol and (C) confocal microscopic image showing the localisation of NBD-cholesterol assimilated by LAB
It is necessary to characterize and classify neural stem cells (NSCs) and differentiated cells (DCs) for potential use of NSC to treat neurodegenerative diseases. We therefore performed an analysis of NSCs and DCs using gas chromatography mass spectrometry (GC-MS) and direct infusion mass spectrometry (DI-MS) with elaborate multivariate statistical analysis for the characterization and classification of rat NSCs and DCs. GC-MS and DI-MS detected a total of 92 metabolites and lipids in NSCs and DCs, and the levels of 72 of them differed significantly between NSCs and DCs. The optimal model for partial least squares (PLS) discriminant analysis was constructed by applying 3 and 2 PLS components with a unit-variance scaling method for classifying NSCs and DCs based on the data obtained in the GC-MS and DI-MS analyses, respectively. The obtained results from PCA and PLS-DA suggest that creatinine, lactic acid, lysine, glutamine, glycine, pyroglutamic acid, PG 18:1/20:2, PS 18:0/20:2, PI 18:0/20:3, PC 16:0/20:4, PI 16:0/20:4, and PI 18:1/20:4 were the main contributors that provided distinct characteristics of NSCs and DCs. The results of this study suggest objective and complementary criteria for the characterization and classification of NSCs and DCs for potential clinical applications.
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Choosing an appropriate ion source is a crucial step in liquid chromatography mass spectrometry (LC/MS) method development. In this paper, we compare four ion sources for LC/MS analysis of 40 pesticides in tomato and garlic matrices. We compare electrospray ionisation (ESI) source, thermally focused/heated electrospray (HESI), atmospheric pressure photoionisation (APPI) source with and without dopant, and multimode source in ESI mode, atmospheric pressure chemical ionisation (APCI) mode, and combined mode using both ESI and APCI, i.e. altogether seven different ionisation modes. The lowest limits of detection (LoDs) were obtained by ESI and HESI. Widest linear ranges were observed with the conventional ESI source without heated nebuliser gas. In comparison to HESI, ESI source was significantly less affected by matrix effect. APPI ranked second (after ESI) by not being influenced by matrix effect; therefore, it would be a good alternative to ESI if low LoDs are not required.
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Liquid-core waveguide (LCW) has many advantages such as the elimination of optical artifacts typically exhibited in systems employing lenses and filters. However, due to the effect of temporal dispersion, LCWs are typically employed in steady-state fluorescence detection microsystems rather than in fluorescence lifetime measurement (FLM) systems. In this paper, we present a compact liquid-core waveguide time-correlated single-photon counting (LCW-TCSPC) sensor for FLM. The propagation of excitation within the LCW is analyzed both analytically and in simulations, with results in agreement with experimental characterization. Results reveal an optimal region within the LCW for highly accurate FLM. The proposed prototype achieves excellent excitation rejection and low temporal dispersion as a result of optimization of the propagation length of the excitation within the LCW. The prototype achieves a detection limit of 5 nM for Coumarin 6 in dimethyl sulfoxide with < 3% lifetime error. The techniques proposed for analyzing the LCW for TCSPC based FLM and prototype demonstration pave the way for developing high-performance fluorescence lifetime measurement for microfluidics and point-of-care applications.
A compact liquid-core waveguide time-correlated single-photon counting (LCW-TCSPC) sensor for fluorescence lifetime measurement (FLM) is presented. Results reveal an optimal propagation length region within the LCW for highly accurate FLM. The prototype achieves a detection limit of 5 nM for Coumarin 6 in dimethyl sulfoxide with < 3% lifetime error.
Evaluation of post-translational modifications of protein molecules is important for both basic and applied biomedical research. Mass spectrometric quantitative studies of modifications, which do not change the mass of the protein, such as isomerization of aspartic acid, do not necessarily require the use of isotope-labelled standards. However, the accurate solution of this problem requires a deep understanding of the relationship between the mole fractions of the isomers and the peak intensities in the mass spectra. In previous studies on the isomerization of aspartic acid in short beta-amyloid fragments, it has been shown that calibration curves used for such quantitative studies often have a non-linear form. The reason for the deviation in the shape of the calibration curves from linearity has not yet been established. Here, we propose an explanation for this phenomenon based on a probabilistic model of the fragmentation process and present a general approach for the selection of fragments that can be used for quantitative studies of the degree of isomerization.
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