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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In this work, a straightforward analytical approach based on headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry was developed for the analysis of salivary volatile organic compounds without any prior derivatization step. With a sample volume of 500 μL, optimal conditions were achieved by allowing the sample to equilibrate for 10 min at 50 °C and then extracting the samples for 10 min at the same temperature, using a carboxen/polydimethylsiloxane fibre. The method allowed the simultaneous identification and quantification of 20 compounds in sample headspace, including short-chain fatty acids and their derivatives which are commonly analysed after analyte derivatization. The proof of applicability of the methodology was performed with a case study regarding the analysis of the dynamics of volatile metabolites in saliva of a single subject undergoing 5-day treatment with rifaximin antibiotic. Non-stimulated saliva samples were collected over 3 weeks from a nominally healthy volunteer before, during, and after antibiotic treatment. The variations of some metabolites, known to be produced by the microbiota and by bacteria that are susceptible to antibiotics, suggest that the study of the dynamics of salivary metabolites can be an excellent indirect method for analysing the gut microbiota. This approach is novel from an analytical standpoint, and it encourages further studies combining saliva metabolite profiles and gut microbiota dynamics.
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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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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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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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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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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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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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The rapid and simultaneous detection of DNA and protein biomarkers is necessary to detect the outbreak of a disease or to monitor a disease. For example, cardiovascular diseases are a major cause of adult mortality worldwide. We have developed a rapidly adaptable platform to assess biomarkers using a microfluidic technology. Our model mimics autoantibodies against three proteins, C-reactive protein (CRP), brain natriuretic peptide (BNP), and low-density lipoprotein (LDL). Cell-free mitochondrial DNA (cfmDNA) and DNA controls are detected via fluorescence probes. The biomarkers are covalently bound on the surface of size- (11–15 μm) and dual-color encoded microbeads and immobilized as planar layer in a microfluidic chip flow cell. Binding events of target molecules were analyzed by fluorescence measurements with a fully automatized fluorescence microscope (end-point and real-time) developed in house. The model system was optimized for buffers and immobilization strategies of the microbeads to enable the simultaneous detection of protein and DNA biomarkers. All prime target molecules (anti-CRP, anti-BNP, anti-LDL, cfmDNA) and the controls were successfully detected both in independent reactions and simultaneously. In addition, the biomarkers could also be detected in spiked human serum in a similar way as in the optimized buffer system. The detection limit specified by the manufacturer is reduced by at least a factor of five for each biomarker as a result of the antibody detection and kinetic experiments indicate that nearly 50 % of the fluorescence intensity is achieved within 7 min. For rapid data inspection, we have developed the open source software digilogger, which can be applied for data evaluation and visualization.
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We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 μg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 μg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range.
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As synthetic cannabinoids are extensively metabolized, there is an urgent need for data on which metabolites can be used for successful urine screening. This study examines the in vitro metabolism of EG-018 and its 5F-analogue EG-2201 by means of comparing three different in vitro models: pooled human liver microsomes, cytochrome P450 isoenzymes, and a fungal approach utilizing the filamentous fungus Cunninghamella elegans LENDNER, which is known for its ability to mimic human biotransformation of xenobiotics. In addition, this study includes the screening of two authentic urine samples from individuals with proven EG-018 consumption, for the evaluation of in vitro–in vivo extrapolations made in the study. Incubation with pooled human liver microsomes yielded 15 metabolites of EG-018 belonging to six different metabolite subgroups, and 21 metabolites of EG-2201 belonging to seven different metabolite subgroups, respectively. Incubation with cytochrome P450 isoenzymes incubation yielded a further three EG-018 and five EG-2201 metabolites. With reference to their summed metabolite peak abundancies, the isoenzymes CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were shown to contribute most to the microsomal metabolism of EG-018 and EG-2201. CYP2B6 was shown to make the lowest contribution, by far. As the phase I metabolism of both synthetic cannabinoids was shown to be distributed over a substantial number of different cytochrome P450 isoenzymes, it was concluded that it is likely to not be significantly affected by co-consumption of other drugs. Although fungal incubation with Cunninghamella elegans yielded an additional three EG-018 and four EG-2201 metabolites not observed after microsomal incubation, metabolites generated by Cunninghamella elegans were in good correlation with those generated by microsomal incubations. The fungal model demonstrated its ability to be an independent in vitro model in synthetic cannabinoid metabolism research. The three tested in vitro models enable sufficient predictive in vitro–in vivo extrapolations, comparable to those obtained from hepatocyte incubation published in the literature. In addition, with regard to the screening of authentic urine samples and comparison with the literature, one monohydroxylated EG-018 metabolite and two monohydroxylated EG-2201 metabolites can be recommended as urinary targets, on the basis of the tested in vitro models.
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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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Single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) is an emerging technology. In this work, we have developed a novel SC-ICP-MS method to quantify metal ions in individual cells of a toxic cyanobacterial species, Microcystis aeruginosa (M. aeruginosa), without complicated post-dosing sample preparation, and applied this method to study the treatment effectiveness of copper-based algaecides (cupric sulfate and EarthTec®) on the toxic algae M. aeruginosa. The developed SC-ICP-MS method uses new intrinsic metal element magnesium to determine real transport efficiency and cell concentration. The cell viability and microcystin-LR release by algaecide treatment were studied by flow cytometry and ultra-fast liquid chromatography-tandem mass spectrometry, respectively. The results showed that this novel method was very rapid, highly sensitive (detection limits of intracellular copper and magnesium were 65 ag/cell and 98 ag/cell, respectively), and reproducible (relative standard deviation within 12%). The algaecide effectiveness study further demonstrated that copper in the forms of cupric sulfate and copper-based algaecide EarthTec® successfully diminished M. aeruginosa populations. The higher the copper concentration used to treat the cells, the faster the speeds of copper uptake and cell lysis in the copper concentrations ranged from 0 to 200 μg/L of copper-based algaecide. The cells exhibit obvious heterogeneity in copper uptake. The result suggests that M. aeruginosa cells uptake and cumulate copper followed by cellular lysis and microcystin-LR release. These novel results indicated that though the copper-based algaecides could control this type of harmful algal bloom, further treatment to remove the released algal toxin from the treated water would be needed.
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We report on the successful application of carboxyl-rich plasma polymerized (PP) films as a matrix layer for bioreceptor immobilization in surface plasmon resonance (SPR) immunosensing. Composition and chemical properties of the carboxyl-rich PP films deposited from a mixture of maleic anhydride and acetylene were investigated. Changes in the films stored in air, water, and buffer were studied and the involved chemical changes were described. Performance in SPR immunosensing was evaluated on interactions of human serum albumin (HSA) with a specific monoclonal antibody. The comparison with the mixed self-assembled monolayer of mercaptoundecanoic acid and mercaptohexanol (MUA/MCH) and one of the most widely used surfaces for SPR, the 2D and 3D carboxymethylated dextran (CMD), was presented to show the efficacy of plasma polymerized matrix layers for biosensing. The PP film-based SPR immunosensor provided a similar detection limit of HSA (100 ng/mL) as MUA/MCH- (100 ng/mL) and 3D CMD (50 ng/mL)-based sensors. However, the response levels were about twice higher in case of the PP film-based immunosensor than in case of MUA/MCH-based alternative. The PP film surfaces had similar binding capacity towards antibody as the 3D CMD layers. The response of PP film-based sensor towards HSA was comparable to 3D CMD-based sensor up to 2.5 μg/mL. For the higher concentrations (> 10 μg/mL), the response of PP film-based immunosensor was lower due to inaccessibility of active sites of the immobilized antibody inside the flat PP film surface. We have demonstrated that due to its high stability and cost-effective straightforward preparation, the carboxyl-rich PP films represent an efficient alternative to self-assembled monolayers (SAM) and dextran-based layers in label-free immunosensing.
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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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An ion chromatography and solid-phase extraction method has been applied for the separation and detection of morpholinium cations in environmental water samples. The water samples were purified and enriched by a UF-SCX sulfonic acid extraction column and eluted with 0.5 mol L−1 phosphoric acid/sodium dihydrogen phosphate buffer solution/55% methanol. The target compounds were separated on a carboxylic acid cation exchange column with 5.0 mmol L−1 methane sulfonic acid/2% acetonitrile as the mobile phase and direct conductivity detection. The method has been successfully applied to extract morpholinium cations from spiked water samples of Songhua River, Hulan River, East Lake, and Mopanshan Reservoir in China with the recoveries ranging from 75.0% to 98.3%. The relative standard deviations of intraday precision and interday precision are 2.1% and 5.9% or less, respectively. Using this method it is possible to preconcentrate water samples to 0.01–0.04 mg L−1. The results show that the method is applicable to detection of morpholinium ionic liquid cations in environmental water samples and provides a new approach for monitoring ionic liquids in environmental water.
The analysis procedure of morpholinium ionic liquids in environmental water samples.
A new class of magnetic ionic liquids (MILs) with metal-containing cations was applied in in situ dispersive liquid–liquid microextraction (DLLME) for the extraction of long and short double-stranded DNA. For developing the method, MILs comprised of N-substituted imidazole ligands (with butyl-, benzyl-, or octyl-groups as substituents) coordinated to different metal centers (Ni2+, Mn2+, or Co2+) as cations, and chloride anions were investigated. These water-soluble MILs were reacted with the bis[(trifluoromethyl)sulfonyl]imide anion during the extraction to generate a water-immiscible MIL capable of preconcentrating DNA. The feasibility of combining the extraction methodology with anion-exchange high-performance liquid chromatography with diode array detection (HPLC-DAD) or fluorescence spectroscopy was studied. The method with the Ni2+- and Co2+-based MILs was easily combined with fluorescence spectroscopy and provided a faster and more sensitive method than HPLC-DAD for the determination of DNA. In addition, the method was compared to conventional DLLME using analogous water-immiscible MILs. The developed in situ MIL-DLLME method required only 3 min for DNA extraction and yielded 1.1–1.5 times higher extraction efficiency (EFs) than the conventional MIL-DLLME method. The in situ MIL-DLLME method was also compared to the trihexyl(tetradecyl)phosphonium tris(hexafluorocetylaceto)nickelate(II) MIL, which has been used in previous DNA extraction studies. EFs of 42–99% were obtained using the new generation of MILs, whereas EFs of only 20–38% were achieved with the phosphonium MIL. This new class of MILs is simple and inexpensive to prepare. In addition, the MILs present operational advantages such as easier manipulation in comparison to hydrophobic MILs, which can have high viscosities. These MILs are a promising new class of DNA extraction solvents that can be manipulated using an external magnetic field.
Magnetic ionic liquids with metal-containing cations are applied in in situ dispersive liquid–liquid microextraction for the extraction of long and short double-stranded DNA
Common gaseous fuels are mixtures of several components. As the properties of the fuels can vary with the composition, but combustion needs to be stable, reliable analytical methods are highly sought after. Raman spectroscopic methods have proved their suitability for the characterization of diverse gaseous mixtures. They have the potential to overcome existing limitations of established technologies, since they are fast, non-consumptive, and accurate. Here, we demonstrate a gas sensor based on fiber-enhanced Raman spectroscopy (FERS) for fuel gas monitoring. Online detection of all gas components, including alkanes, carbon dioxide (CO2), nitrogen (N2), and hydrogen sulfide (H2S), for varying concentration ranges from tens of vol% down to the ppm level enables a comprehensive characterization of the fuels. The developed sensor system features a pinhole assembly which sufficiently reduces the background signal from the fiber to enable the detection of C2–C4 alkanes occurring in low concentrations. Detection limits in the low ppm region were achieved for the minor components of fuel gases, which allow the online monitoring of necessary purification steps, e.g., for biogas. The obtained results indicate that fiber-enhanced Raman sensors have the potential for comprehensive online and onsite gas sensing for fuel gas quality control.
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