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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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 Surface-enhanced Raman spectroscopy (SERS) method based on gold nanoparticles as SERS substrate was investigated for the label-free detection and quantification of probiotic bacteria that are widely used in various pharmaceutical formulations. Indeed, the development of a simple and fast SERS method dedicated to the quantification of bacteria should be very useful for the characterization of such formulations in a more convenient way than the usually performed tedious and time-consuming conventional counting method. For this purpose, uncoated near-spherical gold nanoparticles were developed at room temperature by acidic treatment of star-like gold nanoparticle precursors. In this study, we first investigated the influence of acidic treatment conditions on both the nanoparticle physicochemical properties and SERS efficiency using Rhodamine 6G (R6G) as “model” analyte. Results highlighted that an effective R6G Raman signal enhancement was obtained by promoting chemical effect through R6G-anion interactions and by obtaining a suitable aggregation state of the nanoparticles. Depending on the nanoparticle synthesis conditions, R6G SERS signals were up to 102–103-fold greater than those obtained with star-like gold nanoparticles. The synthesized spherical gold nanoparticles were then successfully applied for the detection and quantification of Lactobacillus rhamnosus GG (LGG). In that case, the signal enhancement was especially due to the combination of anion-induced chemical enhancement and nanoparticle aggregation on LGG cell wall consecutive to non-specific interactions. Both the simplicity and speed of the procedure, achieved under 30 min, including nanoparticle synthesis, sample preparation, and acquisition of SERS spectra, appeared as very relevant for the characterization of pharmaceutical formulations incorporating probiotics.
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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 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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The presence of Salmonella in natural freshwater and drinking water is a leading cause of intestinal illness all over the world; thus, the detection of Salmonella in water is of great importance to public health. The objective of this study is to develop a rapid screening method for the detection of Salmonella enterica serovar Enteritidis in water involving surface-enhanced Raman spectroscopy (SERS), aptamers, and filtration. SERS offers a great alternative to traditional methods of pathogen detection, with a simplified detection assay and shortened detection time. The specific capturing and labeling of Salmonella Enteritidis are realized by a specific single-stranded DNA aptamer, which is modified with an additional chain of adenine and fluorescein (FAM) and used as presence/absence indicator of Salmonella Enteritidis. By incorporating a vacuum filtration system, bacterial cells recognized by the specific aptamer are concentrated onto a membrane. With additional filtration of gold nanoparticles, the aptamer signals were captured and used to construct a SERS mapping indicating the presence and absence of target bacterial strains with potential quantitative capability. The specificity of the method was validated by using other strains of bacteria such as Escherichia coli and Listeria monocytogenes. The sensitivity of the method goes down to 103 CFU/mL for 1 mL of sample with a total detection and analyzing time within 3 h. This study demonstrates the capability of the filtration-based SERS platform for detecting Salmonella Enteritidis in various aqueous matrices such as distilled water and rinsing water from fresh produce with high selectivity and sensitivity.
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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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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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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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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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The preparation and fractionation of oligomeric proanthocyanidins (OPCs) are particularly important for the application of tannins in the biomedical field. By use of two different methods—gel filtration chromatography (GFC) with Sephadex LH-20 and progressive solvent precipitation—the OPCs were prepared and fractionated from mangosteen pericarp. The fractions were compared by reversed-phase and normal-phase high-performance liquid chromatography–electrospray ionization mass spectrometry and gel permeation chromatography. GFC directly purified oligomers (monomer to pentamer) with polydispersity values close to 1 and generated fractions with a higher level of total phenols (800.59 mg gallic acid equivalents per gram) but a lower yield (7.72%). Progressive solvent precipitation rapidly prepared and fractionated OPCs with a lower level of total phenols (609.57 mg gallic acid equivalents per gram) but a higher yield (24.74%) and higher polydispersity. Additionally, we found pronounced structural and quantitative differences among different tannin-rich fractions, and fractions obtained by GFC better reflected the structural diversity and complexity of OPCs from mangosteen pericarp. This study presents different ways of preparing and fractionating OPCs in the biomedical field.
Identification and quantification of microplastics (MP) in environmental samples is crucial for understanding the risk and distribution of MP in the environment. Currently, quantification of MP particles in environmental samples and the comparability of different matrices is a major research topic. Research also focusses on sample preparation, since environmental samples must be free of inorganic and organic matrix components for the MP analysis. Therefore, we would like to propose a new method that allows the comparison of the results of MP analysis from different environmental matrices and gives a MP concentration in mass of MP particles per gram of environmental sample. This is possible by developing and validating an optimized and consistent sample preparation scheme for quantitative analysis of MP particles in environmental model samples in conjunction with quantitative 1H-NMR spectroscopy (qNMR). We evaluated for the first time the effects of different environmental matrices on identification and quantification of polyethylene terephthalate (PET) fibers using the qNMR method. Furthermore, high recovery rates were obtained from spiked environmental model samples (without matrix ~ 90%, sediment ~ 97%, freshwater ~ 94%, aquatic biofilm ~ 95%, and invertebrate matrix ~ 72%), demonstrating the high analytical potential of the method.
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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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This work reports on further development of an inhibition electrochemical sensor array based on immobilized bacteria for the preliminary detection of a wide range of organic and inorganic pollutants, such as heavy metal salts (HgCl2, PbCl2, CdCl2), pesticides (atrazine, simazine, DDVP), and petrochemicals (hexane, octane, pentane, toluene, pyrene, and ethanol) in water. A series of DC and AC electrochemical measurements, e.g., cyclic voltammograms and impedance spectroscopy, were carried out on screen-printed gold electrodes with three types of bacteria, namely Escherichia coli, Shewanella oneidensis, and Methylococcus capsulatus, immobilized via poly l-lysine. The results obtained showed a possibility of pattern recognition of the above pollutants by their inhibition effect on the three bacteria used. The analysis of a large amount of experimental data was carried out using an artificial neural network (ANN) programme for more accurate identification of pollutants as well as the estimation of their concentration. The results are encouraging for the development of a simple and cost-effective biosensing technology for preliminary in-field analysis (screening) of water samples for the presence of environmental pollutants.
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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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Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic–neutral conditions, SP possesses a prevalent β-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular β-strands with a parallel alignment, which favour the self-assembly of SP in β-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of β-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs.
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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.
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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