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.
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.
Graphical abstract
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.
Graphical abstract
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.
Graphical abstract
Starting from simple graphite flakes, an electrochemical sensor for sunset yellow monitoring is developed by using a very simple and effective strategy. The direct electrochemical reduction of a suspension of exfoliated graphene oxide (GO) onto a glassy carbon electrode (GCE) surface leads to the electrodeposition of electrochemically reduced oxide at the surface, obtaining GCE/ERGO-modified electrodes. They are characterized by cyclic voltammetry (CV) measurements and field emission scanning electron spectroscopy (FE-SEM). The GCE/ERGO electrode has a high electrochemically active surface allowing efficient adsorption of SY. Using differential pulse voltammetry (DPV) technique with only 2 min accumulation, the GCE/ERGO sensor exhibits good performance to SY detection with a good linear calibration for concentration range varying 50–1000 nM (R2 = 0.996) and limit of detection (LOD) estimated to 19.2 nM (equivalent to 8.9 μg L−1). The developed sensor possesses a very high sensitivity of 9 μA/μM while fabricated with only one component. This electrochemical sensor also displays a good reliability with RSD value of 2.13% (n = 7) and excellent reusability (signal response change < 3.5% after 6 measuring/cleaning cycles). The GCE/ERGO demonstrates a successful practical application for determination of sunset yellow in commercial soft drinks.
Graphical abstract
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.
Graphical Abstract
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.
Graphical abstract
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.
Graphical abstract
This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies’ integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7 ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7 min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing 153Eu-labelled polyclonal antibodies.
Graphical abstract
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.
Graphical abstract
We report the development of a fast and accurate fluorescence-based assay for amidine linked to cellulose membranes and Sepharose gel. The assay is founded on the glyoxal reaction, which involves reaction of an amidine group with glyoxal and an aromatic aldehyde, leading to the formation of a fluorophore that can be analyzed and quantified by fluorescence spectroscopy and imaging. While the assay has been reported previously for aromatic amidine estimation in solution phase, here we describe its adaptation and application to amidine linked to diverse forms of solid matrices, particularly benzamidine Sepharose and benzamidine-linked cellulose membranes. These functionalized porous matrices find important application in purification of serine proteases. The efficacy of a protein separation device is determined by, among other factors, the ligand (amidine) density. Hence, a sensitive and reproducible method for amidine quantitation in solid phase is needed. The glyoxal reaction was carried out on microbead-sized Sepharose gel and cellulose membranes. Calibration curves were developed for each phase, which established linearity in the range of 0–0.45 μmol per mL amidine for free amidine in solution, 0–0.45 μmol amidine per mL Sepharose gel, and 0–0.48 μmol per mL cellulose membrane. The assay showed high accuracy (~ 3.4% error), precision (RSD < 2%), and reproducibility. Finally, we show how this fluorescent labeling (glyoxal) method can provide a tool for imaging membranes and ligand distribution through confocal laser scanning microscopy.
Graphical abstract
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.
A facile single hydrothermal method was developed to synthetize P,N codoped carbon dots (P,N/CDs), which show strong and stable fluorescence, good water solubility, low toxicity and good biocompatibility. Hence, a novel and efficient “off-on” P,N/CDs fluorescent probe was developed for the highly sensitive detection of lipoic acid (LA) for the first time. The fluorescence of the P,N/CDs was quenched by Cu2+ forming a P,N/CDs-Cu2+ complex, which acted as the “off” process, but Cu2+ could be removed by LA, due to stronger chelating between LA and Cu2+, forming a more stable complex, which recovered the fluorescence of the P,N/CDs, in order to achieve the “on” process. Under optimal conditions, the concentration of LA and the increased fluorescence intensity of the P,N/CDs-Cu2+ complex displayed a good linear relationship within the range of 0.05–28 μM, with a detection limit (S/N = 3) of 0.02 μM. The established “off-on” fluorescent probe was successfully applied to the analysis of LA in urine samples. The average recoveries were in the range of 98.3–101.5%, with a relative standard deviations of less than 3.1%. In addition, the P,N/CDs were also successfully applied to cellular dual-color imaging of live T24 cells. The results show that the P,N/CDs have great application potential in clinical diagnosis, bioassay and bioimaging.
Graphical abstract
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.
Graphical abstract
Hydrophilic and hydrophobic deep eutectic solvents (DESs) as “green” solvents were applied in this study for the microextraction of environmental samples. A series of DESs (five hydrophilic and three hydrophobic) were synthesized and characterized by Fourier transform infrared spectroscopy. Physicochemical property parameters of eight DESs including water solubility, density, conductivity, and freezing point were assessed. Compared with the performance of five hydrophilic DESs in water phase, the three hydrophobic DESs were more suitable for application in dispersive liquid-liquid microextraction for the determination of sulfonamides in water sample. In dispersive liquid-liquid microextraction process, analytical parameters including type and volume of extraction solvent, extraction time, and pH of water sample were investigated. Under optimum conditions, 60 μL of hydrophobic DESs was used for extraction for 2 min in pH = 7.0 sample. The linear ranges were 0.05–5.0 μg/mL for the four sulfonamides with the correlation coefficients in the range of 0.9991–0.9999. The limits of detection were in the range of 0.0005–0.0009 μg/mL and the limits of quantification were in the range of 0.0019–0.0033 μg/mL. The recoveries of the analytes of the proposed method for the spiked samples were 80.17–93.5%, with the relative standard deviation less than 6.31%. The results indicated that three hydrophobic DESs showed commendable performance for extraction of sulfonamides, and hydrophobic DES-based microextraction method was successfully applied for monitoring sulfonamides in water samples.
Graphical abstract
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.
Graphical abstract
A novel colorimetric sensing platform based on the peroxidase activity of hemin regulated by oligonucleotide and pesticide was reported for the ultrasensitive and selective detection of isocarbophos. Oligonucleotides can accumulate on the surface of hemin in acid condition and temporarily inhibit its catalytic activity, which results in the loss of one electron of TMB molecule and produce the blue products. With the addition of isocarbophos, the pesticide molecules can interact with oligonucleotides to form some complexes, which relieve the inhibition of ssDNA to hemin and further enhance its catalytic activity. Thus, the TMB molecules are further oxidized to lose another electron and produce the yellow product in a few minutes, which has the characteristic absorption peak at 450 nm. The color change of the sensing system is related to the amount of isocarbophos, so this method can quickly discriminate whether the target pesticide exceeds the maximal residue limit just by naked eyes. To improve the performance of sensing platform, some important parameters like buffer condition and ssDNA have been investigated, and the peroxidase activity of hemin was further studied to verify the catalytic mechanism. The proposed sensing platform has a detection limit as low as 0.6 μg/L and displays good selectivity against other competitive pesticides. Moreover, the developed sensing platform also exhibits favorable accuracy and stability, indicating that it has potential applications in the detection of pesticide residues in agricultural products.
A novel colorimetric sensing platform based on oligonucleotides and pesticide regulation; the peroxidase catalytic activity of hemin was firstly reported for the ultrasensitive and selective detection of isocarbophos pesticide.
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.
Graphical abstract
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.
Graphical abstract