Analytical Methods最新文献

A simple, accurate method for measuring ricin activity was developed by detecting depurinated nucleic acid stem-loops and adenine using a commercially available hydrophilic interaction liquid chromatography (HILIC) column and a quadrupole-Orbitrap tandem mass spectrometer. Ricin in beverages was isolated using magnetic beads conjugated with ricin B-chain antibodies, and then incubated with a 14 mer RNA or a 12 mer RNA/DNA chimera, in which adenosine at the depurination site of RNA was replaced by deoxyadenosine. The adenine and depurinated nucleic acids were separated by HILIC and both analytes were detected by high-resolution mass spectrometry. The depurinated RNA was detectable at concentrations as low as 100 pM (6.5 μg mL^-1) in orange juice and coffee, and 10 pM (0.65 μg mL^-1) in milk and sake after incubation with the RNA substrate for 4 h. Free adenine was detectable at 10 pM in all matrices, although free adenine was also detected in all blanks and could not be distinguished from the coffee and orange juice blanks at 10 pM. When using the chimera as the substrate, the depurinated chimera and adenine were detected up to concentrations of 10 pM as larger peaks. However, since the depurinated chimera and adenine were also detected in blanks, careful judgment was needed to determine whether they were active. Following the assay, the captured ricin could be analyzed by enzymatic digestion and nano liquid chromatography-high-resolution mass spectrometry. The ricin A chain-specific T7A peptide was detectable at 10 pM for sake and at 100 pM for milk, orange juice, and coffee. Using the present method, a toxicity assay and qualitative analysis of ricin were feasible with a 0.2 mL beverage sample.

Over the last several decades, multiple microfluidic platforms have been used for measurement of hormone secretion from islets of Langerhans. Most have used continuous flow systems where mixing of hormones with assay reagents is governed by diffusion, leading to long mixing times, especially for biomolecules like peptides and proteins which have large diffusion coefficients. Consequently, dispersion of rapidly changing signals can occur, reducing temporal resolution. Droplet microfluidic systems can be used to capture reagents into individual reactors, limiting dispersion and improving temporal resolution. In this study, we integrated a fluorescence anisotropy (FA) immunoassay (IA) for insulin into a droplet microfluidic system. Insulin IA reagents were mixed online with insulin and captured quickly into droplets prior to passing through a 200 mm incubation channel. Double etching of the glass device was used to increase the depth of the incubation channel compared to the IA channels to maintain proper flow of reagents. The droplet system produced highly precise FA results with relative standard deviations < 2% at all insulin concentrations tested, whereas the absolute fluorescence intensity precisions ranged between 5 and 6%. A limit of detection of 3 nM for insulin was obtained, similar to those found in conventional flow systems. The advantage of the system was in the increased temporal resolution using the droplet system where a 9.8 ± 2.6 s response time was obtained, faster than previously reported continuous flow systems. The improved temporal resolution aligns with continued efforts to resolve rapid signaling events in pancreatic islet biology.

A retention time (RT)-independent strategy for nontargeted screening of pesticide residues in herbs was exploited using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF MS). The core of this strategy is a fingerprint database coupled with a data-independent acquisition (DIA) scan mode of all ion fragmentation (AIF). In the fingerprint database, a total of 150 pesticides with quasimolecular ions and fragment ions at five-level collision energies were collected as qualified ions for screening. During the data acquisition, the AIF scan was performed via real unbiased full-spectrum MS/MS acquisition. Six herb matrices spiked with 30 banned pesticides were used to evaluate the applicability of the strategy in real samples. The use of the narrow ion mass extraction window (10 mDa) and the narrow RT window (0.1 min) enabled the effective extraction of spectra from noisy backgrounds and the discovery of suspected pesticides via similarity matching of filtered qualified ions. On average, more than 11/30 of pesticides at 1 ng mL^-1 and more than 23/30 of pesticides at 10 ng mL^-1 or lower could be screened out in each matrix using at least two qualified ions. In addition, the AIF mode exhibited superior anti-interference capability compared to data-dependent acquisition (DDA) and sequential window acquisition of all theoretical mass spectra (SWATH), as determined by comparing the limits of screening (LOSs) of 30 banned pesticides spiked into Isatidis Folium. Finally, the developed strategy was applied to screen pesticide residues in extracts of Ganoderma and Foeniculi Fructus. Phorate-sulfone and phorate-sulfoxide were found in Ganoderma, as well as terbufos-sulfone and terbufos-sulfoxide were found in Foeniculi Fructus. In conclusion, the developed RT-independent strategy based on a fingerprint database and AIF acquisition with LC-QTOF MS seems to be one of the most efficient tools for the analysis of nontargeted pesticide residues in complicated matrices.

Tuberculosis is a highly infectious bacterial disease caused by Mycobacterium tuberculosis. The spread of this agent has caused serious health problems worldwide, and the rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the trans cleavage ability of CRISPR-Cas12a against single-stranded DNA was combined with hybridization chain reaction and chemiluminescent signal to establish an imaging sensor for the hypersensitive detection of M. tuberculosis DNA. We observed linear relationships between the concentration of M. tuberculosis DNA and the output signal over the ranges of 10 to 200 pM and 200 to 800 pM DNA. The equations of the standard curves were y = 56.08x + 3303, with R² = 0.9916 for the lower range and y = 15.69x + 10 685, with R² = 0.9929 for the higher range. The limit of detection was as low as 0.83 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 1 copy per μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from sputum of hospitalized tuberculosis patients. The sensitivity and specificity of the proposed sensor is combined with a long shelf-life and a low cost of materials. This study introduces a new method for tuberculosis detection and broadens the application of CRISPR-Cas12a-based sensors in clinical diagnosis.

This article presents a numerical platform for predicting the performance of paper-based analytical devices. The capillary flow, reaction, dissolution, and other physicochemical phenomena associated with device operation are accounted for using Darcy's law, Richard's equation and other transport equations. The platform can be used for different paper substrates, biorecognition methods, detection systems (such as optical and electrochemical detection), device patterns and dimensions, and ways in which the device is operated such as the input method of the body fluid. The device performance is quantified using indicators such as assay time, signal strength and product cost. The predictive capability of this numerical tool is verified with devices reported in the literature. It is shown that the platform can be used to identify possible improvements to these existing devices. More importantly, it can also serve as a numerical tool for synthesizing new paper-based analytical devices with minimum experimental effort.

The isolation and analysis of chiral isomers are critical parts of the drug development process to ensure effective and safe drug administration to patients. Indirect chiral ligand exchange chromatography (ICLEC) was developed to separate and determine tenofovir alafenamide fumarate (TAF) and its diastereoisomer GS-7339, with a hypothesized separation mechanism. The effect of using a chiral column versus a standard C18 column on the separation of the TAF chiral isomer mixture was investigated. Various factors in ICLEC, including ligand type, ligand ratio, mobile phase composition, and column temperature, were optimized. The separation of TAF and GS-7339 was successfully achieved by selecting L-phenylalanine as the chiral selective agent and Cu(II) as the central metal ion, using a C18 column as the analytic column and a mobile phase of 20 mM ammonium dihydrogen phosphate buffer (pH = 4.0)-acetonitrile (79 : 21, v/v). The corresponding linearity range for TAF and GS-7339 indicated a good correlation with R² > 0.9960. The average recoveries of TAF and GS-7339 ranged from 98.2% to 106.9%. None of the eight manufacturers detected GS-7339, and the percentage of TAF-labeled amounts in the drugs ranged from 95.0% to 98.5%. TAF tablets from eight manufacturers were of satisfactory quality. The separation mechanism of TAF and GS-7339 by ICLEC is due to the different spatial configurations of the two ternary complexes formed by the two chiral isomers, leading to differences in their thermodynamic stability and retention behavior. The established ICLEC method is economical, simple, and flexible, providing an effective strategy for studying chiral drug separation and analysis.

To ensure the effectiveness of traditional Chinese medicine (TCM) and drive the advancement of high-quality Chinese medicine, the establishment of a scientifically sound quality evaluation system is particularly important. The objective of this study was to develop a straightforward and practical comprehensive evaluation method for quality control of Xiaohuoluo Pills (XHLP) by employing diverse fingerprint technologies. Firstly, Fourier transform infrared spectroscopy fingerprint (FT-IRFP), ultraviolet spectral fingerprint (UVFP) and fusion fingerprints based on five-wavelength matched average fusion fingerprint (FWFFP) were established for 21 samples. Secondly, the Comprehensive Quantified Ratio Fingerprinting Method (CQRFM) was used to conduct a comprehensive evaluation of the fingerprint profiles of 21 batches of samples and the results showed that all samples met the quality requirements. Then to further evaluate the samples, the External Standard Method (ESM) was employed to measure the content of three indicator components in XHLP, and this was done with the intention of enhancing the quality control system of the samples. Furthermore, antioxidant activity was also measured, and a model for analyzing the relationship between antioxidant data and fingerprint information from the three spectra was established using Orthogonal Projection to Latent Structures (OPLS), resulting in an accurate spectral efficacy relationship diagram. In conclusion, this study utilized spectral and chromatography techniques to comprehensively assess the quality of XHLP, incorporating qualitative and quantitative aspects. Additionally, a correlation between spectral profiles and efficacy was established by evaluating the antioxidant capacity of the samples. This method offers valuable insights for quality control not only of XHLP but also for other TCM.

This study introduces a novel deconvolution method for accurately quantifying oil or water signals in the NMR spectra of oil-water-sand mixtures. The algorithm employs a parametric model fitting approach that incorporates prior conditions to set parameters. To validate the approach, the widely overlapping NMR spectra of the complex, synthetic oil-water-sand mixes were separated, and their water and oil contents were determined. The results demonstrated high correlation values and maximum errors of 2.11% and 3.90% for water and oil contents, respectively. The proposed deconvolution method provides an innovative solution for simultaneously measuring the content of each fluid in complex porous media containing multiple fluids by NMR spectroscopy and has the potential to facilitate research in the field.

Enzyme-free signal amplification fluorescent aptasensors depending on multi-component freely diffusing probes have become indispensable tools for antibiotic detection in food, but they suffer from low detection efficiency and tedious operation steps. Herein, an all-in-one enzyme-free fluorescent aptasensor integrating localized catalyzed hairpin assembly (L-CHA) was designed for antibiotic detection with improved detection efficiency. In the designed aptasensor, a double-stranded DNA reactant containing an antibiotic aptamer and a primer as well as two paired hairpin DNA reactants were immobilized on one spatial-confinement DNA scaffold (that is a DNA tetrahedron). Upon addition of the target antibiotic kanamycin, the activated primer initiated L-CHA, generating an amplified fluorescence signal. Compared with previously reported enzyme-free signal amplification fluorescent aptasensors, the designed aptasensor integrated the functions of target recognition, signal transduction, and L-CHA signal amplification into a single probe. In this all-in-one design, the reactants in this aptasensor were confined to a compact space for a higher local concentration, which improved detection efficiency. In particular, this aptasensor achieved sensitive detection of kanamycin within 60 min with a low detection limit of 0.019 ng mL^-1. Additionally, the designed aptasensor depended on a single probe rather than multi-component probes, leading to simplified operation steps. Furthermore, this aptasensor was employed for detecting kanamycin in spiked milk samples with recoveries of 96.00% to 108.60%, indicating an acceptable accuracy. Therefore, this L-CHA-based all-in-one enzyme-free fluorescent aptasensor offers a prospective tool for antibiotic detection in the field of food safety.

A simple turn-off fluorescent probe, 5-(diethylamino)-2-(hydrazonomethyl)phenol (DHP), is designed and synthesized for the sensitive and selective detection of Cu²⁺. The bright green fluorescence of DHP is quenched after the addition of Cu²⁺. The probe DHP exhibits good anti-interference performance against Cu²⁺ in the presence of multiple metal ions. The fluorescence intensity of DHP (10 μM) at 522 nm is well linearized with the Cu²⁺ concentration at 0-5 μM, and it has a detection limit of 0.29 nM (R² = 0.9949). The complexation ratio of the probe DHP to Cu²⁺ is 2 : 1 and the complexation constant is 3.44 × 10⁴ M^-1 (R² = 0.9974). In addition, the probe DHP can be recovered using EDTA and Cu²⁺ can be effectively monitored at pH 5-11, with good results in dipstick experiments and actual water samples. HepG-2 cells remained viable in excess of 90% after being exposed to DHP (50 μM) for 24 h, which demonstrates the extremely low toxicity of DHP, and it can be used for in vivo cell imaging.