Analytical and Bioanalytical Chemistry最新文献

The development of feasible, low-cost, and long-persistent chemiluminescence (CL) systems is highly desirable for enhancing detection reproducibility and accuracy. Here, we present a space-confined CL system based on the LHC@G capsules, which are prepared via a one-pot ultrasound-assisted method to co-encapsulate glucose oxidase (GOx), hemin (a peroxidase mimic), and luminol within an oil-in-water structure. The LHC@G capsules are used for producing glow-type CL emission. Specifically, glucose is catalyzed by GOx on the capsule surface, yielding H₂O₂. The H₂O₂ then diffuses into the oil core and undergoes hemin-catalyzed in situ blue emission of luminol. The LHC@G capsule-based CL system exhibits intensive and prolonged blue emission for over 1500 s, with a nearly threefold enhancement in CL intensity compared to that of luminol solution. Mechanistic studies reveal that the excellent CL performance stems from a highly efficient cascade reaction and regulated diffusion. The proposed capsule CL system exhibits excellent reproducibility and storage stability, and has been successfully developed for simple, fast, and sensitive visual glucose detection with a LOD of 1.21 μM. This work provides a convenient strategy for constructing long-persistent and accurate CL systems in the field of point-of-care testing (POCT).

The species of Theobroma grandiflorum, commonly known as cupuassu, is widely used in the production of various cosmetic and food products. However, cupuassu seed shell (CSS), a major agro-industrial residue generated during the processing of its seeds, remains largely underexplored. In this study, a dual-extraction strategy was developed to valorize CSS using green chemistry principles, emphasizing waste recovery, reduced solvent consumption, and lower environmental impact. This methodology was applied to the extraction of theobromine, a methylxanthine of growing interest in the development of nutraceuticals and functional foods. In the first step, lipids were extracted using the energized dispersive guided extraction (EDGE) system, an automated and sustainable alternative to conventional methods. Fatty acids analysis revealed an oil profile like that of commercial cupuassu seed oil, reinforcing the potential of CSS oil as a functional ingredient in food, pharmaceutical, or cosmetic applications. In the second step, defatted CSS solid residue was subjected to theobromine extraction using the same EDGE system, this time employing a natural deep eutectic solvent (NADES) composed of choline chloride and glycerol (1:3). Extraction conditions were optimized using a BBD (Box-Behnken Design), with the best results achieved at 150 °C, 15 min, and 80% NADES, yielding 0.62 mg g^-1 in dry basis (d.b.) of theobromine. This study demonstrates a sustainable and efficient approach to convert a low-value byproduct into high-value bio-based compounds, highlighting the versatility of the EDGE system and reinforcing the principles of a circular economy and green analytical chemistry.

Herbal remedies contain various phenolic compounds. However, it remains difficult to identify the most important bioactive components and compare their effectiveness in different plant species. In this study, effect-directed analysis has been applied to five European medicinal plants. Angelica archangelica, Angelica sylvestris, Agrimonia eupatoria, Sambucus ebulus, and Sambucus nigra have been analyzed to unravel and compare their phenolic profiles and antioxidant potential. Plant extracts obtained by a sustainable microwave-assisted extraction method were fractionated using semi-preparative liquid chromatography to yield continuous fractions, and a miniaturized ABTS radical scavenging assay of the fractions was used to screen for antioxidant activity. Highly active fractions were selected for a second HPLC fractionation and analyzed with a quadrupole time-of-flight mass spectrometer using a non-targeted workflow that successfully linked antioxidant effects to specific compounds or compound classes. In the richest antioxidant fractions, flavan-3-ol oligomers such as procyanidin C1 were found in A. eupatoria, the flavonol glycoside rutin and other co-eluting phenolics in S. nigra. In contrast, A. archangelica showed a distinct metabolite profile rich in coumarins (e.g., bergapten, umbelliferone), but they contributed less to antioxidant activity compared to the flavonoid-dominated profiles of the other species. Overall, leaves and flowers contained the highest diversity and quantity of phenolic antioxidants among the plants studied. The effect-directed analysis of multiple European medicinal plants demonstrated its utility in exploring the major antioxidant compounds and highlighted significant differences in phenolic composition and antioxidant activity between species and plant parts.

Mass spectrometry combined with stable isotope labeling is a powerful technique for detecting disease-related changes in glycosylation patterns and identifying potential biomarkers. However, stable isotope labeling reagents that simultaneously offer high sensitivity, low cost, and stable sialic acid modifications remain scarce. In this study, we developed a convenient and cost-effective microwave-assisted method for synthesizing a stable isotopic quaternary phosphonium hydrazide labeling reagent pair, ¹⁴N/¹⁵N-P₄HZD, for the quantitation difference analysis of N-glycans using HPLC-ESI-HRMS with high sensitivity and convenience. This strategy features high labeling efficiency, excellent reproducibility, and strong linearity (R² = 0.9984) within a dynamic range spanning two orders of magnitude. The reagent pair is compatible with multiple ion source mass spectrometers and front-end chromatographic separation technologies. In particular, it enhances the ionization efficiency of sialylated N-glycans and facilitates their detection. The relative quantification method has been effectively applied to analyze the variations in N-glycomic profiles from two muscular atrophy models induced by simulated microgravity, specifically the C2C12 cell and hindlimb unloading mouse serum. We discover that these variations display characteristic relevance in both models. N-Glycans Man₃GlcNAc₃Fuc₁ and Man₃GlcNAc₄Gal₁Fuc₁Sia₁ exhibit their potential as biomarkers for the early diagnosis of muscular atrophy. The mass spectrometry method based on the ¹⁴N/¹⁵N-P₄HZD reagent pair offers a convenient and feasible strategy for the difference analysis of N-glycomics, demonstrating significant potential for application in the discovery of clinical biomarkers.

A hydrophilic interaction liquid chromatography-data-independent acquisition mass spectrometry (HILIC-DIA-MS) workflow was developed for simultaneous targeted semi-quantification of polar peptides and untargeted profiling of both peptides and other polar compounds in complex food matrices. The method uses a zwitterionic HILIC column optimized for separation of short polar peptides that are challenging to retain on reversed-phase columns. Many of these peptides contain charged amino acids and contribute to basic taste modalities such as umami and saltiness. Both targeted peptide analysis and comprehensive untargeted profiling were achieved by applying DIA-MS detection. This data acquisition mode was shown to be reproducible and sensitive while enabling retrospective data processing. High-resolution MS1 scans (60.000 FWHM), combined with fast MS2 scans and DIA mass windows of 15 m/z yielded highly repeatable and selective LC-MS profiles, allowing differentiation of structural isomers (e.g., alpha-glutamyl (umami) and gamma-glutamyl (kokumi)). The method was validated using taste-relevant dipeptides, demonstrating low detection limits (0.1-0.9 µM), good intra-day and inter-day precision, and high recovery (96%) in commercial soy sauce and yeast extract matrices. The workflow was further applied to the relative quantification of peptides and the untargeted profiling of characteristic molecular features in cheese, ham, and extracts from dried food ingredients. The integration of targeted and untargeted analyses demonstrates the suitability of HILIC-DIA-MS for comprehensive characterization of polar compounds in food systems.

The opioid crisis remains a significant public health concern, necessitating the development of sensitive and reliable analytical methods for drug detection. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of fentanyl, buprenorphine, oxycodone, morphine, tramadol, and tapentadol in plasma and oral fluid. The method was validated according to FDA guidelines, assessing selectivity, linearity, precision, accuracy, matrix effect, extraction efficiency, stability, carryover, and dilution integrity. The lower limits of quantification (LLOQs) were established at 0.1 ng/mL for fentanyl, 1.2 ng/mL for tramadol, and 0.6 ng/mL for the remaining opioids, demonstrating high sensitivity. The method exhibited excellent precision and accuracy, with coefficients of variation below 15% for intra-day, inter-day, and intermediate precision analyses. Extraction efficiencies exceeded 90% for most analytes, and matrix effects remained within acceptable limits. Real-world application to authentic plasma and oral fluid samples confirmed the method's robustness and reliability. Oral fluid concentrations were detectable across all target opioids, although plasma-oral fluid ratios showed some compound-dependent variability. These findings highlight the potential of oral fluid as a non-invasive complementary matrix to plasma for opioid monitoring, with relevant implications for forensic toxicology and clinical drug monitoring.

Virgin olive oil quality assessment is critical for consumer protection and industry regulation. Fraudulent activities involving virgin olive oil are common worldwide. In Brazil, recent nationwide inspections have revealed recurrent cases of fraud, including the sale of adulterated or mislabeled olive oils. As a result, the Brazilian federal agency has suspended the commercialization of numerous brands and seized large quantities of products that failed to meet the official quality requirements. This study presents an automated analytical workflow for classifying Brazilian virgin olive oils based on their volatile organic compound (VOC) profiles. Headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) were used to analyze 215 certified virgin olive oil samples, categorized as either defective (VOO, virgin and LVOO, lampante virgin) or non-defective (EVOO, extra virgin), and 21 undisclosed oils (UNKN). Chemometric modeling using partial least squares-discriminant analysis (PLS-DA) was applied to the resulting data, which comprised 108 recurring VOCs. The PLS-DA model successfully differentiated between the two quality classes (defective and non-defective) with a predictive accuracy of 91% based on the external validation set. Key chemical markers driving the classification were identified. Non-defective oils were characterized by higher levels of C₅ and C₆ aldehydes and alcohols from the lipoxygenase (LOX) pathway, such as (E)-2-hexenal, associated with positive green and fruity notes. Conversely, defective oils showed higher levels of compounds such as nonanal and acetic acid, linked to rancidity and other off-flavors. Finally, the model was used to predict the quality of 21 previously undisclosed samples. This instrumental approach demonstrates a powerful and reliable alternative for forensic analysis of olive oils, generating models that can be interpreted in sensory terms.

Mimotopes of the porcine epidemic diarrhea virus (PEDV) spike protein (SP) were screened from a Fv-antibody library, and the neutralizing activity of screened mimotopes was analyzed using plaque assay and docking simulation. The screened Fv-antibody (mimotopes) corresponded to the variable region of the heavy chain of immunoglobulin G, composed of three complementarity-determining regions (CDRs) and four framework regions. The Fv-antibody library was constructed by randomizing the amino acid sequence of CDR3 and expressed on the outer membrane of E. coli using auto-display technology. Monoclonal anti-PEDV SP antibody was used as probes to screen Fv-antibodies mimicking PEDV SP from the library. Two screened Fv-antibodies (mimotopes of PEDV SP) with binding affinity to the monoclonal antibody were expressed as soluble proteins, and their binding affinity was estimated using a surface plasmon resonance biosensor. The neutralizing activity of PEDV SP mimotopes to prevent PEDV infection was calculated using a plaque assay based on the cytopathic effect. Additionally, molecular docking simulations were performed to examine the interactions of PEDV SP mimotopes with ACE2 receptor as well as APN which had been considered infection-related receptors for coronavirus.

Meat adulteration poses significant social concerns, including the infringement of consumer rights, potential risks of food allergies, and conflicts with religious dietary practices. Therefore, a pressing need exists for the creation of swift and highly sensitive techniques to verify the authenticity of meat products. A portable and quantitative detection assay for pork identification in food was established by integrating a personal glucose meter (PGM) with a heparin-mediated one-pot RPA/Cas12a (called hRPA/SCas12a-PGM). In the hRPA/SCas12a-PGM assay, the target DNA sequence initiates the heparin-enhanced RPA/Cas12a reaction in a single tube, subsequently activating Cas12a's DNase function. Once activated, Cas12a cleaves sucrase-labeled DNA probes immobilized on the electrode, which are labeled with sucrase. Sucrase-labeled DNA fragments are released into the solution through this cleavage process. Sucrase then catalyzes the conversion of sucrose into glucose, producing a quantifiable signal that is detected by the PGM. The integrated system exhibited a broad dynamic range (10 pg/μL to 100 ng/μL), a low detection limit (10 pg/μL), and strong specificity against non-target samples. The hRPA/SCas12a-PGM assay provides a powerful, field-deployable solution for meat authenticity testing, offering significant potential for application in food safety surveillance and regulatory enforcement.

Examining the changes in microRNA expression in vivo enhances the understanding of their tumor-associated functions and facilitates clinical assays based on their roles as disease markers. However, due to the low abundance and high homology, their detection remains challenging. Herein, we developed an exponential amplification method by combining a symmetric dumbbell-type probe (SDTP) and toehold-initiated hyperbranched rolling circle amplification (HRCA) to obtain high sensitivity and selectivity. The toehold region of the dumbbell-type probe specifically binds to the target, triggering a toehold-mediated strand displacement reaction and thus activating SDTP into a circular structure. In the presence of phi29 DNA polymerase, primer P2, and dNTPs, the HRCA reaction proceeds continuously using the circular structure as a template, enabling exponential amplification of let-7a. When the concentration of let-7a ranges from 1.00 fmol·L^-1 to 1.00 nmol·L^-1, the fluorescence intensity obtained by this method exhibits a linear correlation with the logarithm of let-7a concentration (unit: pmol·L^-1), with a detection limit of 214 amol·L^-1. This method can distinguish homologous miRNAs with single-base mismatches. Serum spiking recovery experiments verified the accuracy of the method in actual serum sample detection; meanwhile, the simplicity was demonstrated by the detection of let-7a in different cell lysates without RNA extraction. Therefore, our work exhibited a simple, sensitive, and specific detection of miRNAs in biological samples, which holds promise as a potential tool for miRNA analysis in clinical testing.