Analytical Methods最新文献

Early detection of cancer biomarkers is crucial for improving patient survival rates. Herein, a highly sensitive label-free electrochemical immunosensor for prostate-specific antigen (PSA) was constructed based on in situ grown Ag₂S nanoparticle-decorated MoS₂ nanosheets (Ag₂S@MoS₂). The Ag₂S@MoS₂ nanocomposite synergistically combined the large surface area of MoS₂ with the high conductivity of Ag₂S, significantly amplifying the electroactive surface area and enhancing electron transfer. Upon formation of the insulating PSA-antibody immunocomplex, electron transfer was hindered, leading to a measurable current decrease. Under the optimal conditions, the sensor demonstrated a wide logarithmic linear range from 0.5 pg mL^-1 to 50 ng mL^-1 and an ultralow detection limit of 0.17 pg mL^-1 (based on 3σ/S). More importantly, the immunosensor demonstrated high selectivity, reproducibility, and, crucially, reliable accuracy in the detection of PSA in spiked human serum samples. This work not only provided a highly sensitive tool for PSA detection but also established a versatile and powerful sensing platform based on synergistic interface engineering, with broad potential for the detection of other disease biomarkers.

CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U mL^-1 for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.

Accurate monitoring of cellular energy metabolism is crucial for understanding exercise physiology and optimizing athlete training and recovery. However, current assessments mainly rely on macroscopic indicators such as heart rate and blood lactate, providing limited insight into intracellular glucose utilization-the key process governing energy supply and fatigue during exercise. Herein, we report the in situ synthesis of boronic acid-functionalized near-infrared (NIR) carbon dots (B-OH-CDs) from 1-methylisoquinoline and 4-formylbenzeneboronic acid under green and mild conditions. The resulting B-OH-CDs feature a graphitic carbon core and a borate-rich surface shell, exhibiting excitation-independent red emission (615 nm), high quantum yield (3.2%), and excellent photostability. Abundant boronic acid sites confer selective and sensitive fluorescence responses toward glucose (100 nM-200 µM, detection limit 85 nM). Confocal imaging reveals dual targeting of mitochondria and lysosomes, enabling real-time visualization of intracellular glucose dynamics related to cellular energy metabolism. This sustainable nanoplatform offers a new tool for exploring glucose-dependent metabolic regulation and holds potential for precision monitoring of energy balance, fatigue, and metabolic adaptation in exercise physiology.

Accurate quantification of aldosterone is crucial for screening and diagnosing primary aldosteronism. In clinical laboratories, chemiluminescence is commonly used for aldosterone measurement. However, significant variability exists between different measurement systems. A complete reference system is needed to achieve comparable results across laboratories. Therefore, we developed a candidate reference measurement procedure based on liquid chromatography-tandem mass spectrometry for the quantitative determination of aldosterone in serum and urine. In this study, aldosterone-d₈ was used to prepare standard solutions. Serum and urine aldosterone samples were extracted via liquid-liquid extraction and solid-phase extraction, and the steps in the pre-treatment process were optimised. Chromatographic separation was performed on a ZORBAX Eclipse XDB-C18 column, and quantitative detection was carried out in negative mode via an electrospray ionization source. The performance of the method was also evaluated. Serum aldosterone exhibited linearity in the range of 4.5-3902 pg mL^-1, with a limit of quantification (LOQ) of 3.1 pg mL^-1. The coefficient of variation (CV) ranged from 0.5% to 1.2%. Urine aldosterone showed linearity in the range of 10-998 pg mL^-1, with a LOQ of 9.9 pg mL^-1. The CV ranged from 1.1% to 1.4%. The recoveries of both samples were within the range of 95-105%. This method meets the analytical requirements of a reference measurement procedure, reduces the use of hazardous chemicals during sample preparation, and may serve as a candidate reference method for the accurate quantification of aldosterone in serum and urine samples in the laboratory.

Atomic force microscopy (AFM) images often suffer from random fluctuations, periodic interference, and scanning artifacts. We propose DARA-Net, a denoising framework combining a dual U-shaped GAN architecture with residual attention blocks (RABs) in the decoder stage. Two parallel predictors-one for structural content and one for noise-enable effective feature disentanglement, while the GAN framework enhances detail realism. The fused output benefits from the RAB's ability to suppress complex noise and preserve nanoscale topographical features. A dataset of 1000 in-house AFM images with synthetic degradation was used for training and evaluation. The results show that DARA-Net outperforms classical and state-of-the-art methods in PSNR, SSIM, and RMSE and achieves lower errors in four physical surface metrics (perimeter, height, roughness, and volume), demonstrating superior generalization and structural preservation for nanoscale imaging.

This study presents a dynamic multiple reaction monitoring (dMRM) method for the simultaneous analysis of 122 polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs, halogenated PAHs, heterocyclic PACs, and halogenated HPACs using gas chromatography-tandem mass spectrometry (GC-MS/MS). Conventional MRM methods for these complex mixtures (including the one used as our benchmark) require multiple GC injections and time segments to maintain sufficient MS dwell and cycle times. The dMRM developed here captured all our analytes in a single GC-injection. The analytical performance characteritsitc of the dMRM method was compared to our conventional time-segmented MRM methods using matrices of increasing complexity, including calibration standards, certified sediment and mussel reference materials, and an in-house fortified egg reference material. Instrument detection limits were similar for both methods and ranged from 0.1 to 1.3 pg µL^-1. The dMRM method achieved comparable or improved precision and accuracy compared to conventional MRM for less complex matrices, such as standard solutions and biota. Negative systematic biases were observed for a subset of analytes in the sediment matrix for both approaches and are attributed to to non-exhaustive extractions rather than limitations of the MS methods. Provided that sample preparation is carefully optimized for challenging matrices, the dMRM technique offers a powerful tool for high-throughput environmental analysis of PACs, enabling a single GC injection to streamline laboratory workflows and enhance analytical efficiency.

Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB_L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aurum, and Escherichia coli. The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB_L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB_L378R mutation.

In this work, a novel Ti-based MOF (NH₂-MIL-125) bonded open-tubular (OT) column was firstly prepared via a one-step bonded growth method for capillary electrochromatography (CEC). The stationary phase was characterized by Fourier transform-infrared spectroscopy, scanning electron microscopy, X-ray diffraction spectroscopy, nitrogen adsorption-desorption isotherm measurements and zeta potential measurements. The results showed that the stationary phase exhibited a large specific surface area (1247.57 m² g^-1) with microporous and mesoporous structure and no obvious changes in the morphology/size inside and outside the column. Baseline separation of three β-adrenergic receptor blockers (Prop, Sot, and Lab), three β-adrenergic receptor agonists, and four sulfonamide antibiotics was obtained under the optimized CEC conditions with the shortest analysis time of 2.52 min and a maximum resolution of 11.52. The separation mechanisms were mainly attributed to the polarity and electrophoretic mobility of the analytes, as well as π-π interactions and hydrogen bonding interaction between the stationary phase and the analytes. A quantitative detection method for the three β-adrenergic receptor blockers in pork samples was established using the NH₂-MIL-125 bonded OT column. Good linearity (R² > 0.999) was obtained over the concentration range of 0.01-1.00 mg mL^-1 with limits of detection of 0.0044-0.0084 mg mL^-1 and recoveries of 90.24-106.74%. Thus, the developed method was simple, rapid and highly efficient, and could be applied for the simultaneous separation and detection of the three β-adrenergic receptor blockers in real samples.

In this study, a novel type of coal-based carbon dots (CDs) was synthesized using Inner Mongolia lignite as the carbon source and H₂O₂ as the oxidizing agent, and it was successfully applied for the detection of Fe³⁺ ions. The morphology and structure of CDs were characterized using TEM, XPS and FT-IR techniques. The results revealed that the CDs exhibited a relatively regular triangular star-like morphology with an average diameter of approximately 9.21 nm. The surface was abundant in oxygen-containing functional groups, including hydroxyl and carboxyl groups. Under ultraviolet illumination, the samples exhibited bright blue fluorescence, with a fluorescence quantum yield of up to 23.49%. When the concentration of Fe³⁺ is within the range of 6-670 µmol L^-1, a well-defined linear relationship is observed, with a detection limit of 0.123 µM. Furthermore, SiO₃^2- acts as a fluorescence restorer in the CDs-Fe³⁺ system, enabling the construction of a fluorescence quenching-recovery system based on CDs, which has been successfully applied to the detection of Fe³⁺ in soil and vegetables samples. These novel coal-based CDs exhibit promising application potential in the environmental analysis of Fe³⁺ ions.

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most harmful pathogenic bacteria derived from seafood, and it can cause great damage to aquaculture and human health, even at low concentrations. The rapid and accurate detection of V. parahaemolyticus in clinical samples is essential but remains a challenge. Herein, we have developed a highly sensitive, simple and visual method for V. parahaemolyticus detection. The capture probe is composed of a V. parahaemolyticus-specific aptamer, with its blocking strands designated as the initiator (I), and magnetic beads (MB). In the presence of V. parahaemolyticus, the aptamer on the probe specifically recognizes and binds to V. parahaemolyticus, leading to the separation of the I strand. Next, the I strand sequentially initiates a hybridization chain reaction (HCR) for signal amplification, releasing the G-quadruplex, which was blocked in hairpin H2, and recovers its peroxidase-mimicking activity. Thus, the results can be visualized through the color variation of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS^2-), which displays green for V. parahaemolyticus-containing samples and colorless for negative samples. With the signal amplification of HCR, this method can achieve a detection limit of 0.82 CFU mL^-1 with a wide linear detection range from 10⁰ to 10⁶ CFU mL^-1. It also exhibited excellent specificity against common non-target bacteria in aquaculture and performed well in the detection of V. parahaemolyticus-spiked Pacific white shrimp. Furthermore, this method realized the accurate detection of V. parahaemolyticus in shrimp seedlings from six different breeding bases in Wenchang, Hainan Province, which indicates tremendous potential for early disease diagnosis and prevention.