Analytical and Bioanalytical Chemistry最新文献

CRISPR-Cas12a-driven nucleic acid diagnostics offer considerable potential for highly specific and rapid detection. However, their practical applications are limited by the necessity for pre-amplification of target DNA to enhance sensitivity. To overcome this limitation, we developed Auto-catalyst, a novel one-pot, amplification-free DNA detection platform employing a two-stage autocatalytic Cas12a cascade. This approach integrates a positive feedback amplification mediated by a circular crRNA-DNA nanostructure with an asymmetric CRISPR reaction driven by split crRNA. Without external amplification, this system detects DNA fragments at concentrations as low as 80 aM within 30 min at room temperature and maintains high specificity, accurately distinguishing single-base mutations down to 1 fM. Clinical validation demonstrated successful detection of pathogenic DNA in cerebrospinal fluid samples from patients with intracranial infections, highlighting its potential for rapid bedside diagnostics essential for timely clinical decision-making. Additionally, Auto-catalyst accurately identified the clinically significant isocitrate dehydrogenase 1 (IDH1) gene R132H mutation from glioma tissue samples. This integrated two-stage autocatalytic Cas12a strategy represents a powerful, convenient, and promising diagnostic tool suitable for point-of-care applications.

Potato early blight, caused by Alternaria solani, presents a significant threat to the potato industry. Existing detection methods for A. solani often fail to simultaneously achieve simplicity and accuracy. A gold-platinum (AuPt) nanozyme-assisted CRISPR/Cas12a system, termed the nanoparticle enzyme-assisted CRISPR detection (NACD assay) was developed. By integrating the precise target recognition of CRISPR with the enzyme-like activity of AuPt nanozymes, this system achieves simple, sensitive, and visual detection of A. solani. The NACD assay provided visual results through a distinct color change produced by the substrate catalyzed by the AuPt nanozyme. It can detect 100 copies/μL of the target dsDNA (A. solani 5.8S rRNA gene) and 10⁻³ ng/μL A. solani genomic DNA. This detection method demonstrates high specificity, with no cross-reactivity observed with three other pathogens. Moreover, the incorporation of a filter paper-based readout enables straightforward visual detection by the naked eye, making it particularly suitable for on-site testing. Overall, these features make it an effective on-site diagnostic tool, allowing the potato industry to manage early diseases more efficiently.

Herein, a colorimetric and smartphone dual-channel sensor has been constructed for the sensitive detection of acetone based on a chemically driven redox-cycling reactive system. In the redox-cycling system, o-phenylenediamine (OPD) acted as the colorimetric substrate. Initially, colorless OPD was oxidized by Cu²⁺ ions to generate Cu⁺ ions and light yellow 2,3-diaminophenazine (DAP). The Fenton-like reaction between hydrogen peroxide (H₂O₂) and generated Cu⁺ ions initiated the production of Cu²⁺ ions and hydroxyl radicals (·OH). The resulting ·OH and reproduced Cu²⁺ ions could further catalyze other OPD molecules, leading to the formation of more light yellow DAP molecules, with a distinct ultraviolet absorption peak appearing at 440 nm. And this process continued until all OPD was completely consumed in the cycling reaction system. Interestingly, acetone was introduced into the redox-cycling system, leading to a colorless solution and a reduction of absorbance intensity at 440 nm. Based on this phenomenon, a colorimetric sensor with excellent sensitivity and selectivity has been designed for the visual detection of acetone. The sensor exhibited a linear correlation with acetone concentrations within the range of 2 μM to 5 mM, achieving a detection limit of 0.5 μM. Notably, RGB color space analysis was performed for the quantitative detection of acetone with the assistance of a smartphone. The results were in good agreement with the data obtained via UV-visible absorption spectroscopy. Furthermore, the strategy realized the rapid detection of acetone in real tap water, river water, and lake water samples.

Microfluidics offer a useful platform for cell studies by coupling enhanced fluidic control with real-time imaging, but traditional fabrication methods require expensive equipment and advanced training. In this work, the fabrication and implementation of a low-cost and simple-to-use microfluidic device for cell studies were completed using digital light processing resin 3D printing to produce molds for polydimethylsiloxane (PDMS) devices. Contact angle analysis assessed surface characteristics during fabrication. Tape was used to reversibly seal devices, permitting reuse without damage or adhesion loss. Additionally, tape-sealed devices could withstand an excess of 4 times the internal pressure compared to PDMS devices reversibly sealed on glass. A treatment solution was developed to enable adherent culture of U-87 human glioblastoma cells on the adhesive surface and was confirmed via fluorescence-based viability and morphology monitoring. A miniature incubator, programmable fluidic system, and heating pad were incorporated to maintain conditions for long-term experiments and continuous flow. By employing this unique fabrication method, design prototyping and performance optimization can be rapidly completed for bioanalytical applications of microfluidic-based cell studies. This work provides a foundation, especially for those unfamiliar with microfluidics, to develop microfluidic platforms for cellular analysis using an accessible and less expensive approach.

In this study, a new liquid phase microextraction method was developed by combining micelle-mediated extraction of aristolochic acids (AAs) in traditional Chinese medicines with polyfluorinated alcohol solutions, and subsequent preconcentration via deep eutectic solvent (DES)-induced supramolecular solvent (SUPRAS) formation. The preparation of SUPRASs with polyfluorinated alcohols functioning as amphiphiles and the DESs acting as coacervation agents is demonstrated for the first time. Different polyfluorinated alcohols and DESs were examined for the extraction and preconcentration of AAs. The SUPRAS prepared from hexafluorobutanol (HFB) and a DES based on (1) octanol:(2) fenchyl alcohol achieved the highest extraction efficiency. Compared to traditionally used surfactants such as octanol, octanoic acid, and octylamine, HFB exhibited better extraction efficiency of AAs. The optimized method provided good linearity in the range of 0.14-500 µg g^-1 for AA-I and 0.1-600 µg g^-1 for AA-II with determination coefficients higher than 0.9961. The limits of detection were 0.03-0.04 µg g^-1, which were lower than those of the limits stipulated by the Chinese Pharmacopoeia (0.001%, w/w). The extraction recoveries ranged between 89.2 and 107.8%. The conventional methods for extracting analytes from solid samples typically involve an initial extraction phase utilizing hazardous organic solvents. The developed method can be applied to extract and preconcentrate a wide range of trace analytes from different solid samples by eliminating the need for harmful organic solvents. The developed method is efficient and reliable and can be applied to determine AAs in natural plants.

Microplastic (MP) pollutes our terrestrial and aquatic ecosystems due to their uncontrolled discharge into our environment. The analysis of MP contamination is still a challenge, although significant improvements are made for different environmental matrices. Using mass-based particle analysis methods such as thermal extraction and desorption-gas chromatography/mass spectroscopy (GC/MS) or pyrolysis GC/MS, essential parameters such as the MP's morphology, size, and shape cannot be obtained, which are indispensable to assess the hazard of the respective particles. Raman, micro-Fourier transform infrared, and attenuated total reflectance spectroscopy are particle-based analysis methods, which are time-consuming due to the high purification effort. Thus, novel, reliable, and time-efficient methods for MP analysis are required. Previously, studies showed the potential of frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to identify plastics' type, shape, size, and morphology, and distinguishing these from natural materials. However, only pure plastic granules were investigated, omitting that commodity plastics accumulating in our environment contain various additive, filler, or dye concentrations. To circumvent the dependency of additive, filler, and dye concentrations, we investigated the fluorescence spectra and lifetimes of three plastic types, individually composed with two fillers, three additives, and two dyes in six different concentrations. We heuristically modeled the dependency of the concentration on plastics' fluorescence lifetime using a logarithmic model with a high correlation and showed that identifying the plastic types is hardly possible when fillers, additives, or dyes are added in various concentrations because of their superimposing fluorescence lifetimes. However, further research has to be conducted to investigate different emission states of fluorescence to optimize the FD-FLIM method, as only one excitation wavelength and emission band was used for the investigations.

The analytical determination of critical quality attributes (CQAs) of monoclonal antibodies (mAbs) is often time-consuming. Hence, increasing analysis throughput can shorten biopharmaceutical development timelines and enables rapid decision-making. Microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) allows fast analysis of charge variants and glycoforms of mAb samples. In a recently published MCE-MS method, we used a microfluidic chip with a 22-cm separation channel length, permitting analysis of 96 samples per day. To further increase the throughput of the method and better meet the needs of the biopharmaceutical industry, we propose the use of a chip with a distinctly shorter channel length (10 cm). However, a substantial drop in electrophoretic resolution was observed due to the increased laminar flow caused by the pressure put on the shortened channel during analysis. To mitigate this phenomenon, the MCE setup was modified to allow independent adjustment of the gas pressure on the separation channel inlet. The mAb charge-variant resolution significantly improved by reducing the inlet pressure from 2.0 to 0.75 psi. The performance of the modified setup was assessed by analysis of mAb samples from cell lines, mimicking a typical biosimilar clone screening. The original (22-cm channel, 2.0 psi on inlet) and modified (10-cm channel, 0.75 psi on inlet) setups yielded comparable separation profiles and number of MS-identified proteoforms across all clone mAbs. Overall, the new MCE-MS method reduces total analysis time per sample by 3.3-fold, which conceptually facilitates the analysis of more than 300 samples per day with minimal loss in separation performance.

This study explores the feasibility of using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) to analyse the volatilome of human melanoma A375 cells. The goal was to identify and characterise the volatile organic compounds (VOCs) and monitor alterations induced by treatment with thapsigargin (TG), a drug known to disrupt calcium homeostasis, thereby inducing endoplasmic reticulum stress and ultimately triggering cell death. Reproducibility of experimental conditions is a major issue in biological experiments, which presents intrinsic variability of the samples to be analysed. In our case, initial analysis revealed a significant batch effect, accounting for 56.88% of the total variance. To address this, external parameter orthogonalisation (EPO) was applied, which successfully reduced the batch variance to just 0.16%. After this correction, the treatment factor became the dominant source of variation, explaining 47.12% of the total variance with strong statistical significance (p-value = 0.001). A supervised classification model using partial least squares discriminant analysis (PLS-DA) was developed and validated to characterise the differences between treated and untreated cells. The model achieved a mean overall accuracy of 92.21% and an area under the curve (AUC) of 0.974, indicating excellent discrimination between the two classes. The robustness of these findings was confirmed by repeated double cross-validation and permutation testing, which showed that the model's predictive ability was not due to random chance. The results demonstrate that TG treatment induces a reproducible and highly discriminant volatilome signature in A375. This suggests that VOCs could potentially serve as biomarkers for monitoring cellular responses to drug treatments.

Fusion genes are a series of typical tumor biomarkers that can induce dysregulated gene expression and generate oncogenic proteins, both of which contribute to malignant transformation. Consequently, their detection is crucial for early cancer diagnosis, treatment selection, and prognostic evaluation. However, the existing fusion gene detection techniques remain constrained by time-consuming protocols, labor-intensive sample processing, and dependence on sophisticated instrumentation. To overcome these challenges, we present a rapid and portable photothermal biosensing platform utilizing DNA sandwich nanozymes (DSNs). The DSN integrates dual functionalities: a highly specific recognition probe for the BCR-ABL fusion gene, and a peroxidase-mimetic nanozyme that catalyzes the 3,3',5,5'-tetramethylbenzidine (TMB)-H₂O₂) redox reaction, producing both visible colorimetric signals and quantifiable photothermal effects. This strategy enables sensitive detection of the BCR-ABL fusion gene, providing a valuable tool for the early diagnosis and minimal residual disease monitoring of chronic myeloid leukemia.

Adiponectin serves as a critical biomarker for metabolic disorders, with its concentration in human plasma providing predictive value for the risk of prediabetes. In this work, we report a rapid and reliable electrochemical immunosensor designed for point-of-care testing of adiponectin. The immunosensor was fabricated using a gold-sputtered silver screen-printed electrode as the substrate, on which sulfhydrylated antibodies were directly immobilized via Au-S covalent bonding. The fabrication process was characterized and optimized using electrochemical impedance spectroscopy and differential pulse voltammetry. Under optimal conditions, the proposed immunosensor exhibited a wide linear range (0.3-15 μg/mL) with a detection limit of 0.15 μg/mL. It also demonstrated excellent reproducibility and high selectivity for adiponectin detection. Additionally, the proposed immunosensor can be fabricated within 1.5 h and enables accurate quantification in human plasma within 15 min. The practical applicability of the sensor was validated through analysis of human plasma samples, and the comparison results with commercial ELISA kit are satisfactory.