Analytical Chemistry最新文献

Lateral flow immunochromatography (LFIA) with gold nanoparticles (AuNPs) is widely used in the biomedical field as a rapid and simple in vitro detection technique. However, the conventional AuNP-LFIA has limitations in sensitivity and detection range. In this study, nonprecious metal iron-based bimetallic FeWO₄ nanomaterials with convenient and excellent enzyme-mimetic catalytic activities were synthesized by a one-pot hydrothermal method. Here, FeWO₄ nanomaterials were combined with C-reactive protein (CRP) detection antibodies to form a novel signal-enhancing probe for the analysis of CRP. The probe further achieves significant signal amplification by catalyzing the oxidation reaction of 3-amino-9-ethylcarbazole in the LFIA assay, thereby improving the sensitivity and accuracy of the assay. The application of FeWO₄-based LFIA improved the limit of detection of CRP to 19.38 ng/mL after catalytic amplification, which is approximately 30-fold lower than that of the conventional AuNP-LFIA method. In addition, the method demonstrated good stability and reproducibility, providing a promising and prospective strategy for the early diagnosis of inflammatory diseases.

Interferon-γ release assay (IGRA) is one of the most important diagnostic tools for tuberculosis (TB) infection. Despite its high accuracy, conventional IGRA has several drawbacks, including complicated procedures, large blood volume requirements, lengthy incubation times, and difficulties in parallel testing. Efforts have been made to develop miniaturized and highly sensitive biosensors for interferon-γ or to evaluate the specific immune response through microfluidic platforms. However, the need for sophisticated consumables and equipment, as well as the partial experimental design, has limited the application of these advanced techniques in TB diagnosis and disease control. Here, we report the development of a tip optofluidic immunoassay (TOI)-based consolidated microscale IGRA (CM-IGRA) for the dynamic and parallel evaluation of TB infection, refining both the blood incubation and interferon-γ quantification processes. The TOI system comprises 12 microfluidic immuno-reactors and a portable chemiluminescent imaging station, capable of quantifying interferon-γ with high sensitivity (8.00 pg/mL in plasma) and a wide detection range (∼10⁴). The results generated with CM-IGRA achieved 98.39% agreement with the standard IGRA while reducing blood sample consumption to 50 μL per assay (20-fold reduction) and significantly shortening the incubation time from 20 to 10 h. This diagnostic method simplifies operations and improves efficiency for the parallel assays required in IGRA, providing a promising solution for TB screening in patients for whom current methods are inconvenient, such as children and older adults.

Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, and potential compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer with a self-activated CRISPR-Cas12a biosensor (DESCRIBER) for integrated nucleic acid detection. This platform features varying balance points to minimize interference between RPA and Cas12a in one pot and maximize their activity at different stages. Initially, the reaction focused on RPA, while Cas12a was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, the activator, was generated during the RPA process, which linearizes C-crRNA to activate Cas12a and transfer the equilibrium toward signal readout. Meanwhile, activated Cas12a can further linearize C-crRNA to promote self-activation and accelerate equilibrium transfer. According to this principle, highly sensitive detection of the HIV-1 genome, as low as 500 CPs/mL, was achieved within 1 h while maintaining universality in detecting common subtypes and specificity against opportunistic infectious pathogens. Compared with qRT-PCR, it also exhibited good accuracy in detecting 35 spiked samples. Overall, we believe that the proposed strategy will enhance existing CRISPR systems to promote their practical applications in clinical diagnosis.

Thanks to the plummeting costs of continuously evolving omics analytical platforms, research centers collect multiomics data more routinely. They are, however, confronted with the lack of a versatile software solution to harmoniously analyze single-omics and interpret multiomics data. We have developed iSODA, a web-based application for the analysis of single- and multiomics data. The tool emphasizes intuitive interactive visualizations designed for user-driven data exploration. Researchers can access a variety of functions ranging from simple visualization like volcano plots and PCA to advanced functional analyses like enrichment analysis and lipid saturation analysis. For integrated multiomics, iSODA incorporates multi-omics factor analysis and similarity network fusion. The ability to adapt the data on-the-fly allows for tasks, such as the removal of outlier samples or failed features, imputation, or normalization. All results are presented through interactive plots, the modular design supports extensions, and tooltips and tutorials provide comprehensive guidance for users. iSODA is accessible under http://isoda.online/.

Revealing changes in the tumor microenvironment is crucial for understanding cancer and developing sensitive methods for precise cancer imaging and diagnosis. Intracellular hydrogen peroxide (H₂O₂) and microenvironmental factors (e.g., viscosity and polarity) are closely linked to various physiological and pathological processes, making them potential biomarkers for cancer. However, a triple-response theranostic probe for precise tumor imaging and therapy has not yet been achieved due to the lack of effective tools. Herein, we present a mitochondria-targeting near-infrared (NIR) fluorescent probe, VPH-5DF, capable of simultaneously monitoring H₂O₂, viscosity, and polarity through dual NIR channels. The probe specifically detects H₂O₂ via NIR emission (λ_em = 650 nm) and shows high sensitivity to microenvironmental viscosity/polarity in the deep NIR channel (λ_em ≈ 750 nm). Furthermore, the probe not only monitors mitochondrial polarity, viscosity, and fluctuations in endogenous/exogenous H₂O₂ levels but also distinguishes cancer cells from normal cells through multiple parameters. Additionally, VPH-5DF can be employed to monitor alterations in H₂O₂ levels, as well as changes in viscosity and polarity, during drug-induced pyroptosis in living cells. After treatment with VPH-5DF, chemotherapy-induced oxidative damage to the mitochondria in tumor cells activated the pyroptosis pathway, leading to a robust antitumor response, as evidenced in xenograft tumor models. Thus, this triple-response theranostic prodrug offers a new platform for precise in vivo cancer diagnosis and anticancer chemotherapy.

Even though mAbs have attracted the biggest interest in the development of therapeutic proteins, next-generation therapeutics such as single-domain antibodies (sdAb) are propelling increasing attention as new alternatives with appealing applications in different clinical areas. These constructs are small therapeutic proteins formed by a variable domain of the heavy chain of an antibody with multiple therapeutic and production benefits compared with their mAb counterparts. These proteins can be subjected to different bioconjugation processes to form single-domain antibody-drug conjugates (sdADCs) and hence increase their therapeutic potency, and akin to other therapeutic proteins, nanobodies and related products require dedicated analytical strategies to fully characterize their primary structure prior to their release to the market. In this study, we report for the first time the extensive sequence characterization of a conjugated anti-EGFR 14 kDa sdADC by using state-of-the-art top-down mass spectrometry strategies in combination with liquid chromatography (LC-TD-MS). Mass analysis revealed a highly homogeneous sample with one conjugated molecule. Subsequently, the reduced sdADC was submitted to different fragmentation techniques, namely, higher-energy collisional dissociation, electron-transfer dissociation, and electron-transfer higher-energy collision dissociation, allowing to unambiguously assess the conjugation site with 24 diagnostic fragment ions and 85% of global sequence coverage. The sequence coverage of the nonreduced protein was significantly lower (around 16%); however, the analysis of the fragmentation spectra corroborated the presence of the intramolecular disulfide bridge along with the localization of the conjugation site. Altogether, our results pinpoint the difficulties and challenges associated with the fragmentation of sdAb-derived formats in the LC time scale due to their remarkable stability as a consequence of the intramolecular disulfide bridge. However, the use of complementary activation techniques along with the identification of specific ion fragments allows an improved sequence coverage, the characterization of the intramolecular disulfide bond, and the unambiguous localization of the conjugation site.

Obtaining high-quality matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) images and the reproducibility of the technique depend strongly on the sample preparation protocol. The most crucial part is the application of the MALDI matrix, which often relies on expensive spraying or sublimation coaters. In this work, we present a new dual-polarity matrix for MALDI mass spectrometry imaging (MSI): Basic Blue 7 (BB7), which belongs to the group of triarylmethane dyes. Thanks to its good solubility in water, this matrix allows a quick and simple sample preparation protocol without the need for sophisticated spraying or sublimation instrumentation: dipping the glass with tissue into the dye solution. This technique closely resembles the staining methods employed in classical histopathology. The technique is demonstrated on MSI of lipids in mouse brain sections in positive and negative ion modes using a subatmospheric pressure MALDI source coupled with an orbital trap mass spectrometer. The results are compared with traditional matrices, such as 2,5-dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN). BB7 excels, especially in negative ion mode, offering low background signals and high signal intensities of many lipid classes. Furthermore, the stained tissue can simply be inspected visually and allows basic histopathology annotation prior to MSI. Here, we demonstrate that staining offers excellent image quality, reproducible sample preparation, and the potential for automation and utilization for high spatial resolution MSI.