Analytical Chemistry最新文献

Cryptococcus neoformans (C. neoformans) infections pose a critical threat to global health, with high mortality rates observed among immunocompromised individuals. Rapid, cost-effective diagnostics are essential for preventive treatment and reducing mortality in resource-scarce settings, yet the development of selective fluorescent probes for C. neoformans remains challenging. The Prp8 intein, a unique self-splicing protein in C. neoformans, represents an ideal target for developing fluorescent probes and antifungal drugs. In this study, a series of fluorescent probes integrating Prp8 intein inhibitors with an environment-sensitive fluorophore was developed for selective covalent binding to the Prp8 intein. Among these probes, H2 enables specific detection of C. neoformans with minimal interference from Candida species. A dual-staining approach combining capsule staining with intracellular Prp8 intein labeling significantly improves the accuracy of C. neoformans detection. Probe H2 was also successfully applied for fluorescence imaging of fungal infections in a Galleria mellonella model, demonstrating potential for assessing fungal infections in vivo. Furthermore, the fluorescence intensity of the probe exhibited a dose-dependent relationship with Prp8 inhibitor activity, enabling rapid screening of intein-splicing inhibitors within 2 h, thereby improving efficiency and reducing assay time.

Accurate disease diagnosis requires analyzing multiple biomarkers in a single sample to improve diagnostic precision and guide treatment. However, the simultaneous detection of three or more proteases in a sample, especially with a single probe, remains a major challenge. Herein, we developed a triple-response peptide probe for monitoring three proteases via a single aerolysin nanopore. The T-shaped probe contains three independent arms each with a target-specific cleavage sequence. Upon protease recognition, short peptide fragments are released and generate characteristic current signatures during nanopore translocation. The enzymes themselves are sterically excluded, ensuring signals originate solely from cleavage products. Each peptide yields a distinct signature, enabling simultaneous, label- and amplification-free quantification with detection limits extending to the femtomolar range. Integrated with a machine learning classifier, the system achieves 97.7% accuracy in signal discrimination. This strategy enables accurate and multiplexed quantification of multiple enzymes in complex biological fluids, such as serum, which is important for disease diagnosis based on liquid biopsies.

The critical binding domain (CBD) is typically identified via tedious affinity screening of truncated and mutated sequences of an aptamer. We report here a wet-dry-wet experiment strategy, which enables the isolation of rapamycin-binding aptamers and the identification of the CBD at the same time without the need for affinity testing of numerous rationally designed sequences. In the first wet-experimental module, the pre-enriched library was obtained via 15 rounds of Capture-SELEX. In the dry-experimental module, the two key binding architectures, a three-stem-three-loop (3S3L) motif and a 3S3L-A motif (containing an additional adenosine in the second loop of 3S3L), were identified by comprehensively analyzing the high-throughput sequencing data using K-mer assembly, unsupervised learning (RBM), and mFold structure simulation. The structure-confined mixed secondary library designed based on the two structures exhibited high affinity, validating the importance of structure. In the second wet-experimental module, enriched library 2R6 was obtained after six rounds of second Capture-SELEX. A series of aptamers with nanomolar dissociation constants and high specificity were obtained, along with the identification of 11-nt CBD via analyzing the high-throughput sequencing result of 2R6. The decreased or completely lost binding affinity of the mutated sequences of seed aptamer 1R15-2 confirmed the CDB. A strand-displacement fluorescence sensor was constructed and capable of the detection of rapamycin spiked in 10% human serum with a nanomolar limit of detection. This study provides an efficient method for simultaneous aptamer isolation and CBD identification and can be applied to other targets.

Rapid and accurate identification of respiratory tract infections is a critical clinical need, with current diagnostic approaches predominantly relying on culture isolation, immunological testing, and molecular biology techniques. Although PCR-based methods have gained clinical acceptance due to their specificity and sensitivity, they are limited by the signal acquisition capabilities. Therefore, breaking through the limitations of signal acquisition capability and developing a high-throughput pathogen identification method remains an urgent challenge. In this study, we propose 3D-FLIP9 (3D Fluorescent Fingerprint Integrated PCR for Simultaneous Detection of 9 Pathogens), a high-throughput platform that combines 3D fluorescent fingerprinting and PCR. This method enables the detection of 9 respiratory pathogens in a single tube with significant efficiency and convenience. 3D-FLIP9 has made a breakthrough in signal acquisition and processing, allowing for accurate pathogen identification based on distinctive data points, with each pathogen presenting a unique fingerprint. The fluorescence signal is detectable at the target identification position even at 10 copies/μL. Moreover, 3D-FLIP9 has demonstrated substantial effectiveness in a total of 136 clinical samples (overall AUC = 97%). This work underscores the critical role of 3D-FLIP9 in the simultaneous detection of multiple respiratory pathogens, offering significant application value for the prevention and control of respiratory infections.

This study demonstrates the transformative potential of quantum technologies for healthcare diagnostics by developing a new analytical method, the quantum-enabled microfiltration immunoassay (QEMFIA). QEMFIA integrates the strengths of dot blot and enzyme-linked immunosorbent assays, enabling rapid, sensitive, and quantitative detection of clinically relevant antigens using nanoscale quantum sensors in a high-throughput format. The assay leverages fluorescent nanodiamonds (FNDs) with nitrogen-vacancy centers as reporters, combined with magnetically modulated fluorescence (MMF) for background-free detection of optically addressable spin defects. Additionally, to achieve high-throughput operation, the assays are performed on a 24-well microfiltration manifold, with target antigens captured by antibodies immobilized on a nitrocellulose membrane, followed by detection using antibody-conjugated FNDs. Finally, retained FNDs are directly analyzed on the membrane via MMF under a fluorescence microscope. The limits of detection for disease markers, such as C-reactive protein and interleukin-6, are below 100 fM within 1 h. The method is compatible with standard 96-well plates and conventional lab workflows. It also supports integration with automation platforms for high-throughput analysis across a broad range of target antigens using the FND quantum sensors.

Uric acid, a vital circulating metabolite, is a key biomarker for various health conditions including gout, preeclampsia, and kidney disorders. This underscores the need for noninvasive, rapid, sensitive, and cost-effective methods for monitoring uric acid to enable early preventive interventions. This study introduces a simple and sensitive separation-free "mix-and-detect" method for the direct surface-enhanced Raman scattering (SERS) detection of uric acid in urine, using bimetallic gold-silver nanostars functionalized with sodium dodecyl sulfate (BGNS@SDS). The SDS capping layer facilitates efficient uric acid capture through multiple hydrogen bonds under alkaline conditions, as confirmed by density functional theory (DFT) analysis. Using the optimized nanostar morphology (BGNS-3@SDS), uric acid was detected in water and spiked artificial urine samples with limits of detection of 2.2 and 3 μg/mL, respectively. These detection limits are substantially lower than the clinically relevant concentration range of uric acid in urine and well below the pathological threshold (∼750 μg/mL). The platform also successfully quantified uric acid levels in urine from ten healthy volunteers without sample pretreatment, enabling differentiation between healthy individuals and those at risk. This straightforward and sensitive SERS strategy holds strong promise for rapid, point-of-care diagnostics targeting low-affinity biomarkers.

Fluorescence imaging is widely applied in oncology owing to its cost-effectiveness, noninvasiveness, and real-time imaging capability. Many activatable fluorescent probes targeting tumor biomarkers, such as β-galactosidase (β-gal) and viscosity, have been developed. However, the reliance on a single-response mechanism limits their ability to capture the dynamic alterations within tumors during cancer progression and chemotherapy. In this study, we rationally designed and developed ZW-gal, a dual-locked near-infrared (NIR) probe activated by both β-gal activity and viscosity. ZW-gal exhibited favorable photophysical properties, such as a large Stokes shift (125 nm), rapid enzymatic activation (within 2 min), and a strong viscosity-dependent fluorescence enhancement (up to 24.6-fold). Leveraging this dual-responsiveness, ZW-gal successfully distinguished cancer from normal cells, visualized doxorubicin-induced cancer cell senescence, and monitored cell death. In a mouse model of liver cancer, ZW-gal enabled precise tumor localization and identified senescent tumors. Moreover, through in situ spraying, ZW-gal provided real-time surgical navigation, facilitating complete tumor resection. Building on these advantages, ZW-gal represents a powerful tool with broad potential to advance both basic cancer research and personalized clinical applications.

Nanozymes with multienzymatic activities provide synergistic effects for diverse biosensing applications. However, precise manipulation of their functionalities without cross-reactivity for advanced multimode sensing remains challenging. Herein, we employed a cytochrome c (Cyt c)-templated pyrolysis strategy to construct Fe single-atom nanozymes (FeSAN) featuring Fe-N₅ moieties with pH-switchable multienzymatic activities. Notably, the oxidase and peroxidase-like activities of FeSAN exhibit acid-dependent catalytic behavior, enabling a self-supplying oxidative catalytic cascade in acidic conditions. Conversely, in alkaline media, FeSAN demonstrates superoxide dismutase and catalase-like activity, forming a complementary antioxidant pathway for superoxide anion scavenging. The enzyme-mimicking mechanism and potential cascade pathways were investigated through comprehensive experiments and theoretical calculations. Capitalizing on the divergent pH requirements of colorimetric (acidic) and electrochemiluminescence (alkaline) systems, this pH-switchable dual-cascade catalytic platform enables amplified colorimetric response via TMB oxidation in acidic media while suppressing electrochemiluminescence intensity in alkaline circumstances. Using an organophosphorus pesticide (e.g., trichlorfon) as a proof-of-concept target, this platform with inverse signal correction achieved at least 10-fold higher sensitivity and improved accuracy compared to conventional methods. This work establishes a novel paradigm for advanced biosensing by fully utilizing the multienzymatic functionalities of single-atom nanozymes to construct dual-cascade catalysis in dual-mode platforms.

Lateral flow immunochromatographic assay (LFIA) features advantages such as a rapid analysis process, interpretable detection results, and low cost, making it an effective method for on-site detection of pesticides. However, the existing LFIA technologies still face bottleneck issues, including strong background fluorescence interference from the substrate and insufficient detection sensitivity for low pesticide residue levels. Here, long-lived room-temperature phosphorescent carbon dots embedded in silica (CDs@SiO₂) were developed to overcome background fluorescence interference. Fe₃O₄ coated with dopamine (Fe₃O₄@PDA) was utilized for the enrichment of target pesticide molecules. Meanwhile, by virtue of the quenching effect of Fe₃O₄@PDA on CDs@SiO₂, colorimetric and phosphorescent dual-mode LFIA technology was designed for the detection of the pesticide acetamiprid. Through the integration of the advantages of CDs@SiO₂ and Fe₃O₄@PDA, the limit of detection (LOD) for acetamiprid was as low as 0.271 ng/mL, and satisfactory recoveries were achieved in real samples. This research breaks through the millisecond-level lifetime limitation in traditional time-resolved fluorescence immunoassays, providing novel insight for the development of labeling materials for LFIA.