Analytical Chemistry最新文献

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.

The conventional photoacoustic effect is based on the photothermal mechanism in semiconductors, where light absorption is converted to heat within photoacoustic materials. In this study, we introduce a novel, heat-independent phototriggered vibration on a quartz plate under a broad spectrum of light irradiation. Through a series of experiments, we elucidate the underlying mechanism and demonstrate its potential as an innovative photodetector and photoacoustic sensor. This approach enables applications in small-molecule self-assembly, DNA hybridization, and protein interactions, offering superior sensitivity and accuracy. These findings highlight the promise of this unconventional photoacoustic effect for the development of highly sensitive biosensors.

The precise detection of norepinephrine (NE) is critically important for understanding depression pathology and improving clinical diagnosis. Current strategies for designing NE-responsive photoacoustic (PA) probes, such as the "protect-deprotect" and the "hunting-shooting", are limited by slow kinetics, susceptibility to interference, or poor generalizability. Here, we report a novel "Recognizing-Releasing" strategy for designing NE-responsive photoacoustic (PA) probes and present QSH-NE as a proof-of-concept probe to demonstrate the generalizability of this strategy for in vivo imaging of depression. This rationally designed probe incorporates a quinolinium-derived sulfur-substituted hemicyanine (QSH-OH) as the PA reporter and a 2-hydroxy-5-(hydroxymethyl) benzaldehyde-derived unit as the recognition moiety. The PA activation mechanism initiates with aldehyde-amino recognition to form a five-membered ring, followed by a releasing step through carbamate cleavage and 1,6-elimination. QSH-NE exhibited favorable sensitivity, high activation fold, and a good response rate for NE in vitro. Notably, QSH-NE enabled noninvasive monitoring of depression progression and drug intervention efficacy through quantitative PA signal analysis in live animal brains. This general design strategy not only advances NE detection specificity but also provides a versatile platform for depression research and clinical diagnosis.