In this work, a fluorescent aptasensor was constructed for enrofloxacin (ENR) detection by combining the polyvalent aptamer (PA) structure, apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification and mesoporous silica-encapsulated perovskite quantum dot (CsPbBr3@MSN) nanocomposites. With their multiple recognition sites, polyvalent aptamers bonded to ENR and released the trigger for the strand displacement reaction. Meanwhile, a highly sensitive FRET-based sensor was constructed using the CsPbBr3@MSN probe and gold nanoparticles (Au NPs). In the presence of the trigger and APE1, the fluorescence was recovered by separating the CsPbBr3@MSN probe and Au NPs via a cyclic strand displacement reaction. Coupled with the signal amplification strategy, this system achieved the linear detection of ENR in the range of 0.1 pg mL-1 to 50 ng mL-1 with a detection limit of 0.073 pg mL-1. This sensor offers a novel analytical strategy for detecting antibiotic residues and holds significant potential for applications in food safety monitoring and environmental analysis.

Olaparib (Lynparza®) is a highly selective poly(ADP-ribose) polymerase (PARP) inhibitor used in the advanced treatment of ovarian, breast, and prostate cancers. An effective, environmentally friendly, rapid, and reliable UPLC-MS/MS method was established to quantify OLA in human liver microsomes (HLMs) and was used to evaluate the in vitro metabolic stability of OLA. The UPLC-MS/MS method was validated in accordance with the US-FDA bioanalytical method validation standards. The current UPLC-MS/MS method showed a high degree of greenness, as evidenced by a ComplexMoGAPI value of 66.0 and an AGREEprep tool value of 0.63. The StarDrop software package (WhichP450 and DEREK modules) was used to assess metabolic lability and characterize in silico alerts regarding the OLA chemical structure. The current UPLC-MS/MS method showed a linearity range of 1 to 4000 ng mL-1, ultra-fast separation in 1 min, and exhibited precision and accuracy unaffected by HLMs. Chromatographic separation of OLA and dasatinib (internal standard) was performed using a reversed-phase Eclipse Plus 1.8 µm C8 column (50 mm × 2.1 mm), with the mobile phase consisting of 0.1% HCOOH in water (pH 3.2) at 60% and 0.1% HCOOH in ACN (40%). The intra- and inter-day evaluations of the accuracy and precision of the UPLC-MS/MS approach ranged from 0.79% to 11.67% and -0.86% to 10.33%, respectively. The in vitro half-life (t1/2) of OLA was 43.7 min, and its intrinsic clearance (Clint) was 18.55 mL min-1 kg-1, confirming the low metabolic clearance (high metabolic stability). In silico studies suggest that minor structural modifications of the phthalazin-1-one (52%) and piperazine (24%) moieties during drug design may improve the safety profile and metabolic stability of new derivatives compared with OLA; however, experimental confirmation is needed.

Small extracellular vesicles (sEVs) hold immense potential for liquid biopsy given the wealth of biological information they carry. Currently, the clinical application of these methods is limited due to their low abundance and the complexities associated with traditional isolation techniques. To address this, we developed a strategy integrating cholesterol-mediated capture with a Self-Protected DNAzyme Walker for the rapid and simultaneous specific isolation and quantification of small extracellular vesicles (sEVs). Upon specific binding to CD63, the blocker strand is released, which activates the DNAzyme catalytic core, leading to substrate cleavage, which triggers the specific release of sEVs from magnetic beads and the generation of a fluorescent signal. Importantly, the circular DNA Shield design provides remarkable stability to the system by safeguarding the DNAzyme core from nuclease degradation. Furthermore, the cyclic cleavage mechanism allows for highly sensitive detection, achieving a limit of detection (LOD) as low as 361 particles per μL. In addition, by leveraging the lipid bilayer structure for sEV enrichment, this strategy effectively eliminates interference from free proteins. Furthermore, the clinical feasibility of this assay was validated by successfully distinguishing Stage I breast cancer patients from healthy individuals with high statistical significance (p < 0.001), highlighting its promise for early cancer diagnosis. This work presents a robust paradigm for sEV analysis and lays a solid foundation for their downstream biomedical applications.

The sensitive detection of hydrogen peroxide is important for many applications, including biomedical diagnostics and environmental monitoring. In this work, we report a simple turn-on fluorescent platform based on conjugated polyelectrolytes (CPEs), specifically poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV), complexed with polyvinylpyrrolidone (PVP) for the detection of hydrogen peroxide (H2O2). MPS-PPV alone exhibits weak fluorescence enhancement in response to the incremental addition of H2O2. However, when PVP is added, a sixfold increase in fluorescence is observed. Mechanistically, this enhancement is attributed to the dual role of PVP: (1) its complexation with hydrogen peroxide increases the local oxidant concentration, promoting oxidative attack on the CPE backbone, and (2) it stabilizes the resulting oxidized polymers, thus reducing aggregation and enhancing their emission. Changes in the ionic strength further confirm that the CPE conformation and its complexation with PVP are key to the observed fluorescence enhancement. Time-resolved fluorescence measurements in the presence of ascorbic acid, a radical scavenger, demonstrate that hydroxyl radicals are the active species responsible for the sensing response. Finally, as a proof of concept, the sensor was successfully applied to the enzymatic detection of glucose, enabling quantitative detection over a physiologically relevant range (3.5-10.5 mM). This work demonstrates how microenvironmental control of conjugated polyelectrolytes can play a critical role in developing CPE-based fluorescent sensors and enhancing their sensitivity.

Long COVID is characterized by persistent symptoms, including fatigue, cognitive impairment, and respiratory issues, affecting a considerable number of individuals post-infection. The underlying mechanism is not fully understood, but it has been proposed to involve the reactivation of virus, which subsequently induces immune dysregulation. In this proof-of-concept study, we developed a paper-based immunoassay for the detection of nucleocapsid (N) protein, which, due to its stability and low mutation rate, is a valuable biomarker for detecting the presence of residual virus. By utilizing reporter antibodies conjugated to cleavable ionic probes through dendrimer chemistry, we were able to analyze the immunoassay results with ambient mass spectrometry using on-chip paper spray ionization. The used dendrimer enhanced mass spectrometry sensitivity by enabling the attachment of multiple ionic probes to a single reporter antibody. The method presented here achieved a limit of detection of 2.4 pM for N protein detection from paper. Unlike traditional sensitive COVID tests that are only accessible to hospitalized individuals, our paper-based assay has potential to enable long COVID to be detected under resource-limited settings. Our method was applied to analyze 20 human plasma samples, including 10 from individuals with long COVID and 10 from healthy controls with no history of SARS-CoV-2 infection. We observed a significantly higher MS signal-by up to two orders of magnitude-for samples collected from long COVID patients compared to controls. The ability to use the paper device in remote locations was tested by evaluating the stability of the assay, which showed that after 30 days of storage at room temperature, the device retained sufficient analytical performance. Given its robustness, we believe that our platform will be suitable for direct-to-consumer testing, enabling individuals with low viral loads to be screened in a timely fashion.

Spontaneous hypertension (SH) is a prevalent chronic cardiovascular disorder characterized by the synergistic elevation of hydrogen peroxide (H2O2) levels and tyrosine hydroxylase (TH) activity in the brainstem nucleus tractus solitarius (NTS). Traditional detection techniques lack the specificity and spatiotemporal resolution to monitor the dynamic interplay of these two core pathological biomarkers, hindering the in-depth exploration of SH pathogenesis. Herein, a series of novel cascade-activated fluorescent probes (PPTHs) were rationally designed and synthesized based on a purine core, which achieve specific fluorescence responses only upon sequential activation by H2O2 and TH. In vitro assays demonstrated that the probes exhibited high sensitivity toward H2O2 in SH-SY5Y cell lysates, with a reliable limit of detection (LOD) and ideal anti-interference capability. Live-cell imaging further confirmed that purine-based molecules not only successfully mitigated probe adsorption on the cell membrane but also effectively improved the imaging signal-to-noise (S/N) ratio. Notably, PPTH-2-assisted confocal imaging clearly distinguished the differential fluorescence signals between normotensive control and SHR groups, which correlated with endogenous H2O2 level and TH activity in the NTS region. Our study presents a robust fluorescent probe platform for the synchronous detection of H2O2 and TH, offering a promising molecular tool for the early diagnosis and elucidation of the pathological mechanisms of SH.

Historical production of per- and polyfluoroalkyl substances (PFAS) via electrochemical fluorination has resulted in complex mixtures of linear (L) and branched (Br) isomers, yet most environmental studies still treat them as single compounds. Emerging research highlights that isomer-specific differences critically shape PFAS environmental fate, bioaccumulation, and toxicity. These distinctions are particularly critical for aquatic organisms, which experience continuous exposure to PFAS and serve as sentinels of ecosystem health. A comprehensive review of literature from January 2000 to December 2025 reveals that most studies on PFAS in aquatic species overlook isomer resolution, constraining insights into mixture behavior. The relatively few studies that report isomer profiles across fish, sharks, marine mammals, aquatic insects, seabirds, alligators, and polar bears primarily focus on PFOS (perfluorooctane sulfonic acid), leaving substantial knowledge gaps for other PFAS classes. Evidence also indicates that precursor compositions strongly influence isomer-specific bioaccumulation; several studies show that L-isomers tend to bioaccumulate more than their Br counterparts, suggesting potential differences in environmental stability and metabolism. Advancing knowledge on PFAS isomer distribution requires broader use of orthogonal separation techniques. Ion mobility spectrometry can resolve L- and Br-isomers based on differences in their collision cross-sections. Other techniques that can separate L- and Br-isomers include gas chromatography/mass spectrometry with derivatization, and supercritical fluid chromatography/mass spectrometry, capable of efficient separation of isomers based on differences in partition coefficients between two phases. Integrating these techniques into current conventional PFAS analytical methods is essential for uncovering the PFAS structure-environmental behavior and for enhancing future ecological risk assessments.

The inherently weak Raman signals from molecules with small scattering cross-sections pose a significant challenge for surface-enhanced Raman scattering (SERS), a technique that is further limited by its reliance on costly precious metal substrates and exogenous labeling strategies. To address these limitations, this study constructs a Ti3C2-CoFe2O4 heterostructure by anchoring oxidase (OXD)-like CoFe2O4 nanoparticles (NPs) on two-dimensional (2D) conductive Ti3C2 MXene nanosheets. The resulting interface forms a Mott-Schottky junction, which facilitates rapid charge transfer and synergistically enhances both catalytic and SERS performance. Structurally, the 2D Ti3C2 framework provides abundant anchoring sites for the uniform dispersion of CoFe2O4 NPs. This effectively prevents particle aggregation and maximizes the exposure of catalytic active sites, thereby enhancing both stability and catalytic activity. Additionally, the Ti3C2-CoFe2O4 heterojunction effectively suppresses the recombination of charge carriers and promotes the separation of photogenerated charges, generating abundant superoxide anion radicals that oxidize 3,3',5,5'-tetramethylbenzidine (TMB) for catalytic signal amplification. Therefore, the ingenious combination of nanozymes and SERS technology enables the generation of SERS-active reporters via nanozyme-catalyzed reactions, thus avoiding the need for external labeling modifications. The strategy simultaneously enhances Raman signals through the synergistic effect of photoinduced charge transfer and localized surface plasmon resonance. This Ti3C2-CoFe2O4 heterojunction exhibits integrated OXD-like activity and SERS enhancement, enabling sensitive glutathione (GSH) detection in human serum samples. Through catalytic oxidation of TMB to oxidized TMB, a distinct Raman peak emerges at 1615 cm-1, with its intensity reduction quantitatively correlating with GSH concentration via competitive reactive oxygen species scavenging. Quantitative analysis demonstrates a linear response range of 0.50-200 μmol L-1 and a detection limit of 0.073 μmol L-1, with serum sample recoveries ranging from 94.7%-115%. This study provides a paradigm for designing non-precious metal nanozyme materials with integrated catalytic and SERS capabilities, demonstrating significant potential for practical applications in clinical diagnostics and biosensing.

This study presents, for the first time, a direct quantitative comparison between the binding affinities and selectivities of antibodies and their molecularly imprinted polymer (nanoMIP) counterparts for a target protein antigen. NanoMIPs were synthesized upon protein functionalised magnetic nanoparticles (MNPs) using bovine haemoglobin as a target protein. This solid-phase synthesis process gave nanoMIP yields of 10 ± 2 mg produced in less than 1 h. Physical characterization of nanoMIPs by dynamic light scattering (DLS) revealed an average particle diameter of 121 ± 53 nm, consistent with nanoparticle tracking analysis (NTA) results, confirming uniform particle formation and comparable concentrations to antibody preparations. Antibody and nanoMIP affinities were characterized using surface plasmon resonance (SPR), the current gold-standard technique, as well as using a newly developed electrochemical method based on electrochemical impedance spectroscopy (EIS). This dual approach enables direct comparison and standardization of nanoMIPs as synthetic alternatives to conventional antibodies. NanoMIP binding affinities of 34.7 ± 2 pM (EIS) and 3.06 pM (SPR) were obtained, with a selectivity factor of 130 : 1 (target : non-target) based on the electrochemical method. In contrast, the corresponding polyclonal antibody for haemoglobin (pAb) demonstrated contrasting affinities of 51.9 ± 0.74 pM (EIS) and 48.7 nM (SPR) and with a substantially lower selectivity factor of 1 : 1.1. These results indicate that while the two sensor techniques are ideal for nanoMIP characterisation, further harmonisation is required for antibody binding characterisation. We demonstrate that the developed nanoMIPs not only rival but can surpass traditional animal-derived antibodies in both affinity and molecular discrimination. Overall, these findings highlight nanoMIPs as a robust and reproducible alternative to antibodies, offering superior selectivity and comparable affinity for next-generation bioanalytical and diagnostic applications.

Microfluidics and nanofluidics have contributed much to the fields of chemistry and biochemistry. The small sizes of micro and nanochannels provide short diffusion lengths, resulting in highly efficient reactions with control over channel size, flow and temperature. In this review, progress in chemical and biochemical reactors based on micro and nanochannels is summarized. Various types of reactors such as homogeneous and heterogeneous catalytic reactors based on wall-coated, packed-bed or monolithic column designs are examined. The ultra-small spaces provided by micro and nanochannels allow rapid mixing and promote interactions between different phases. As such, faster reaction rates and better yields can be obtained using systems that are easy to operate. In addition, unique reaction mechanisms can be achieved based on the specific properties exhibited only by nanospaces. Although it remains challenging to balance high efficiency with a suitable production volume, new super-high-performance reactors allowing well-controlled processes with suitable productivity are anticipated in future. This advanced technology will represent significant progress in the areas not only of analytical chemistry and bioanalytical chemistry, but also chemical and biochemical engineering.