The increasing global prevalence of pollen-induced respiratory allergy highlights the need for precise and accessible methods of allergen identification. In this study, we developed a sensing platform based on carbon dots (CDs) for the detection of Art v1, the primary allergen in mugwort (Artemisia) pollen. Biomass-derived CDs were synthesized from Xanthii Fructus using a microwave-assisted hydrothermal method. The synthesis process was carefully designed to retain the specific bioactive components from the plant precursor, which endowed the CDs with inherent molecular recognition toward Art v1. The correlation between the retained component content and the recognition capability of the CDs was established through FTIR and XPS analyses. Subsequently, a ratiometric fluorescent probe was constructed by integrating the CDs with a Ru(bpy)3Cl2·6H2O reference signal into a portable test strip sensor. This probe allows visual assessment through distinct color shifts under UV light. Furthermore, when coupled with a smartphone-based colorimetric readout system, the platform facilitates accurate and highly selective identification and quantification of Art v1 within 5 min. The detection range was 0.5-80nM, with a limit of detection of 0.186nM. The sensor demonstrated robust on-site detection capability in complex biological and environmental matrices, with recovery rates ranging from 90.5% to 104.5%. This work not only presents a cost-effective, user-friendly strategy for allergen detection but also illustrates a pathway to confer targeted molecular recognition to sensing materials through rational biomass precursor selection and process design.

PIWI-interacting RNAs (piRNAs) are well-recognized as promising diagnostic biomarkers for cancer, yet their quantitative detection remains a great challenge owing to their short sequences, low cellular abundance, high degradation susceptibility, and significant sequence homology among family members. Herein, we developed an ultrasensitive and highly specific biosensor for the detection of piRNA-54265-a colorectal cancer (CRC)-associated piRNA-by integrating strand displacement amplification (SDA) with the CRISPR/Cas12a system. After systematic optimization, the biosensor exhibited remarkably enhanced amplification efficiency and target specificity, achieving an ultra-low limit of detection (LOD) of 57.54 aM for piRNA-54265. Notably, this CRISPR-SDA platform enabled accurate discrimination of CRC cells from other cancer cells via high-fidelity intracellular imaging of piRNA-54265 and also realized reliable detection of the target in complex biological matrices with favorable recovery. Benefiting from its simple sequence design, user-friendly operation, and isothermal reaction conditions, the developed biosensor not only overcomes the inherent technical bottlenecks in piRNA detection but also shows great potential for applications in cellular imaging and early clinical diagnosis of CRC.

The rapid development of recombinase polymerase amplification (RPA) has driven a growing demand for UvsX, the key DNA recombinase for this technique. During bioreactor cultivation for UvsX production inEscherichia coli, biomass and substrate are critical factors affecting its efficient expression. However, the real-time monitoring techniques for these parameters remain immature, leading to delayed process control,hindering stable high-level production and precise process optimization. Using an online Raman spectrometer, this study developed synchronous quantitative prediction models for critical process parameters by partial least squares (PLS). Robust multivariate regression models showed strong correlations between predicted and measured values for biomass (R2 = 0.994), glucose (R2 = 0.979), UvsX (R2 = 0.989), and total product (R2 = 0.983). Based on these predictive models, the study implemented online monitoring and feedback control of critical fermentation parameters, successfully raising UvsX and total product titer to 8.23g/L and 9.17g/L, representing increases of 33.17% and 27.54% compared with conventional strategies. This study achieved a marked improvement in UvsX titer and production efficiency by applying Raman spectroscopy as a simple and rapid tool for process monitoring and optimization, providing dependable technical support for scaling up and quality control of complex recombinant proteins.

Cystinosis is a rare, lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, resulting in lysosomal cystine accumulation, the phenotypic hallmark of cystinosis, and progressive cellular dysfunction. Accurate quantification of cystine levels is therefore essential for assessing lysosomal transport deficiency and treatment response. In vitro cell models provide a controlled platform to investigate disease mechanisms and to evaluate emerging therapeutic strategies. To determine intracellular cystine concentrations in these models, adequate sample preparation, storage, and highly sensitive analytical methods are essential. In this work, a rapid hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for the direct determination of cystine in cellular extracts. Use of a PEEK-lined HILIC-Z column proved essential to minimize metal-induced peak tailing and improve chromatographic performance. The total run time of 5min enabled high-throughput analysis, facilitating efficient screening of novel therapeutic approaches in cellular systems. Validation demonstrated excellent linearity (R2 ≥ 0.9986) and a lower limit of quantification (LLOQ) of 12.5nM, representing a > 8-fold improvement over reported reversed-phase LC methods. Addition of N-ethylmaleimide (NEM) prior to cell lysis effectively limited cysteine oxidation and maintained sample stability at 4°C. Applicability was demonstrated in an isogenic laboratory HEK293T cell model, where CTNS-knockout (KO) cells exhibited significantly elevated intracellular cystine levels compared to wild-type (WT) cells. As additional controls, elevated levels were successfully restored following cysteamine treatment or lentiviral-vector (LV)-mediated CTNS protein re-expression. The developed method hence provides a sensitive and reliable analytical platform for in vitro evaluation of novel therapeutic strategies in cystinosis research.

The increasing global consumption of pharmaceuticals and illicit drugs, particularly new psychoactive substances, has resulted in their continuous release into aquatic environments, raising concerns regarding potential adverse effects on ecosystems and human health. In this study, a dispersive micro-solid-phase extraction using customized extraction devices was developed in combination with liquid chromatography-high-resolution mass spectrometry for target and suspect screening, followed by sensitive quantification by liquid chromatography-tandem mass spectrometry. The extraction procedure was optimized using experimental design. Optimal conditions comprised 50mg of Oasis MCX sorbent, 100mL of sample (pH 2, containing 7.5% NaCl, w v⁻1), and an extraction time of 30min. Under these conditions, limits of quantification ranged from 4.67 to 29.3ng L⁻1, and extraction recoveries varied between 73.0% and 108% for most target analytes. Application to real wastewater samples included an initial suspect screening step by liquid chromatography-high resolution mass spectrometry using data-dependent acquisition and an untargeted data-mining workflow. The screening results revealed a broad range of compounds, including central nervous system stimulants, cardiovascular drugs, opioid analgesics, and sedatives. Quantification of selected target analytes was subsequently performed by liquid chromatography-tandem mass spectrometry. Among the investigated illicit drugs, THC and cocaine were the predominant compounds detected, whereas fentanyl was present at trace levels (6.17-14.4ng L⁻1). The proposed workflow enables robust, sensitive monitoring of illicit drugs and pharmaceuticals in wastewater for epidemiological and environmental surveillance applications.

An interlaboratory comparison study was conducted among five laboratories for determining 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The Centers for Disease Control and Prevention (CDC), Imperial College Healthcare NHS Trust, University College Cork, University of Liège, and University of Washington analyzed 50 single-donor samples and two new Standard Reference Materials (SRMs®). The results from each laboratory were compared with target values assigned by the National Institute of Standards and Technology (NIST) using a reference measurement procedure (RMP) and evaluated using Ordinary Deming linear regression and Bland-Altman analysis. Three of the five laboratory methods provided results that were in good agreement with the NIST RMP results showing linear regression slopes ranging from 0.972 to 1.003 and Bland-Altman mean bias of -0.092nmol/L, 0.025nmol/L, and 0.035nmol/L. Two laboratories demonstrated a significant positive bias with linear regression slopes of 1.158 and 1.214 and Bland-Altman mean bias of 0.162nmol/L and 0.708nmol/L. The CDC method is currently used to assign "information only" values for 24,25(OH)2D3 in the quarterly distributions of the Vitamin D External Quality Assessment Scheme (DEQAS). Given the agreement (linear regression slope = 0.984, R2 = 0.996 and mean bias of -0.092nmol/L) between the CDC method and the NIST RMP observed in this study, the CDC-assigned 24,25(OH)2D3 values in DEQAS may provide a more accurate reference than the current participant consensus mean values.

Sensitive and highly selective analysis of ochratoxin A (OTA) plays an important role in food safety. In this paper, an aptamer sensing system based on rolling circle amplification (RCA) technology is proposed for the ultrasensitive fluorescence polarization detection of OTA. The binding of OTA to its aptamer releases the primer S, which rapidly hybridizes with the dumbbell-shaped ring template (HH) to initiate RCA. The effects of the complementary strand length, the hybridization ratio of aptamer (AP) to primer S, the target recognition time, and the reaction temperature on the sensing performance were systematically investigated. Under the optimal experimental conditions, this sensing method demonstrated a well-defined linear relationship with OTA concentrations ranging from 1 × 10-4 to 10 ng/mL, with a detection limit of 0.032 pg/mL. The developed assay was applied to OTA detection in different samples. These results clearly highlight the potential of the aptamer sensing system for food safety monitoring.

Traditional fluorescence polarization immunoassay (FPIA) requires costly monoclonal antibodies. This work develops a cost-effective fluorescence polarization enzyme assay (FPEA) for quercetin, employing α-amylase as a significantly cheaper recognition agent. Based on competitive binding with a fluorescent zearalenone tracer, the assay achieves a detection limit of 1.7 mg/mL, a working range of 2.3-6.4 mg/mL, and completes analysis within 5 min, offering a substantial time saving versus typical HPLC runs (~ 20 min). It demonstrates high specificity, with only minimal cross-reactivity to rutin (0.1%) and none to dihydroquercetin, gallic acid, and acarbose. FPEA analysis yielded a quercetin*α-amylase binding affinity constant of 3.9 × 103 L/mol. Successful application to onion peel extract and a dietary supplement yielded results consistent with liquid chromatography with diode-array detection (LC-DAD). The proposed FPEA provides a rapid, simple, and economical alternative for quality control of sources with high quercetin content.

The accurate diagnosis of anxiety disorders remains challenging, as current detection methods primarily rely on subjective questionnaires. In this study, a novel electrochemical biosensor for the detection of fibroblast growth factor-2 (FGF-2), a biomarker associated with anxiety disorders, was developed for the first time. The sensing principle was based on monitoring the impedimetric signal changes generated by the interaction between anti-FGF-2 and the FGF-2 antigen, using a carbon screen-printed electrode as the transducer. To enhance the sensitivity and overall performance of the biosensor, a multi-walled carbon nanotube-gold-platinum (MWCNT-Au-Pt) hybrid nanocomposite was incorporated into the immunosensor structure. Critical parameters-including the amount of MWCNT-Au-Pt, the concentration, incubation time, and temperature of anti-FGF-2, as well as the incubation time and temperature for the anti-FGF-2/FGF-2 interaction-were systematically optimized. Under optimized conditions, the developed impedimetric FGF-2 immunosensor exhibited a linear response in the range of 10-100ng/mL, with a limit of detection of 1.01ng/mL and a limit of quantification of 3.10ng/mL. The specificity of the sensor was confirmed in the presence of potential interfering substances. For real sample analysis, human saliva samples (n = 30) were diluted appropriately and spiked with FGF-2 at concentrations of 20, 50, and 75ng/mL. The obtained recovery values demonstrated that the developed impedimetric FGF-2 immunosensor is highly suitable for real-sample applications and holds significant potential as a reliable tool for the detection of anxiety disorders.