Exhaled breath condensate (EBC) analysis, a promising noninvasive respiratory monitoring method, has emerged as a pivotal technique for assessing the health status of patients with respiratory disorders and is widely used in clinical research and daily health management. However, conventional analytical methods face challenges in real-time in situ detection of physicochemical indicators and active EBC collection in a power-free way. Herein, a wearable multimodal detection system (WMDS) with efficient collection and real-time analysis of EBC is developed. Specifically, the WMDS consists of a bio-inspired collector, an electrochemical sensor (EBC analysis), a respiratory sensor (humidity and respiratory rate), a temperature sensor, and a flexible printed circuit board. The miniature-sized collector with a cactus spine-like structure can actively harvest 4.1 μL of EBC within 1 min without power consumption. Leveraging self-developed sensors and wireless data transmission circuitry, the WMDS enables real-time in situ monitoring of multimodal EBC analytes (hydrogen peroxide, nitrite, urea) and respiratory parameters (temperature, humidity, and rate). Remarkably, the WMDS exhibits dual-range detection capability covering both physiological and pathological conditions: the low-concentration range of 0-500 μmol/L is applicable for routine health monitoring and early disease screening, with detection limits (LODs) of 0.209, 0.155, and 0.573 μmol/L and sensitivities of 2.7 × 10-2, 3.4 × 10-2, and 9.0 × 10-3 μA/(μmol/L) for EBC analytes; the high-concentration range exceeding 500 μmol/L is designed for severe pathological condition detection, where LODs and sensitivities are 302.2, 278.7, 325.2 μmol/L and 1.9 × 10-2, 1.3 × 10-2, 3.9 × 10-3nA/(μmol/L) respectively. As a proof-of-concept, the WMDS is applied to on-body respiratory monitoring, validating its potential application in real-time in situ health monitoring.

Common electrochemical aptamer-based (E-AB) biosensors employing methylene blue (MB) redox reporters suffer from significant pH-induced signal variations. Consequently, dynamic pH fluctuations in biofluids, such as in sweat, can greatly distort the measured signal and lead to inaccurate target quantification. Here, we introduce a self-correction strategy that enables accurate analyte quantification by compensating for pH-induced signal variations in MB-based aptasensors using the Nernstian shift of the MB peak potential. The highly reproducible reversible pH dependence of the MB peak potential enables real-time monitoring of the sample pH that provides a continuously synchronized built-in compensation of the pH interference, thereby eliminating the need for external pH sensors or additional measurement steps. This precise yet simple and effective self-correction of pH effects ensures that the Square Wave Voltammetry (SWV) signal reliably reflects real-time analyte variations, as demonstrated using a cortisol MB-labeled E-AB biosensor in both in vitro and on-body settings. In vitro measurements in artificial sweat across pH 5.5-7.5 showed excellent correlation with real-time pH and cortisol changes, confirming the reliability of the peak potential-based correction. On-body measurements using an epidermal wearable patch showed cortisol changes in sweat that would have been missed without this signal-correction method. This approach is broadly applicable to other MB-labeled E-AB biosensors and biofluids, providing a robust strategy for continuous on-body monitoring.

Capillary microfluidics offer an attractive route to pump-free point-of-care (POC) diagnostics, yet most existing platforms remain constrained by continuous flow, short residence times (<2 min), and mandatory wash cycles that prevent integration with many affinity-based assays. We present CapSense-Flex, a laser-cut, roll-to-roll-manufacturable capillary chip that enables wash-free, diffusion-dominated molecular sensing with programmable incubation windows of 6-30 min, matching the binding kinetics required for molecularly imprinted polymer (MIP)-based electrochemical detection. The platform autonomously transports ≤25 μL of sweat, saliva, plasma, or whole blood without pumps, valves, or user actuation, achieving fluid-front arrival times within <5% of finite-element transport predictions while preventing bubble formation via fiber-assisted wicking. Embedded with a cortisol-selective MIP electrode, CapSense-Flex provides log-linear quantification in buffer (PBS) and human saliva from 1 to 1000 ng mL-1 (R2 = 0.9835), a 0.1 ng mL-1 limit of detection, an imprinting factor of ≈5.0, and <3.1% RSD (n = 5). The sensor retained an ∼90% signal after 60 days at 4 °C and directly quantified cortisol in unprocessed saliva samples (1.6-5.0 ng mL-1) with <12% signal loss relative to PBS standards. Each capillary chip is fabricated in <7 min at <$0.40 material cost and supports plug-and-replace electrode modules for alternative MIP targets. By decoupling capillary transport from binding kinetics and eliminating external pumps or wash steps, CapSense-Flex establishes a universal architecture for scalable MIP-based biosensing across handheld, wearable, and decentralized diagnostic formats.

Bioluminescent luciferases have emerged as powerful tools for bioimaging, enabling to image biological systems without external excitation light, reducing thus phototoxicity and eliminating background autofluorescence. Advanced imaging requires luciferases that deliver high photon output for enhanced sensitivity, tunable emission colors for multicolor imaging, and red-shifted emission for effective deep tissue imaging. Here, we introduce LumiFAST, a small tunable luciferase engineered by fusing the bright blue-light-emitting NanoLuc with the tunable chemogenetic fluorescent reporter pFAST. pFAST binds and stabilizes the fluorescent state of a variety of synthetic fluorogenic chromophores (also called fluorogens). Its proximity to NanoLuc leads to efficient bioluminescence resonance energy transfer (BRET), enabling customizable red-shifted emission. Thanks to the small size of pFAST, LumiFAST maintains a compact structure, while its modular design allows emission color to be tuned from cyan to green, yellow, orange, and red simply by changing the fluorogen. Systematic optimization of the fusion topology and linker length yielded an optimal variant with apparent BRET efficiencies reaching up to 90%. The red-shifted emission of LumiFAST enables dual-color microscopy imaging when used alongside NanoLuc and allows imaging through thick scattering media. Beyond imaging, our insights into the structural factors governing efficient BRET allowed us to engineer biosensors based on NanoLuc and pFAST for the visualization of protease activity and protein-protein interactions in live cells.

Photoacoustic (PA) sensing has garnered growing interest as a volumetric optical-acoustic transduction modality that mitigates optical scattering and enables sensitive detection in complex matrices. However, conventional PA assays rely on diffusion-dominated biochemical reaction kinetics and endpoint-based signal acquisition, which limit their capacity for rapid and real-time monitoring. Here, we report a magnetic-actuation-enhanced indirect PA sensing platform that integrates rotating magnetic fields (RMFs) with real-time PA detection to accelerate and continuously monitor operationally homogeneous immunoassays (without separation or washing steps). Functionalized magnetic nanoparticles (MNPs) were actuated by the RMF into dynamic chained or clustered structures, thereby enhancing collision frequency with target analytes and promoting target-induced aggregation. The resultant MNP aggregates caused interparticle light shielding, enhanced light scattering of the particle system, and underwent rapid sedimentation, thereby modulating the PA signal amplitude, which enabled continuous volumetric PA monitoring. Using cardiac troponin I as a clinical target, the platform achieved detection limits of 0.9 pM (22 pg/mL) in buffer and 2 pM (48 pg/mL) in 50% serum, outperforming conventional passive or endpoint assays. Clinical serum validation showed strong concordance with a clinical reference method, underscoring the platform's utility in point-of-care diagnostics.

Early and precise detection of cytokine release is essential for mitigating cytokine release syndrome, a life-threatening complication of CAR T-cell therapy. However, existing immunoassays fall short in sensitivity, dynamic range, and temporal resolution, limiting their ability to capture the subtle, transient cytokine fluctuations that mark the earliest phase of immune activation. To address this unmet need, we developed an ultrasensitive nanoplasmonic digital immunoassay that integrates rationally engineered antibody-derived peptide aptamers (ADPAs), plasmonic gold nanospheres for digital dark-field imaging, and convolutional neural network signal quantification. This platform enables ultrasensitive cytokine detection down to tens of fg/mL, with quantitative coverage across the tested concentration range spanning approximately six orders of magnitude using only microliter-scale sample volumes. We applied this technology to a physiologically relevant in vitro model of CAR T-cell-induced cytokine release, enabling high-frequency cytokine monitoring at early time points previously inaccessible to conventional assays. More broadly, this platform enables continuous, ultra-sensitive monitoring of cytokine dynamics at the onset of inflammation, opening new opportunities for early diagnosis and precision-guided intervention in a wide range of immune-mediated conditions.

Ferroptosis is an iron-dependent form of regulated cell death driven by dysregulated iron metabolism and subsequent lipid peroxidation, which contributes significantly to cardiovascular pathogenesis. While maintaining iron homeostasis is crucial for cardiac function, excessive iron accumulation initiates ferroptotic cell death, leading to cardiomyocyte damage. However, conventional biological methods lack the ability to dynamically and continuously monitor this process. To overcome this limitation, we developed a new biosensing platform centered on a custom 32-channel microelectrode array, which is designed for the non-invasive, label-free, and quantitative detection of electrophysiological signatures characteristic of cardiomyocyte ferroptosis. Using this platform, we successfully captured the drug concentration- and time-dependent electrophysiological alterations during Erastin-induced ferroptosis. Importantly, these detected electrophysiological changes closely aligned with traditional biochemical assays, accurately reflecting the progression of cardiomyocyte ferroptosis. The platform further demonstrated high specificity by recording the rescue effects of ferrostatin-1. Overall, our findings establish the feasibility of visualizing cardiomyocyte ferroptosis through electrophysiological monitoring, enabling continuous tracking of the entire process. This integrated biosensing system provides not only a powerful tool for investigating ferroptosis mechanisms but also a promising platform for drug screening and studying ferroptosis-related cardiac pathologies.

Circulating tumor cells (CTCs) in peripheral blood serve as valuable indicators of cancer progression and important biomarkers for the early diagnosis and prognosis of the disease. Unfortunately, CTCs are typically present at extremely low concentrations, and the challenge of enriching and isolating rare cells is often insurmountable. Herein, we present a fluorescence-based active cell sorting system designed to isolate rare cells from peripheral blood. Our platform is based on the principle of positive selection, where cell-surface markers are specifically labelled with fluorescent antibodies, ranked by aliquots, and subsequently sorted. The developed system comprises a multilayer microfluidic device that incorporates 10 parallel channels equipped with pneumatically actuated control valves. A one-dimensional complementary metal-oxide-semiconductor image sensor integrated with a field-programmable gate array enables real-time fluorescence detection across all channels, with the system processing 0.5 mL of minimally processed whole blood per minute. The platform is validated by spiking cancer cells into 5-fold diluted, minimally processed whole blood, at a ratio of 1:108 cancer cells to blood cells. Remarkably, after only three rounds of sorting, cancer cells are enriched by 8 orders of magnitude with a recovery rate exceeding 90%. To showcase the utility of the platform as a clinical tool, we assay clinical blood samples from cancer patients, successfully isolating CTCs at an average concentration of 7.6 CTCs/mL. Such performance metrics confirm immediate utility in the enrichment of CTCs and as a minimally invasive biopsy tool in clinical diagnostics and cancer research.

The development of reliable and field-deployable detection technologies of Salmonella typhimurium (S. typhimurium) throughout the food supply chain is crucial for the early warning and effective control of salmonellosis outbreaks. CRISPR-based biosensors offer excellent specificity, high sensitivity, and portability; however, their practical applications are significantly limited by the poor environmental stability of Cas enzymes, which are highly susceptible to temperature fluctuations and organic solvent interference. Here, a metal-organic framework (MOF) material, named ZIF-L, was employed as a sensing reactor to encapsulate the whole CRISPR sensing system, effectively enhancing its stability against variable external conditions such as temperature fluctuations and organic solvents encountered along the food supply chain. The feasibility of the conventional and encapsulated anti-S. typhimurium CRISPR sensor was confirmed through CLSM imaging, PAGE testing, and fluorescent verification. Importantly, the protective ability of the fabricated sensing reactor was precisely regulated by optimizing the pore size, ligand ratio, and dimensions of the MOFs and then evaluated under extreme detection conditions: (i) different external temperatures (4, 37, 50, 60, and 70 °C) and (ii) different organic solvents (methanol, acetone, and isopropyl alcohol). An impressive sensing performance of over 75% of its bioactivity, with a detection limit of 33 CFU/mL for S. typhimurium, was retained, confirming its detection ability under variable detection conditions. Moreover, recovery rates of 93.2-105.7% in spiked food samples, even when subjected to typical environmental interferences, including low temperatures (4 °C), high temperatures (60 °C), and organic solvent (methanol) exposure were obtained, showcasing its potential for the ultra-stable, on-site detection of S. typhimurium, particularly under variable external conditions.

Accurate and continuous detection of human pulse waves is critical for long-term health monitoring and physiological assessment during physical activity. However, most conventional flexible pulse sensors are single-mode, making them susceptible to interference from both ambient and physiological temperature fluctuations. Developing integrated dual-mode sensors remains challenging due to the difficulty in effectively decoupling pressure and temperature signals. Herein, we report a dual-mode flexible patch sensor featuring a unique "compass-like" structural design. The sensor demonstrates a broad pressure detection range (50 Pa-200 kPa) and rapid response/recovery times (60/96 ms), while its integrated temperature-sensing unit achieves a high resolution of 0.1 °C. Through unique structural design, the sensor achieves robust pressure decoupling of the temperature signal, with resistance fluctuations of only 0.102% under pressures up to 30 kPa. This independent temperature signal is subsequently utilized as a reference for the real-time temperature compensation of the pressure signal. The compensated pressure signal exhibits minimal temperature drift (41 × 10-6 °C-1 across 5-45 °C), enabling stable, interference-resistant monitoring of radial artery pulse waves under dynamic thermal conditions. Furthermore, when integrated with a ResNet-based deep learning model, the sensor successfully classifies diverse pulse signals with an accuracy of 97.6%. This research provides an effective solution for interference-resistant wearable electronics, offering significant potential for intelligent disease diagnosis and sports health management.