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Abstract Rapid and accurate immune monitoring plays a decisive role in effectively treating immune‐related diseases especially at point‐of‐care, where an immediate decision on treatment is needed upon precise determination of the patient immune status. Derived from the emerging clinical demands, there is an urgent need for a cytokine immunoassay that offers unprecedented sensor performance with high sensitivity, throughput, and multiplexing capability, as well as short turnaround time at low system complexity, manufacturability, and scalability. In this paper, a label‐free, high throughput cytokine immunoassay based on a magnet patterned Fe₃O₄/Au core–shell nanoparticle (FACSNP) sensing array is developed. By exploiting the unique superparamagnetic and plasmonic properties of the core–shell nanomaterials, a facile microarray patterning technique is established that allows the fabrication of a uniform, self‐assembled microarray on a large surface area with remarkable tunability and scalability. The sensing performance of the FACSNP microarray is validated by real‐time detection of four cytokines in complex biological samples, showing high sensitivity (≈20 pg mL⁻¹), selectivity and throughput with excellent statistical accuracy. The developed immunoassay is successfully applied for rapid determination of the functional immunophenotype of leukemia tumor‐associated macrophages, manifesting its potential clinical applications for real‐time immune monitoring, early cancer detection, and therapeutic drug stratification toward personalized medicine. 

Although many advanced biosensing techniques have been proposed for cytokine profiling, there are no clinically available methods that integrate high-resolution immune cell monitoring and in situ multiplexed cytokine detection together in a biomimetic tissue microenvironment. The primary challenge arises due to the lack of suitable label-free sensing techniques and difficulty for sensor integration. In this work, we demonstrated a novel integration of a localized-surface plasmon resonance (LSPR)-based biosensor with a biomimetic microfluidic ‘adipose-tissue-on-chip’ platform for an in situ label-free, high-throughput and multiplexed cytokine secretion analysis of obese adipose tissue. Using our established adipose-tissue-on-chip platform, we were able to monitor the adipose tissue initiation, differentiation, and maturation and simulate the hallmark formation of crown-like structures (CLSs) during pro-inflammatory stimulation. With integrated antibody-conjugated LSPR barcode sensor arrays, our platform enables simultaneous multiplexed measurements of pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10 and IL-4) cytokines secreted by the adipocytes and macrophages. As a result, our adipose-tissue-on-chip platform is capable of identifying stage-specific cytokine secretion profiles from a complex milieu during obesity progression, highlighting its potential as a high-throughput preclinical readout for personalized obesity treatment strategies.