In this study, we present a fluorescence imaging cytometer capable of detecting multiple biomarkers using temporally multiplexed illumination. We independently illuminated the classification and quantitative detection channels using multiwavelength LEDs. To quantify the biomarker concentrations, we employed a sandwich immunoassay with Cy5 dye, while the microbeads were internally dyed with three different intensity levels of quantum dots to distinguish among the three cytokines. We conducted separate tests with three cytokines, namely IFN-α, IL-5, and IL-6, at varying concentrations. The detection ranges for these cytokines were determined to be 10 pg/ml-2500 pg/ml, 30 pg/ml-2500 pg/ml, and 30 pg/ml-2500 pg/ml, respectively. The results of the multiplexed experiment, show that our device can independently detect three different biomarkers in a single assay. This validation demonstrates that our device has potential in diagnosing sepsis and other diseases requiring various laboratory results. With further refinement and development, our device holds great promise for enhancing point-of-care medical services.
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