Microneedle–peptide biosensor platform for multiplexed profiling of cyclin-dependent kinase activities in cancer cell lines

• Integration of Microneedle and Fluorescent Peptide Biosensors in a single platform enables direct, minimally invasive sampling and real-time, kinase activity readouts. • Multiplexed assay on a single microneedle array with plate-reader detection in ∼30 min. • Distinct kinase activity signatures resolved across five lung cancer cell lines, providing functional information beyond kinase abundance • Optimized sampling and sensor loading maximize signal without lysis buffers. • Translation path toward point-of-care profiling and therapeutic response monitoring is outlined. Protein kinases are frequently dysregulated in cancer and constitute invaluable yet underexploited biomarkers for point-of-care diagnostics. Indeed, quantification of their relative abundance does not reflect their functional activity, calling for biosensors that report on their catalytic activity. We have developed a highly sensitive optical biosensing platform that combines polycarbonate microneedle arrays for direct sampling of protein biomarkers with CDK-selective fluorescent peptide biosensors for optical transduction, thereby facilitating kinase activity profiling from biological samples. We have optimized sampling of proteins from cultured cancer cells, as well as optical biosensing to achieve robust and multiplexed profiling of CDK1, CDK2, CDK4 and CDK6 activities from a single sample on a 96-well plate reader within 30 min. We have implemented this hybrid platform to resolve distinct CDK activity signatures across five lung cancer cell lines (A549, PC9, H1299, H358, H322). This minimally invasive hybrid biosensing platform, eliminates detergent-based cell lysis for extraction of protein kinases and supports kinetic and quantitative measurement of their functional activities. By providing a scalable route for ex vivo kinase activity profiling, this approach offers perspectives for point-of-care cancer diagnostics and monitoring of therapeutic response.