MultiplexedDetection of Breast Cancer-Specific miRNAand Protein Biomarkers on Exosomes Using a Microfluidic Platform

Exosomes, rich in diverse bioactive molecules, have emerged as a promising "liquid biopsy" approach for early cancer screening. However, relying solely on a single biomarker type can easily lead to missed diagnoses or misjudgments. Therefore, there is an urgent need to develop multiple biomarkers detection strategies to facilitate precise diagnosis. Herein, a microfluidic chip integrating two signals (ALP-driven chemiluminescence and nanobeacons-based fluorescence) into a single device was developed using breast cancer exosomes as a model. A simple sample injection enabled capture and quantitative detection of exosomal surface proteins (CD63, EpCAM, HER2) and internal miRNAs (miRNA-21, miRNA-375). After capture, protein levels were determined by driving magnetic beads and capturing images with a smartphone. Prefabricated nanobeacons were then directly introduced into the chip, enabling miRNA signal detection without RNA extraction. This five-marker biomarker panel achieved a diagnostic accuracy of 99.6% in breast cancer identification, representing an improvement of at least 4.6% over single-marker approaches and an 8.6% increase over any individual biomarker. This multitarget exosome analysis platform may provide a highly sensitive, low-cost, and easily scalable novel tool for breast cancer early diagnosis and treatment efficacy assessment.