Panagiota M. Kalligosfyri, Antonella Miglione, Ada Raucci, Wanda Cimmino, Gabriella Iula, Alessandra Glovi, Sima Singh, Michelino De Laurentiis, Antonio Giordano, Stefano Cinti
Liquid biopsy has revolutionized noninvasive disease diagnosis and monitoring by detecting circulating biomarkers such as tumor-derived DNA, miRNAs, proteins, exosomes, and circulating tumor cells in body fluids. Conventional single-analyte approaches fail to capture disease complexity, limiting diagnostic accuracy. Multifunctional biosensors enable simultaneous detection of multiple biomarkers, representing a paradigm shift in liquid biopsy. This review highlights recent advances in diverse detection strategies, including optical, electrochemical, plasmonic, and clustered regularly interspaced short palindromic repeats-based assays, alongside innovations in hybrid platforms, smart surface engineering, and nanotechnology. We also discuss integration with microfluidics, artificial intelligence, and chemometric analysis to enhance sensitivity, reduce signal interference, and improve scalability. Finally, challenges in clinical translation and standardization are highlighted, underscoring the transformative potential of multifunctional biosensors in precision medicine and early disease detection.