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Transforming microfluidics-based biosensing systems from laboratory research into clinical reality remains an elusive goal despite decades of intensive research. A fundamental obstacle for the development of fully automated microfluidic diagnostic systems is the lack of an effective strategy for combining pumping, sample preparation, and detection modules into an integrated biosensing platform. Herein, we report a universal electrode approach, which incorporates dc electrolytic pumping, ac electrokinetic sample preparation, and self-assembled monolayer based electrochemical sensing on a single microfluidic platform, to automate complicated molecular analysis procedures that will enable biosensing applications in nontraditional health care settings. Using the universal electrode approach, major microfluidic operations required in molecular analyses, such as pumping, mixing, washing, and sensing, can be performed in a single platform. We demonstrate the universal electrode platform for detecting bacterial 16S rRNA, a phylogenetic marker, toward rapid diagnostics of urinary tract infection. Since only electronic interfaces are required to operate the platform, the universal electrode approach represents an effective system integration strategy to realize the potential of microfluidics in molecular diagnostics at the point of care.