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Abstract Controlled molecular transport and separation is of significant importance in various applications. In this work, we presented a novel concept of nanofluidic molecular charge-coupled device (CCD) for controlled DNA transport and separation. By leveraging the unique field-effect coupling in nanofluidic systems, the nanofluidic molecular CCD aims to store charged biomolecules such as DNAs in discrete regions in nanochannels and transfer and separate these biomolecules as a charge packet in a bucket brigade fashion. We developed a quantitative model to capture the impact of nanochannel surface charge, gating voltage and frequency, molecule diffusivity, and gating electrode geometry on the transport and separation efficiency. We studied the synergistic effects of these factors to guide the device design and optimize the DNA transport and separation in a nanofluidic CCD. The findings in this study provided insight into the rational design and implementation of the nanofluidic molecular CCD.