A three-dimensional microfluidic device embedded within a thermal cycler tube for electrokinetic DNA extraction.

Microfluidic devices have been widely used in modern chemical and biological analyses as stand-alone units, typically in series with other equipment such as extraction columns, manual or robotic pipetting, and even advanced next-generation sequencing systems. While microfluidic devices have enhanced various aspects of laboratory workflows, their integration with established commercial assay platforms remains limited. To this end, we developed a three-dimensional microfluidic insert embedded directly into a commercially available polymerase chain reaction (PCR) tube. This integration creates a microfluidic device compatible with conventional thermal cyclers, which support complex temperature cycling and multiplexed fluorescence detection. The integrated system facilitates key bioassay functions like nucleic acid purification through a selective ionic focusing method known as isotachophoresis (ITP), PCR amplification, and real-time fluorescence detection. We validated the performance of the integrated system by purifying nucleic acids from raw human serum samples and detecting exogenous SARS-CoV-2 N gene using FAM-labeled TaqMan probes, with both the DNA extraction and detection carried out within the same PCR tube. We achieved a detection sensitivity of 100 cp μL within a total process time of 60 min in these experiments. Human serum samples processed without purification show no PCR amplification results. This integrated system demonstrates the powerful concept of integrating microfluidic structures into form factors compatible with the highly complex and sensitive operation of current off-the-shelf systems.