Real-time monitoring of DNA immobilization and detection of DNA polymerase activity by a microfluidic nanoplasmonic platform.

DNA polymerase catalyzes the replication of DNA, one of the key steps in cell division. The control and understanding of this reaction owns great potential for the fundamental study of DNA-enzyme interactions. In this context, we developed a label-free microfluidic biosensor platform based on the principle of localized surface plasmon resonance (LSPR) to detect the DNA-polymerase reaction in real-time. Our microfluidic LSPR chip integrates a polydimethylsiloxane (PDMS) channel bonded with a nanoplasmonic substrate, which consists of densely packed mushroom-like nanostructures with silicon dioxide stems (40 nm) and gold caps (22 nm), with an average spacing of 19 nm. The LSPR chip was functionalized with single-stranded DNA (ssDNA) template (T30), spaced with hexanedithiol (HDT) in a molar ratio of 1:1. The DNA primer (P8) was then attached to T30, and the second strand was subsequently elongated by DNA polymerase assembling nucleotides from the surrounding fluid. All reaction steps were detected in-situ inside the microfluidic LSPR chip, at room temperature, in real-time, and label-free. In addition, the sensor response was successfully correlated with the amount of DNA and HDT molecules immobilized on the LSPR sensor surface. Our platform represents a benchmark in developing microfluidic LSPR chips for DNA-enzyme interactions, further driving innovations in biosensing technologies.