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

Abstract

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.

Micro and Nanoscale Technologies for Diagnosis of Viral Infections

“…Reproduced with permission. [ 208 ] Copyright 2019, Elsevier. d,e) Photonic crystal biosensors for viruses’ detection.…”

Recent applications of QCM-D for the design, synthesis, and characterization of bioactive materials

“…Structure/orientation 54,55,76 Complement selectivity 52 Aptamer selectivity [70][71][72] Enzymatic activity [73][74][75] Cell layers Mechanical/rheological properties 17 Adhesion kinetics 38,39,42,43 Cytoskeletal changes 47,48 Apoptosis 49 Oncogenesis 50 into a continuous film depending on the concentration, surface hydration, and particle composition (Figure 2(a) to (c)). Tajik-Ahmadabad et al similarly assessed the deposition of lipid nanoparticles meant for the delivery of siRNA.…”

“…Template directed DNA synthesis with polymerase enzymes has been studied on QCM-D sensors to reveal the time-dependent increases in layer thickness and changes in viscosity and shear modulus of the layer during polymerization. [73][74][75] Mechanical changes have also been observed while operating novel, tetrahedral nucleic acid nanopumps. 76 These pumps were self-assembled using complementary oligonucleotides and stably coupled to a gold QCM-D sensor through a thiol linker.…”

Rapid and simple single-chamber nucleic acid detection system prepared through nature-inspired surface engineering

“…Thermal cycler-free PCR can also be conducted in a microfluidic chip by alternately flowing the fluid across hot and cold regions 12 . In addition to on-chip amplification, recently developed microfluidic devices have also been integrated with highly sensitive sensing platforms such as nano-plasmonic sensors, electrochemical sensors, and terahertz spectroscopy, enabling rapid and accurate NA identification 13 - 15 . However, the multifunctionality built into a single chip often requires multichambers and sophisticated fluidic controls; these present a major technical obstacle to mass-producing devices, particularly via injection molding in plastics, for commercialization.…”