Real-Time FO-SPR Monitoring of Solid-Phase DNAzyme Cleavage Activity for Cutting-Edge Biosensing

Abstract

DNA nanotechnology has a great potential in biosensor design including nanostructuring of the biosensor surface through DNA origami, target recognition by means of aptamers, and DNA-based signal amplification strategies. In this paper, we use DNA nanotechnology to describe for the first time the concept of real-time solid-phase monitoring of DNAzyme cleavage activity for the detection of specific singlestranded DNA (ssDNA) with a fiber optic surface plasmon resonance (FO-SPR) biosensor. Hereto, we first developed a robust ligation strategy for the functionalization of the FO-SPR biosensing surface with ssDNA-tethered gold nanoparticles, serving as the substrate for the DNAzyme. Next, we established a relation between the SPR signal change, due to the cleavage activity of the 10−23 DNAzyme, and the concentration of the DNAzyme, showing faster cleavage kinetics for higher DNAzyme concentrations. Finally, we implemented this generic concept for biosensing of ssDNA target in solution. Hereto, we designed a DNAzyme−inhibitor complex, consisting of an internal loop structure complementary to the ssDNA target, that releases active DNAzyme molecules in a controlled way as a function of the target concentration. We demonstrated reproducible target detection with a theoretical limit of detection of 1.4 nM, proving that the presented ligation strategy is key to a universal DNAzyme-based FO-SPR biosensing concept with promising applications in the medical and agrofood sector.

DNA-only bioassay for simultaneous detection of proteins and nucleic acids

“…To select one for establishing the competitive bioassay, we first tested their interaction with several thrombin concentrations (0, 62, 124, and 248 nM) in Buffer A , Buffer B , and Buffer C . This was done on a FO-SPR sensor, commercialized by FOx Biosystems (a platform based on an in-house developed technology [29,30]), by immobilizing thiol-modified aptamers on the FO-SPR probes (Fig. S2).…”

“…TBA has been selected as an attractive model system due to its extensive characterization and applicability in numerous bioassays [27]. The DNAzyme 1 was recently made in our group for optimal catalysis at the standard room temperature (RT, i.e., 20-25°C) [26], starting from the traditional DNAzyme that cleaves at elevated temperatures (ranging between 37 and 55°C) [28,29]. Aptazyme 2.20-5′ is designed by joining a random NA 2 target recognition sequence with DNAzyme 2 .…”

High-Sensitivity PtSe2 Surface Plasmon Resonance Biosensor Based on Metal-Si-Metal Waveguide Structure

“…Surface plasmon resonance (SPR) found at the interface of two media (such as dielectric and metal) with opposite signs of dielectric constant can be excited by Otto or Kretschmann prism coupling model [ 1 , 2 ]. In recent years, SPR biosensors have been applied in food safety [ 3 , 4 ], environmental detection [ 5 ], medical diagnosis [ 6 , 7 , 8 ] and other fields, due to their outstanding performance of high detection accuracy, real time [ 9 , 10 ] and label free [ 11 ]. Traditional SPR biosensor structures usually contain glass prisms and noble metals which are in direct contact with biomolecules.…”

FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices

“…The FO‐SPR platform can offer multiple advantages to the EV field, such as sensitivity and specificity of prism‐based SPR devices at the fraction of their cost and complexity. Over the years, our team successfully implemented different bioassays on this platform for detecting DNA (Daems et al., 2018; Knez et al., 2015; Peeters et al., 2019), proteins (Lu et al., 2016, 2017) and bacteriophages (Knez et al., 2013). rEVs originate from medium conditioned by HEK293T cells that express the major structural component of HIV‐1 virus particles, the gag polyprotein.…”