Abstract
Functionalized DNA sequences are promising sensing elements to combine with transducers for bio-sensing specific target microbes. As an application example, this paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM). Such hybridization leads to a frequency signal, which is suitable for monitoring real-time LAMP amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface. Target DNA LAMP products cause irreversible frequency shifts on the QCM surfaces during hybridization in the kHz range, which result from both changes in mass and charge on the electrode surface. In order to confirm the LAMP assay working in the QCM sensing system at elevated temperature, the sky blue of positive LAMP products solution was achieved by using the Hydroxy Naphthol Blue (HNB) and agarose gel electrophoresis. Since on-QCM sensing of DNA hybridization leads to irreversible sensor responses, this work shows characterization by X-ray photoelectron spectroscopy (XPS) core spectra of S2p, N1s, Mg1s, P2p and C1s. XPS results confirmed that indeed both DNA and by-products of LAMP attached to the surface. Listeria monocytogenes DNA served to study in-situ detection of amplified LAMP products on DNA-functionalized surfaces.
Highlights
Detection of DNA-based immobilization and hybridization on solid surfaces is the basis of several biotechnological tools and strategies, including DNA microarrays [1,2] and biosensors [3,4]
This paper demonstrates in situ detection of loop-mediated isothermal amplification products by hybridizing them with thiolated-ssDNA covalently anchored on the electrodes of a quartz crystal microbalance (QCM)
Such hybridization leads to a frequency signal, which is suitable for monitoring real-time loop mediated isothermal amplification (LAMP) amplification based on mass-sensing: it detects interactions between the complementary nucleobases of LAMP products in solution and the thiolated-ssDNA probe sequence on the gold surface
Summary
Detection of DNA-based immobilization and hybridization on solid surfaces is the basis of several biotechnological tools and strategies, including DNA microarrays [1,2] and biosensors [3,4]. That paper demonstrates the method for achieving such measurements in principle and strongly focuses on the optimization steps required to ensure optimal hybridization between the immobilized oligonucleotide and a target single-strand DNA. It laid out the fundaments of how to prepare optimal self-assembled monolayers for that purpose. To develop a more in-depth understanding of DNA hybridization and the efficiency of LAMP target capture, we characterized the surfaces after binding with XPS This approach allows for validating the mass signals on QCM and for quantifying the chemical composition of the surface layer at different stages of recognition/sensing. Sterile deionized water (18.2 MΩ cm−1) served for preparing all solutions
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