Amplification and extraction free quantitative detection of viral nucleic acids and single-base mismatches using magnetic signal amplification circuit

Abstract

Established nucleic acid detection assays require extraction and purification before sequence amplification and/or enzymatic reactions, hampering their widespread applications in point-of-care (POC) formats. Magnetic immunoassays based on magnetic particle spectroscopy and magnetic nanoparticles (MNPs) are isothermal, extraction- and purification-free, and can be quantitative and benchtop, making them suitable for POC settings. Here, we demonstrate a Magnetic signal Amplification Circuit (MAC) that combines specificity of toehold-mediated DNA strand displacement with magnetic response of MNPs to a clustering/declustering process. Our MAC assays require neither amplification nor extraction of target nucleic acids, and reveal four times better sensitivity than that of a magnetic circuit without signal amplification. Using MAC, we detect a highly specific 43 nucleotides sequence of SARS-CoV-2 virus. The MAC enables sensing both DNA and RNA targets with varying lengths and resolving single-base mismatches. Our MAC can be a powerful tool for translating research of nucleic acids detection to the clinic.

Cooperative dynamics of DNA-grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami

“…DNA-grafted inorganic nanoparticles (NPs) are versatile building blocks for superstructures of selected geometries and functionalities as well as responsive nanomaterials for biosensing and biocomputing. [1][2][3][4][5] DNA-labelled NPs and nanostructures have been used as molecular nanothermometers, 6 in vitro bio-sensors, 7 and nanoparticle beacons 8 and for intracellular sensing and molecular recognition. 9,10 NP-DNA conjugates offer novel possibilities for further modifications through competitive hybridization, 11 DNA strand displacement, 7 and ligation.…”

“…[1][2][3][4][5] DNA-labelled NPs and nanostructures have been used as molecular nanothermometers, 6 in vitro bio-sensors, 7 and nanoparticle beacons 8 and for intracellular sensing and molecular recognition. 9,10 NP-DNA conjugates offer novel possibilities for further modifications through competitive hybridization, 11 DNA strand displacement, 7 and ligation. 12 The functionalization of Au NPs with ssDNA can be achieved through direct Au-S covalent bonds, making it straightforward and requiring only minimal surface modification steps.…”

“…6 Furthermore, MNPs can change their Brownian magnetic relaxation dynamics by binding to targeting molecules, 24,25 upon which magnetic biosensing has been built for the detection of bacteria, 26 proteins, 27,28 and DNA/ RNA. [29][30][31] Magnetic-based assays offer an enzyme-, wash-, and amplification-free 7 detection workflow, which are highly desirable features for point-of-care detection of nucleic acid biomarkers. A particular challenge stems from the multistep premodification and surface chemistries that are needed for successful DNA labeling of MNPs.…”

Cooperative dynamics of DNA grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami

“…[1][2][3][4][5] Programmed assemblies of gold (Au) nanoparticles (NPs) using Watson-Crick base pairing with complementary single-stranded DNA (ssDNA) strands were introduced two decades ago. 1,2 DNA labelled NPs and nanostructures have been used as molecular nanothermometers, 6 in vitro biosensors, 7 nanoparticle beacons, 8 and as an intracellular sensing platform. 9 NP-DNA conjugates offer novel possibilities for further modifications through competitive hybridization, 10 DNA strand displacement, 7 and ligation.…”

“…1,2 DNA labelled NPs and nanostructures have been used as molecular nanothermometers, 6 in vitro biosensors, 7 nanoparticle beacons, 8 and as an intracellular sensing platform. 9 NP-DNA conjugates offer novel possibilities for further modifications through competitive hybridization, 10 DNA strand displacement, 7 and ligation. 11 The functionalization of Au NPs with ssDNA can be achieved through direct Au-S covalent bonds, making it straightforward and requiring only minimal surface modification steps.…”