Amplification- and Enzyme-Free Magnetic Diagnostics Circuit for Whole-Genome Detection of SARS-CoV-2 RNA.

Polymerase chain reaction (PCR) requires thermal cycling and enzymatic reactions for sequence amplification, hampering their applications in point-of-care (POC) settings. Magnetic bioassays based on magnetic particle spectroscopy (MPS) and magnetic nanoparticles (MNPs) are isothermal, wash-free, and can be quantitative. Realizing them amplification- and enzyme-free on a benchtop device, they will become irreplaceable for POC applications. Here we demonstrate a first-in-class magnetic signal amplification circuit (MAC) that enables detection of whole genome of SARS-CoV-2 by combining the specificity of toehold-mediated DNA strand displacement with the magnetic response of MNPs to declustering processes. Using MAC, we detect the N gene of SARS-CoV-2 samples at a concentration of 104 RNA copies/µl as determined by droplet digital PCR. Further, we demonstrate that MAC can reliably distinguish between SARS-CoV-2 and other human coronaviruses. Being a wash-, amplification- and enzyme-free biosensing concept and working at isothermal conditions (25 °C) on a low-cost benchtop MPS device, our MAC biosensing concept offers several indispensable features for translating nucleic acid detection to POC applications.

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

Magnetic nanoparticles (MNPs) provide new opportunities for enzyme-free biosensing of nucleic acid biomarkers and magnetic actuation by patterning on DNA origami, yet how the DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we performed surface functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry with a tunable grafting density, which enables the encapsulation of single MNPs inside a functional polymeric layer. We used several complementary methods to show that particle translational and rotational dynamics exhibit a sigmoidal dependence on the ssDNA grafting density. At low densities, ssDNA strands adopt a coiled conformation that results in minor alterations to particle dynamics, while at high densities, they organize into polymer brushes that collectively influence particle dynamics. Intermediate ssDNA densities, where the dynamics are most sensitive to changes, show the highest magnetic biosensing sensitivity for the detection of target nucleic acids. Finally, we demonstrate that MNPs with high ssDNA grafting densities are required to efficiently couple to DNA origami. Our results establish ssDNA grafting density as a critical parameter for the functionalization of MNPs for magnetic biosensing and functionalization of DNA nanostructures.

Decoupling the Characteristics of Magnetic Nanoparticles for Ultrahigh Sensitivity.

Immunoassays exploiting magnetization dynamics of magnetic nanoparticles are highly promising for mix-and-measure, quantitative, and point-of-care diagnostics. However, how single-core magnetic nanoparticles can be employed to reduce particle concentration and concomitantly maximize assay sensitivity is not fully understood. Here, we design monodisperse Néel and Brownian relaxing magnetic nanocubes (MNCs) of different sizes and compositions. We provide insights into how to decouple physical properties of these MNCs to achieve ultrahigh sensitivity. We find that tricomponent-based Zn0.06Co0.80Fe2.14O4 particles, with out-of-phase to initial magnetic susceptibility χ″/χ0 ratio of 0.47 out of 0.50 for magnetically blocked ideal particles, show the ultrahigh magnetic sensitivity by providing a rich magnetic particle spectroscopy (MPS) harmonics spectrum despite bearing lower saturation magnetization than dicomponent Zn0.1Fe2.9O4 having high saturation magnetization. The Zn0.06Co0.80Fe2.14O4 MNCs, coated with catechol-based poly(ethylene glycol) ligands, measured by our benchtop MPS show 3 orders of magnitude better particle LOD than that of commercial nanoparticles of comparable size.

Point-of-need detection of pathogen-specific nucleic acid targets using magnetic particle spectroscopy.

The ongoing COVID-19 pandemic stresses the need for widely available diagnostic tests for the presence of SARS-CoV-2 in individuals. Due to the limited availability of vaccines, diagnostic assays which are cheap, easy-to-use at the point-of-need, reliable and fast, are currently the only way to control the pandemic situation. Here we present a diagnostic assay for the detection of pathogen-specific nucleic acids based on changes of the magnetic response of magnetic nanoparticles: The target-mediated hybridization of modified nanoparticles leads to an increase in the hydrodynamic radius. This resulting change in the magnetic behaviour in an ac magnetic field can be measured via magnetic particle spectroscopy (MPS), providing a viable tool for the accurate detection of target nucleic acids. In this work we show that single stranded DNA can be detected in a concentration-dependent manner by these means. In addition to detecting synthetic DNA with an arbitrary sequence in a concentration down to 500 pM, we show that RNA and SARS-CoV-2-specific DNA as well as saliva as a sample medium can be used for an accurate assay. These proof-of-principle experiments show the potential of MPS based assays for the reliable and fast diagnostics of pathogens like SARS-CoV-2 in a point-of-need fashion without the need of complex sample preparation.

Toward Rapid and Sensitive Detection of SARS-CoV-2 with Functionalized Magnetic Nanoparticles.

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global medical systems and economies and rules our daily living life. Controlling the outbreak of SARS-CoV-2 has become one of the most important and urgent strategies throughout the whole world. As of October 2020, there have not yet been any medicines or therapies to be effective against SARS-CoV-2. Thus, rapid and sensitive diagnostics is the most important measures to control the outbreak of SARS-CoV-2. Homogeneous biosensing based on magnetic nanoparticles (MNPs) is one of the most promising approaches for rapid and highly sensitive detection of biomolecules. This paper proposes an approach for rapid and sensitive detection of SARS-CoV-2 with functionalized MNPs via the measurement of their magnetic response in an ac magnetic field. For proof of concept, mimic SARS-CoV-2 consisting of spike proteins and polystyrene beads are used for experiments. Experimental results demonstrate that the proposed approach allows the rapid detection of mimic SARS-CoV-2 with a limit of detection of 0.084 nM (5.9 fmole). The proposed approach has great potential for designing a low-cost and point-of-care device for rapid and sensitive diagnostics of SARS-CoV-2.

Biophysical Characterization of (Silica-coated) Cobalt Ferrite Nanoparticles for Hyperthermia Treatment.

Magnetic hyperthermia is a technique that describes the heating of material through an external magnetic field. Classic hyperthermia is a medical condition where the human body overheats, being usually triggered by a heat stroke, which can lead to severe damage to organs and tissue due to the denaturation of cells. In modern medicine, hyperthermia can be deliberately induced to specified parts of the body to destroy malignant cells. Magnetic hyperthermia describes the way that this overheating is induced and it has the inherent advantage of being a minimal invasive method when compared to traditional surgery methods. This work presents a particle system that offers huge potential for hyperthermia treatments, given its good loss value, i.e., the particles dissipate a lot of heat to their surroundings when treated with an ac magnetic field. The measurements were performed in a low-cost custom hyperthermia setup. Additional toxicity assessments on Jurkat cells show a very low short-term toxicity on the particles and a moderate low toxicity after two days due to the prevalent health concerns towards nanoparticles in organisms.