Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence.

Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly.

Nicking-assisted on-loop and off-loop enzymatic cascade amplification for optomagnetic detection of a highly conserved dengue virus sequence.

Applications of conventional linear ligation-rolling circle amplification (RCA) are restricted by the sophisticated operation steps and unsatisfactory picomolar-level detection limits. We herein demonstrate an RCA-based cascade amplification reaction that converts a side-reaction to secondary amplification, which improves the detection limit and simplifies the operation compared to linear ligation-RCA assays. The proposed nicking-assisted enzymatic cascade amplification (NECA) comprises an on-loop amplification reaction using circular templates to generate intermediate amplicons, and an off-loop amplification reaction using intermediate amplicons as primers for end amplicons.

CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.

False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic.

Integration of rolling circle amplification and optomagnetic detection on a polymer chip.

Rolling circle amplification (RCA) combined with padlock probe recognition of a DNA target is attractive for on-chip nucleic acid testing due to its high specificity and isothermal reaction conditions. However, the integration of RCA on an automated chip platform is challenging due to the different reagents needed for the reaction steps and the temperature sensitivity of the phi29 polymerase. Here, we describe the integration of an RCA assay on a single-use polymer chip platform where magnetic microbeads are used as solid support to transport the DNA target between three connected reaction chambers for (i) padlock probe annealing and ligation, (ii) RCA, and (iii) optomagnetic detection of RCA products.

Integration of microbead DNA handling with optomagnetic detection in rolling circle amplification assays.

Rolling circle amplification (RCA) is a linear isothermal amplification technique that is widely applied in biomolecular assays due to its high specificity. Handling of a target sample using magnetic microbeads (MMBs) in a multi-step assay is appealing as the MMBs enable separation and transportation using an external magnet. Detection of amplicons using optomagnetic measurements of the rotational diffusion properties of magnetic nanoparticles (MNPs) is also appealing as it can be performed on any transparent sample container.

Ultrasensitive Real-Time Rolling Circle Amplification Detection Enhanced by Nicking-Induced Tandem-Acting Polymerases.

Padlock probe ligation-based rolling circle amplification (RCA) can distinguish single-nucleotide variants, which is promising for the detection of drug-resistance mutations in, e.g., ().

MicroRNA Detection through DNAzyme-Mediated Disintegration of Magnetic Nanoparticle Assemblies.

DNA-assembled nanoparticle superstructures offer numerous bioresponsive properties that can be utilized for point-of-care diagnostics. Functional DNA sequences such as deoxyribozymes (DNAzymes) provide novel bioresponsive strategies and further extend the application of DNA-assembled nanoparticle superstructures. In this work, we describe a microRNA detection biosensor that combines magnetic nanoparticle (MNP) assemblies with DNAzyme-assisted target recycling.

Field-dependent dynamic responses from dilute magnetic nanoparticle dispersions.

The response of magnetic nanoparticles (MNPs) to an oscillating magnetic field outside the linear response region is important for several applications including magnetic hyperthermia, magnetic resonance imaging and biodetection. The size and magnetic moment are two critical parameters for the performance of a colloidal MNP dispersion. We present and demonstrate the use of optomagnetic (OM) and AC susceptibility (ACS) measurements vs.

On-Particle Rolling Circle Amplification-Based Core-Satellite Magnetic Superstructures for MicroRNA Detection.

Benefiting from the specially tailored properties of the building blocks as well as of the scaffolds, DNA-assembled core-satellite superstructures have gained increasing interest in drug delivery, imaging, and biosensing. The load of satellites plays a vital role in core-satellite superstructures, and it determines the signal intensity in response to a biological/physical stimulation/actuation. Herein, for the first time, we utilize on-particle rolling circle amplification (RCA) to prepare rapidly responsive core-satellite magnetic superstructures with a high load of magnetic nanoparticle (MNP) satellites.

Quantitative Detection of Trace Level Cloxacillin in Food Samples Using Magnetic Molecularly Imprinted Polymer Extraction and Surface-Enhanced Raman Spectroscopy Nanopillars.

There is an increasing demand for rapid, sensitive, and low cost analytical methods to routinely screen antibiotic residues in food products. Conventional detection of antibiotics involves sample preparation by liquid-liquid or solid-phase extraction, followed by analysis using liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), or gas chromatography (GC). The process is labor-intensive, time-consuming, and expensive.

Shape anisotropy enhanced optomagnetic measurement for prostate-specific antigen detection via magnetic chain formation.

We demonstrate a homogeneous biosensor for the detection of multivalent targets by combination of magnetic nanoparticle (MNP) chains and a low-cost 405nm laser-based optomagnetic system. The MNP chains are assembled in a rotating magnetic field and stabilized by multivalent target molecules. The number of chains remaining in zero field is proportional to the target concentration, and can be quantified by optomagnetic measurements.

Two-dimensional salt and temperature DNA denaturation analysis using a magnetoresistive sensor.

We present a microfluidic system and its use to measure DNA denaturation curves by varying the temperature or salt (Na) concentration. The readout is based on real-time measurements of DNA hybridization using magnetoresistive sensors and magnetic nanoparticles (MNPs) as labels. We report the first melting curves of DNA hybrids measured as a function of continuously decreasing salt concentration at fixed temperature and compare them to the corresponding curves obtained vs.

Characterization of fine particles using optomagnetic measurements.

The remanent magnetic moment and the hydrodynamic size are important parameters for the synthesis and applications of magnetic nanoparticles (MNPs). We present the theoretical basis for the determination of the remanent magnetic moment and the hydrodynamic size of MNPs with a narrow size distribution using optomagnetic measurements. In these, the 2nd harmonic variation of the intensity of light transmitted through an MNP suspension is measured as a function of an applied axial oscillating magnetic field.

Optomagnetic Detection of MicroRNA Based on Duplex-Specific Nuclease-Assisted Target Recycling and Multilayer Core-Satellite Magnetic Superstructures.

Superstructural assembly of magnetic nanoparticles enables approaches to biosensing by combining specially tailored properties of superstructures and the particular advantages associated with a magnetic or optomagnetic read-out such as low background signal, easy manipulation, cost-efficiency, and potential for bioresponsive multiplexing. Herein, we demonstrate a sensitive and rapid miRNA detection method based on optomagnetic read-out, duplex-specific nuclease (DSN)-assisted target recycling, and the use of multilayer core-satellite magnetic superstructures. Triggered by the presence of target miRNA and DSN-assisted target recycling, the core-satellite magnetic superstructures release their "satellites" to the suspension, which subsequently can be quantified accurately in a low-cost and user-friendly optomagnetic setup.

Comparison of optomagnetic and AC susceptibility readouts in a magnetic nanoparticle agglutination assay for detection of C-reactive protein.

There is an increasing need to develop biosensor methods that are highly sensitive and that can be combined with low-cost consumables. The use of magnetic nanoparticles (MNPs) is attractive because their detection is compatible with low-cost disposables and because application of a magnetic field can be used to accelerate assay kinetics. We present the first study and comparison of the performance of magnetic susceptibility measurements and a newly proposed optomagnetic method.

Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads.

We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum of manual steps involved.

Geometrical optimization of microstripe arrays for microbead magnetophoresis.

Manipulation of magnetic beads plays an increasingly important role in molecular diagnostics. Magnetophoresis is a promising technique for selective transportation of magnetic beads in lab-on-a-chip systems. We investigate periodic arrays of exchange-biased permalloy microstripes fabricated using a single lithography step.

Configurational Statistics of Magnetic Bead Detection with Magnetoresistive Sensors.

Magnetic biosensors detect magnetic beads that, mediated by a target, have bound to a functionalized area. This area is often larger than the area of the sensor. Both the sign and magnitude of the average magnetic field experienced by the sensor from a magnetic bead depends on the location of the bead relative to the sensor.

Classification of Magnetic Nanoparticle Systems--Synthesis, Standardization and Analysis Methods in the NanoMag Project.

This study presents classification of different magnetic single- and multi-core particle systems using their measured dynamic magnetic properties together with their nanocrystal and particle sizes. The dynamic magnetic properties are measured with AC (dynamical) susceptometry and magnetorelaxometry and the size parameters are determined from electron microscopy and dynamic light scattering. Using these methods, we also show that the nanocrystal size and particle morphology determines the dynamic magnetic properties for both single- and multi-core particles.

Blu-ray based optomagnetic aptasensor for detection of small molecules.

This paper describes an aptamer-based optomagnetic biosensor for detection of a small molecule based on target binding-induced inhibition of magnetic nanoparticle (MNP) clustering. For the detection of a target small molecule, two mutually exclusive binding reactions (aptamer-target binding and aptamer-DNA linker hybridization) are designed. An aptamer specific to the target and a DNA linker complementary to a part of the aptamer sequence are immobilized onto separate MNPs.

On the importance of sensor height variation for detection of magnetic labels by magnetoresistive sensors.

Magnetoresistive sensors are widely used for biosensing by detecting the signal from magnetic labels bound to a functionalized area that usually covers the entire sensor structure. Magnetic labels magnetized by a homogeneous applied magnetic field weaken and strengthen the applied field when they are over and outside the sensor area, respectively, and the detailed origin of the sensor signal in experimental studies has not been clarified. We systematically analyze the signal from both a single sensor stripe and an array of sensor stripes as function of the geometrical parameters of the sensor stripes as well as the distribution of magnetic labels over the stripes.

Quantification of rolling circle amplified DNA using magnetic nanobeads and a Blu-ray optical pick-up unit.

We present the first implementation of a Blu-ray optical pickup unit (OPU) for the high-performance low-cost readout of a homogeneous assay in a multichamber microfluidic disc with a chamber thickness of 600 μm. The assay relies on optical measurements of the dynamics of magnetic nanobeads in an oscillating magnetic field applied along the light propagation direction. The laser light provided by the OPU is transmitted through the sample chamber and reflected back onto the photo detector array of the OPU via a mirror.

Magnetoresistive sensor for real-time single nucleotide polymorphism genotyping.

We demonstrate a magnetoresistive sensor platform that allows for the real-time detection of point mutations in DNA targets. Specifically, we detect point mutations at two sites in the human beta globin gene. For DNA detection, the present sensor technology has a detection limit of about 160 pM and a dynamic range of about two orders of magnitude.

Microstripes for transport and separation of magnetic particles.

We present a simple technique for creating an on-chip magnetic particle conveyor based on exchange-biased permalloy microstripes. The particle transportation relies on an array of stripes with a spacing smaller than their width in conjunction with a periodic sequence of four different externally applied magnetic fields. We demonstrate the controlled transportation of a large population of particles over several millimeters of distance as well as the spatial separation of two populations of magnetic particles with different magnetophoretic mobilities.