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. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.

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. The whole NECA reaction is homogeneous and isothermal. Amplicons anneal to detection probes that are grafted onto magnetic nanoparticles (MNPs), such that MNP clusters form and can be detected in real-time using optomagnetic measurements. The optomagnetic sensor detects the presence and size increase of MNP clusters by optical transmission measurements in an oscillating magnetic field. A detection limit of 2 fM was achieved with a total assay time of ca. 70 min. By combining optomagnetic readouts of signal phase lag and hydrodynamic size increase of MNPs, NECA-based target quantification provided a wide dynamic detection range of ca. 4.5 orders of magnitude. Moreover, the specificity and the serum detection capability of the proposed method were investigated.

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. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.

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. Two strategies are described for integration of MMB sample handling in an RCA assay with on-chip optomagnetic detection of the amplification products. The first strategy relies on selective and irreversible release of the amplicons from the MMBs so that the binding of functionalized MNPs to the amplicons can be detected optomagnetically. The second strategy relies on the incorporation of MNPs into RCA products during RCA, followed by their separation on MMBs and subsequent optomagnetic detection upon release from the RCA products. Using MMB handling of RCA steps, the limits of detection (LODs) for a synthetic DNA target representative of Victoria Influenza type B were found to be between 4 and 20 pM with total assay times between 2 and 2.5 h. Without magnetic microbead sample handling, the LOD was 200 fM. The findings provide deeper insight into the use of magnetic microbeads as solid substrates to handle a DNA target for integration of RCA as well as other DNA-based assays. Graphical Abstract Schematic illustration of magnetic microbeads transporting a DNA target through the steps in a rolling circle amplification assay. Optomagnetic measurements detect the binding of magnetic nanoparticles to amplicons released from microbeads (top) or the pH-induced release of magnetic nanoparticles trapped in amplicons (bottom).

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. The three chambers were loaded with reagents by sequential filling combined with passive microfluidic structures. After loading, the on-chip assay steps were automated. For an assay in which all steps but the padlock probe annealing on the target were performed on-chip, we found a limit of detection (LOD) for a synthetic influenza target of 2 pM after 45 min of RCA, which is comparable to the corresponding laboratory assay. The entire assay, including padlock probe annealing, could be performed on-chip with an LOD of 20 pM after 45 min of RCA. This LOD can likely be reduced by further optimizing the microbead mixing. The results present important steps towards the integration and automation of RCA and potentially also other complex multi-step assays on a single-use polymer chip for molecular analysis.

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., Mycobacterium tuberculosis (Mtb). However, the clinical application of conventional linear RCA is restricted by its unsatisfactory picomolar-level limit of detection (LOD). Herein, we demonstrate the mechanism of a nicking-enhanced RCA (NickRCA) strategy that allows several polymerases to act simultaneously on the same looped template, generating single-stranded amplicon monomers. Limiting factors of NickRCA are investigated and controlled for higher amplification efficiency. Thereafter, we describe a NickRCA-based magnetic nanoparticle (MNP) dimer formation strategy combined with a real-time optomagnetic sensor monitoring MNP dimers. The proposed methodology is applied for the detection of a common Mtb rifampicin-resistance mutation, rpoB 531 (TCG/TTG). Without additional operation steps, an LOD of 15 fM target DNA is achieved with a total assay time of ca. 100 min. Moreover, the proposed biosensor holds the advantages of single-nucleotide mutation discrimination and the robustness to quantify targets in 10% serum samples. NickRCA produces short single-stranded monomers instead of the DNA coils produced in conventional RCA, which makes it more convenient for downstream operation, immobilization or detection, thus being applicable with different molecular tools and biosensors.

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. The DNA scaffolds of the MNP assemblies contain substrate sequences for DNAzyme and can form cleavage catalytic structures in the presence of target DNA or RNA sequences, leading to rupture of the scaffolds and disintegration of the MNP assemblies. The target sequences are preserved during the cleavage reaction and release into the suspension to trigger the digestion of multiple DNA scaffolds. The high local concentration of substrate sequences in the MNP assemblies reduces the diffusion time for target recycling. The concentration of released MNPs, which is proportional to the concentration of the target, can be quantified by a 405 nm laser-based optomagnetic sensor. For the detection of let-7b in 10% serum, after 1 h of isothermal reaction at 50 °C, we found a linear detection range between 10 pM and 100 nM with a limit of detection of 6 pM. For the quantification of DNA target in buffer solution, a limit of detection of 1.5 pM was achieved. Compared to protein enzyme-based microRNA detection methods, the proposed DNAzyme-based biosensor has an increased stability, a reduced cost and a possibility to be used in living cells, all of which are valuable features for biosensing applications.

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. frequency and magnetic field strength to obtain the size and magnetic moment distributions including the correlation between the distributions. The correlation between the size and the magnetic moment contains information on the morphology and intrinsic structure of the particle. In OM measurements, the variation of the second harmonic light transmission through a dispersion of MNPs is measured in response to an oscillating magnetic field. We solve the Fokker-Planck equations for MNPs with a permanent magnetic moment, and develop analytical approximations to the ACS and the OM signals that also account for the change in the curve shapes with increasing field strength. Further, we describe the influence of induced magnetic moments on the signals, by solving the Fokker-Planck equation for particles, which apart from the permanent magnetic moment may also have an induced magnetic moment and shape anisotropy. Using the results from the Fokker-Planck calculations we fit ACS and OM measurements on two multi-core particle systems. The obtained fit parameters also describe the correlations between the magnetic moment and size of the particles. From such an analysis on a commercially available polydisperse multicore particle system with an average particle size of 80 nm, we find that the MNP magnetic moment is proportional to the square root of the hydrodynamic size.

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. Combined with duplex-specific nuclease-assisted target recycling, the proposed magnetic superstructures hold great promise in sensitive and rapid microRNA detection. The long single-stranded DNA produced by RCA serving as the scaffold of the core-satellite superstructure can be hydrolyzed by duplex-specific nuclease in the presence of target microRNA, resulting in a release of MNPs that can be quantified in an optomagnetic sensor. The proposed biosensor has a simple mix-separate-measure strategy. For let-7b detection, the proposed biosensor offers a wide linear detection range of approximately 5 orders of magnitude with a detection sensitivity of 1 fM. Moreover, it has the capability to discriminate single-nucleotide mismatches and to detect let-7b in cell extracts and serum, thus showing considerable potential for clinical applications.

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. In this study, we developed a new analytical method that combines magnetic molecularly imprinted polymer (MMIP)-based sample preparation with surface-enhanced Raman spectroscopy (SERS)-based detection for quantitative analysis of cloxacillin in pig serum. MMIP microspheres were synthesized using a core-shell technique. The large loading capacity and high selectivity of the MMIP microspheres enabled efficient extraction of cloxacillin, while the magnetically susceptible characteristics greatly simplified sample handling procedures. Low cost and robust SERS substrates consisting of vertical gold capped silicon nanopillars were fabricated and employed for the detection of cloxacillin. Quantitative SERS was achieved by normalizing signal intensities using an internal standard. By coherently combining MMIP extraction and silicon nanopillar-based SERS biosensor, good sensitivity toward cloxacillin was achieved. The detection limit was 7.8 pmol. Cloxacillin recoveries from spiked pig plasma samples were found to be more than 80%.

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. temperature at fixed salt concentration. The magnetoresistive sensor platform provided reliable results under varying temperature as well as salt concentration. The salt concentration melting curves were found to be more reliable than temperature melting curves. We performed a two-dimensional mapping of the melting profiles of a target to probes targeting its wild type (WT) and mutant type (MT) variants in the temperature-salt concentration plane. This map clearly showed a region of optimum ability to differentiate between the two variants. We finally demonstrated single nucleotide polymorphysm (SNP) genotyping using both denaturation methods on both separate sensors but also using a differential measurement on a single sensor. The results demonstrate that concentration melting provides an attractive alternative to temperature melting in on-chip DNA denaturation experiments and further show that the magnetoresistive platform is attractive due to its low cross-sensitivity to temperature and liquid composition.

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. We first show how the measurements of the optomagnetic signal magnitude at a low frequency vs. magnetic field amplitude can be used to determine the MNP moment. Subsequently, we use linear response theory to describe the dynamic non-equilibrium response of the MNP suspension at low magnetic field amplitudes and derive a link between optomagnetic measurements and magnetic AC susceptibility measurements. We demonstrate the presented methodology on two samples of commercially available multi-core MNPs. The results compare well with those obtained by dynamic light scattering, AC susceptibility and vibrating sample magnetometry measurements on the same samples when the different weighting of the particle size in the techniques is taken into account. The optomagnetic technique is simple, fast and does not require prior knowledge of the concentration of MNPs and it thus has the potential to be used as a routine tool for quality control of MNPs.

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. Target miRNAs are preserved in the cleaving reaction and can thereby trigger more cleavage and release of "satellites". For singleplex detection of let-7b, a linear detection range between 10 fM and 10 nM was observed, and a detection limit of 4.8 fM was obtained within a total assay time of 70 min. Multiplexing was achieved by releasing nanoparticles of different sizes in the presence of different miRNAs. The proposed method also has the advantages of single-nucleotide mismatch discrimination and the ability of quantification in a clinical sample matrix, thus holding great promise for miRNA routine multiplex diagnostics.

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. For the comparison we use the C-reactive protein (CRP) induced agglutination of identical samples of 100nm MNPs conjugated with CRP antibodies. Both methods detect agglutination as a shift to lower frequencies in measurements of the dynamics in response to an applied oscillating magnetic field. The magnetic susceptibility method probes the magnetic response whereas the optomagnetic technique probes the modulation of laser light transmitted through the sample. The two techniques provided highly correlated results upon agglutination when they measure the decrease of the signal from the individual MNPs (turn-off detection strategy), whereas the techniques provided different results, strongly depending on the read-out frequency, when detecting the signal due to MNP agglomerates (turn-on detection strategy). These observations are considered to be caused by differences in the volume-dependence of the magnetic and optical signals from agglomerates. The highest signal from agglomerates was found in the optomagnetic signal at low frequencies.

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. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25pM with the same sample-to-answer time (15min 30s) using the two differently sized beads for the two detection methods. In both cases a sample volume of only 10µl is required. The demonstrated automation, low sample-to-answer time and portability of both detection instruments as well as integration of the assay on a low-cost disc are important steps for the implementation of these as portable tools in an out-of-lab setting.

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. Hybridization of the DNA linker and the aptamer induces formation of MNP clusters. The target-to-aptamer binding on MNPs prior to the addition of linker-functionalized MNPs significantly hinders the hybridization reaction, thus reducing the degree of MNP clustering. The clustering state, which is thus related to the target concentration, is then quantitatively determined by an optomagnetic readout technique that provides the hydrodynamic size distribution of MNPs and their clusters. A commercial Blu-ray optical pickup unit is used for optical signal acquisition, which enables the establishment of a low-cost and miniaturized biosensing platform. Experimental results show that the degree of MNP clustering correlates well with the concentration of a target small molecule, adenosine triphosphate (ATP) in this work, in the range between 10µM and 10mM. This successful proof-of-concept indicates that our optomagnetic aptasensor can be further developed as a low-cost biosensing platform for detection of small molecule biomarkers in an out-of-lab setting.

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. Magnetic beads can be continuously moved across such arrays by combining the spatially periodic magnetic field from microstripes with a rotating external magnetic field. By measuring and modeling the magnetophoresis properties of thirteen different stripe designs, we study the effect of the stripe geometry on the magnetophoretic transport properties of the magnetic microbeads between the stripes. We show that a symmetric geometry with equal width of and spacing between the microstripes facilitates faster transportation and that the optimal period of the periodic stripe array is approximately three times the height of the bead center over the microstripes.

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. Consequently, the signal from multiple beads also depends on their locations. Thus, a given coverage of the functionalized area with magnetic beads does not result in a given detector response, except on the average, over many realizations of the same coverage. We present a systematic theoretical analysis of how this location-dependence affects the sensor response. The analysis is done for beads magnetized by a homogeneous in-plane magnetic field. We determine the expected value and standard deviation of the sensor response for a given coverage, as well as the accuracy and precision with which the coverage can be determined from a single sensor measurement. We show that statistical fluctuations between samples may reduce the sensitivity and dynamic range of a sensor significantly when the functionalized area is larger than the sensor area. Hence, the statistics of sampling is essential to sensor design. For illustration, we analyze three important published cases for which statistical fluctuations are dominant, significant, and insignificant, respectively.

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. The presented results are obtained from the four year EU NMP FP7 project, NanoMag, which is focused on standardization of analysis methods for magnetic nanoparticles.

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. We show that the signal from sensor stripes with a uniform protective coating, contrary to conventional wisdom in the field, is usually dominated by the contribution from magnetic labels between the sensor stripes rather than by the labels on top of the sensor stripes because these are at a lower height. We therefore propose a shift of paradigm to maximize the signal due to magnetic labels between sensor stripes. Guidelines for this optimization are provided and illustrated for an experimental case from the literature.