Retrofittable plug-flow reactor for in situ high-temperature vibrating sample magnetometry with well-controlled gas atmospheres.

We have developed an in situ sample-holder-akin to a quartz-based plug-flow reactor-for vibrating sample magnetometry (VSM) in gas-controlled environments at ambient pressure and temperatures up to ∼1000 °C. The holder matches onto a specific type of vibrating sample magnetometer (Lake Shore model 7404-S), but the principles are applicable to other types of VSM. The holder has been tested on powder samples of Co particles on a MgAl2O4 support in both reducing and oxidizing atmospheres. The results show control of gas composition and sample reduction/oxidation. In comparison with conventional sample cups, the in situ holder shows a similar measurement sensitivity but a better repeatability due to the well-controlled gas atmosphere. Moreover, the in situ holder uses a closed gas tubing system such that the active gas only passes by the sample and it is not in contact with the VSM and oven parts. At the outlet, the gas can be collected for analysis and safe handling.

3ω correction method for eliminating resistance measurement error due to Joule heating.

Electrical four-terminal sensing at (sub-)micrometer scales enables the characterization of key electromagnetic properties within the semiconductor industry, including materials' resistivity, Hall mobility/carrier density, and magnetoresistance. However, as devices' critical dimensions continue to shrink, significant over/underestimation of properties due to a by-product Joule heating of the probed volume becomes increasingly common. Here, we demonstrate how self-heating effects can be quantified and compensated for via 3ω signals to yield zero-current transfer resistance. Under further assumptions, these signals can be used to characterize selected thermal properties of the probed volume, such as the temperature coefficient of resistance and/or the Seebeck coefficient.

Effective electrical resistivity in a square array of oriented square inclusions.

The continuing miniaturization of optoelectronic devices, alongside the rise of electromagnetic metamaterials, poses an ongoing challenge to nanofabrication. With the increasing impracticality of quality control at a single-feature (-device) resolution, there is an increasing demand for array-based metrologies, where compliance to specifications can be monitored via signals arising from a multitude of features (devices). To this end, a square grid with quadratic sub-features is amongst the more common designs in nanotechnology (e.g. nanofishnets, nanoholes, nanopyramids, µLED arrays etc). The electrical resistivity of such a quadratic grid may be essential to its functionality; it can also be used to characterize the critical dimensions of the periodic features. While the problem of the effective electrical resistivity ρ eff of a thin sheet with resistivity ρ 1, hosting a doubly-periodic array of oriented square inclusions with resistivity ρ 2, has been treated before (Obnosov 1999 SIAM J. Appl. Math. 59 1267-87), a closed-form solution has been found for only one case, where the inclusion occupies c = 1/4 of the unit cell. Here we combine first-principle approximations, numerical modeling, and mathematical analysis to generalize ρ eff for an arbitrary inclusion size (0 < c < 1). We find that in the range 0.01 ≤ c ≤ 0.99, ρ eff may be approximated (to within <0.3% error with respect to finite element simulations) by: [Formula: see text] [Formula: see text] whereby at the limiting cases of c â†’ 0 and c â†’ 1, α approaches asymptotic values of α = 2.039 and α = 1/c - 1, respectively. The applicability of the approximation to considerably more complex structures, such as recursively-nested inclusions and/or nonplanar topologies, is demonstrated and discussed. While certainly not limited to, the theory is examined from within the scope of micro four-point probe (M4PP) metrology, which currently lacks data reduction schemes for periodic materials whose cell is smaller than the typical µm-scale M4PP footprint.

Automated on-chip analysis of tuberculosis drug-resistance mutation with integrated DNA ligation and amplification.

Detection of a single base mutation in Mycobacterium tuberculosis DNA can provide fast and highly specific diagnosis of antibiotic-resistant tuberculosis. Mutation-specific ligation of padlock probes (PLPs) on the target followed by rolling circle amplification (RCA) is highly specific, but challenging to integrate in a simple microfluidic device due to the low temperature stability of the phi29 polymerase and the interference of phi29 with the PLP annealing and ligation. Here, we utilized the higher operation temperature and temperature stability of Equiphi29 polymerase to simplify the integration of the PLP ligation and RCA steps of an RCA assay in two different strategies performed at uniform temperature. In strategy I, PLP annealing took place off-chip and the PLP ligation and RCA were performed in one pot and the two reactions were clocked by a change of the temperature. For a total assay time of about 1.5 h, we obtained a limit of detection of 2 pM. In strategy II, the DNA ligation mixture and the RCA mixture were separated into two chambers on a microfluidic disc. After on-disc PLP annealing and ligation, the disc was spun to mix reagents and initiate RCA. For a total assay time of about 2 h, we obtained a limit of detection of 5 pM. Graphical abstract.

Optomagnetic Detection of Rolling Circle Amplification Products.

Rolling circle amplification (RCA) of a synthetic nucleic acid target is detected using magnetic nanoparticles (MNPs) combined with an optomagnetic (OM) readout. Two RCA assays are developed with on-chip detection of rolling circle products (RCPs) either at end-point where MNPs are mixed with the sample after completion of RCA or in real time where MNPs are mixed with the sample during RCA. The plastic chip acts as a cuvette, which is positioned in a setup integrated with temperature control and simultaneous detection of four parallel DNA hybridization reactions between functionalized MNPs and products of DNA amplification. The OM technique probes the small-angle rotation of MNPs bearing oligonucleotide probes complementary to the repeated nucleotide sequence of the RCPs. This rotation is restricted when MNPs bind to RCPs, which can be observed as a turn-off of the signal from MNPs that are free to rotate. The amount of MNPs bound to RCPs is found to increase in response to the amplification time as well as in response to the synthetic DNA target concentration (2-40 pM dynamic range). We report OM real-time results obtained with MNPs present during RCA and compare to relevant end-point OM results for RCPs generated for different RCA times. The real-time approach avoids opening of tubes post-RCA and thus reduces risk of lab contamination with amplification products without compromising the sensitivity and dynamic range of the assay.

Dual-Function Cobalt-Nickel Nanoparticles Tailored for High-Temperature Induction-Heated Steam Methane Reforming.

The tailored chemical synthesis of binary and ternary alloy nanoparticles with a uniform elemental composition is presented. Their dual use as magnetic susceptors for induction heating and catalytic agent for steam reforming of methane to produce hydrogen at temperatures near and above 800 °C is demonstrated. The heating and catalytic performance of two chemically synthesized samples of CoNi and Cu⊂CoNi are compared and held against a traditional Ni-based reforming catalyst. The structural, magnetic, and catalytic properties of the samples were characterized by X-ray diffraction, elemental analysis, magnetometry, and reactivity measurements. For induction-heated catalysts, the conversion rate of methane is limited by chemical reactivity, as opposed to the case of traditional externally heated reformers where heat transport limitations are the limiting factor. Catalyst production by the synthetic route allows controlled doping with miniscule concentrations of auxiliary metals.

Sequence-specific validation of LAMP amplicons in real-time optomagnetic detection of Dengue serotype 2 synthetic DNA.

We report on an optomagnetic technique optimised for real-time molecular detection of Dengue fever virus under ideal as well as non-ideal laboratory conditions using two different detection approaches. The first approach is based on the detection of the hydrodynamic volume of streptavidin coated magnetic nanoparticles attached to biotinylated LAMP amplicons. We demonstrate detection of sub-femtomolar Dengue DNA target concentrations in the ideal contamination-free lab environment within 20 min. The second detection approach is based on sequence-specific binding of functionalised magnetic nanoparticles to loops of LAMP amplicons. Melting studies reveal that true positive and spurious amplicons have different melting points and this allows us to discriminate between them. This is found to be in a good agreement with subsequent studies on real-time sequence-specific discrimination of LAMP amplicons. The specific binding causes clustering of magnetic nanoparticles via binding to multiple sites (loops) emerging in the elongation phase of LAMP. Formation of nanoclusters is monitored via the depletion of the optomagnetic signal due to free nanoparticles. After sequence-specific validation, we claim detection of down to 100 fM of Dengue target after 20 min of LAMP with a contamination background.

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

Optomagnetic detection of DNA triplex nanoswitches.

We report on optomagnetic dose-dependent detection of DNA triplex-mediated and pH-switchable clusters of functionalised magnetic nanoparticles.

Denaturation strategies for detection of double stranded PCR products on GMR magnetic biosensor array.

Microarrays and other surface-based nucleic acid detection schemes rely on the hybridization of the target to surface-bound detection probes. We present the first comparison of two strategies to detect DNA using a giant magnetoresistive (GMR) biosensor platform starting from an initially double-stranded DNA target. The target strand of interest is biotinylated and detected by the GMR sensor by linking streptavidin magnetic nanoparticles (MNPs) to the sensor surface. The sensor platform has a dynamic detection range from 40pM to 40nM with highly reproducible results and is used to monitor real-time binding signals. The first strategy, using off-chip heat denaturation followed by sequential on-chip incubation of the nucleic acids and MNPs, produces a signal that stabilizes quickly but the signal magnitude is reduced due to competitive rehybridization of the target in solution. The second strategy, using magnetic capture of the double-stranded product followed by denaturing, produces a higher signal but the signal increase is limited by diffusion of the MNPs. Our results show that both strategies give highly reproducible results but that the signal obtained using magnetic capture is higher and insensitive to rehybridization.

Quantification of NS1 dengue biomarker in serum via optomagnetic nanocluster detection.

Dengue is a tropical vector-borne disease without cure or vaccine that progressively spreads into regions with temperate climates. Diagnostic tools amenable to resource-limited settings would be highly valuable for epidemiologic control and containment during outbreaks. Here, we present a novel low-cost automated biosensing platform for detection of dengue fever biomarker NS1 and demonstrate it on NS1 spiked in human serum. Magnetic nanoparticles (MNPs) are coated with high-affinity monoclonal antibodies against NS1 via bio-orthogonal Cu-free 'click' chemistry on an anti-fouling surface molecular architecture. The presence of the target antigen NS1 triggers MNP agglutination and the formation of nanoclusters with rapid kinetics enhanced by external magnetic actuation. The amount and size of the nanoclusters correlate with the target concentration and can be quantified using an optomagnetic readout method. The resulting automated dengue fever assay takes just 8 minutes, requires 6 µL of serum sample and shows a limit of detection of 25 ng/mL with an upper detection range of 20000 ng/mL. The technology holds a great potential to be applied to NS1 detection in patient samples. As the assay is implemented on a low-cost microfluidic disc the platform is suited for further expansion to multiplexed detection of a wide panel of biomarkers.

The copper binding properties of metformin--QCM-D, XPS and nanobead agglomeration.

Study of the copper binding properties of metformin is important for revealing its mechanism of action as a first-line type-2 diabetes drug. A quantitative investigation of interactions between metformin and L-cysteine-copper complexes was performed. The results suggest that metformin could interact with biological copper, which plays a key role in mitochondrial function.

Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples.

We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.

Novel readout method for molecular diagnostic assays based on optical measurements of magnetic nanobead dynamics.

We demonstrate detection of DNA coils formed from a Vibrio cholerae DNA target at picomolar concentrations using a novel optomagnetic approach exploiting the dynamic behavior and optical anisotropy of magnetic nanobead (MNB) assemblies. We establish that the complex second harmonic optical transmission spectra of MNB suspensions measured upon application of a weak uniaxial AC magnetic field correlate well with the rotation dynamics of the individual MNBs. Adding a target analyte to the solution leads to the formation of permanent MNB clusters, namely, to the suppression of the dynamic MNB behavior. We prove that the optical transmission spectra are highly sensitive to the formation of permanent MNB clusters and, thereby to the target analyte concentration. As a specific clinically relevant diagnostic case, we detect DNA coils formed via padlock probe recognition and isothermal rolling circle amplification and benchmark against a commercial equipment. The results demonstrate the fast optomagnetic readout of rolling circle products from bacterial DNA utilizing the dynamic properties of MNBs in a miniaturized and low-cost platform requiring only a transparent window in the chip.

Turn-on optomagnetic bacterial DNA sequence detection using volume-amplified magnetic nanobeads.

Detection of a Vibrio cholerae DNA-sequence using an optomagnetic read-out exploiting the dynamic behavior of magnetic nanobeads along with two turn-on data analysis approaches is demonstrated. The optomagnetic method uses a weak uniaxial AC magnetic field of varying frequency applied perpendicular to the optical path and measures the modulation of laser light passing through a cuvette containing the sample with oligonucleotide-tagged magnetic beads and macromolecular coils of single-stranded DNA. The DNA coils are formed upon a padlock probe ligation followed by rolling circle amplification (RCA). The presence of target gives rise to a change of the 2nd harmonic component, V2=V2(')+iV2(''), of the transmitted light. We demonstrate that by using the phase angle ξ defined as ξ=arctanV2(')/V2('') in the low-frequency region we obtain a limit of detection of 10pM for an RCA time of only 20min corresponding to a total assay time of 60min. Moreover, we show that the approach based on ξ is significantly more robust than the analysis based on a turn-off of the signal due to free magnetic nanobeads used in previous work (Donolato et al., submitted for publication), where a limit of detection of 10pM was obtained for an RCA time of 60min. The increased robustness and the reduction in total assay time constitute significant steps towards the realization of a low-cost, rapid and sensitive biosensor platform suitable for pathogen detection in both human and veterinary medicine settings.

On-chip detection of rolling circle amplified DNA molecules from Bacillus globigii spores and Vibrio cholerae.

For the first time DNA coils formed by rolling circle amplification are quantified on-chip by Brownian relaxation measurements on magnetic nanobeads using a magnetoresistive sensor. No external magnetic fields are required besides the magnetic field arising from the current through the sensor, which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chip-based readout is an important step towards the realization of field tests based on rolling circle amplification molecular analyses.