Giovanni Rizzi

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. Spectra of the 2nd harmonic photo detector signal vs. the frequency of the applied magnetic field show a characteristic peak due to freely rotating magnetic nanobeads. Beads bound to ~1 μm coils of DNA formed off-chip by padlock probe recognition and rolling circle amplification show a different dynamics and the intensity of the characteristic peak decreases. We have determined the optimum magnetic bead concentration to 0.1mg/mL and have measured the response vs. concentration of DNA coils formed from Escherichia Coli. We have found a limit of detection of 10 pM and a dynamic range of about two orders of magnitude, which is comparable to the performance obtained using costly and bulky laboratory equipment. The presented device leverages on the advanced but low-cost technology of Blu-ray OPUs to provide a low-cost and high-performance magnetic bead-based readout of homogeneous bioassays. The device is highly flexible and we have demonstrated its use on microfluidic chambers in a disc with a thickness compatible with current optical media mass-production facilities.

Measurements of Brownian relaxation of magnetic nanobeads using planar Hall effect bridge sensors.

We compare measurements of the Brownian relaxation response of magnetic nanobeads in suspension using planar Hall effect sensors of cross geometry and a newly proposed bridge geometry. We find that the bridge sensor yields six times as large signals as the cross sensor, which results in a more accurate determination of the hydrodynamic size of the magnetic nanobeads. Finally, the bridge sensor has successfully been used to measure the change in dynamic magnetic response when rolling circle amplified DNA molecules are bound to the magnetic nanobeads. The change is validated by measurements performed in a commercial AC susceptometer. The presented bridge sensor is, thus, a promising component in future lab-on-a-chip biosensors for detection of clinically relevant analytes, including bacterial genomic DNA and proteins.

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.

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. The sensors are based on a new geometry for biological sensing that detects the difference between the amount of beads bound to a sensing pad and a local integrated negative reference pad. The magnetic beads are magnetised by the magnetic field arising from the sensor bias current such that no external magnetic fields are needed. The sensors are integrated in a microfluidic system with temperature control. The local negative reference integrated in the sensor geometry efficiently compensates for sensor offsets, external magnetic fields and a uniform background of magnetic beads, which enables real-time quantification of the specific binding of magnetic beads to the sensor surface under varying experimental conditions.

Planar Hall effect bridge geometries optimized for magnetic bead detection

Novel designs of planar Hall effect bridge sensors optimized for magnetic bead detection are presented and characterized. By constructing the sensor geometries appropriately, the sensors can be tailored to be sensitive to an external magnetic field, the magnetic field due to beads being magnetized by the sensor self-field or a combination thereof. The sensors can be made nominally insensitive to small external magnetic fields, while being maximally sensitive to magnetic beads, magnetized by the sensor self-field. Thus, the sensor designs can be tailored towards specific applications with minimal influence of external variables. Three different sensor designs are analyzed theoretically. To experimentally validate the theoretical signals, two sets of measurements are performed. First, the sensor signals are characterized as function of an externally applied magnetic field. Then, measurements of the dynamic magnetic response of suspensions of magnetic beads with a nominal diameter of 80 nm are performed. Fur...

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.

On-chip measurements of Brownian relaxation of magnetic beads with diameters from 10 nm to 250 nm

We demonstrate the use of planar Hall effect magnetoresistive sensors for AC susceptibility measurements of magnetic beads with frequencies ranging from DC to 1 MHz. This wide frequency range allows for measuring Brownian relaxation of magnetic beads with diameters ranging from 10 nm to 250 nm. Brownian relaxation is measured for six different magnetic bead types and their hydrodynamic diameters are determined. The hydrodynamic diameters are found to be within 40% of the nominal bead diameters. We discuss the applicability of the different bead types for volume-based biosensing with respect to sedimentation, magnetic trapping, and signal per bead. Among the investigated beads, we conclude that the beads with a nominal diameter of 80 nm are best suited for future on-chip volume-based biosensing experiments using planar Hall effect sensors.

On-chip measurements of Brownian relaxation vs. concentration of 40 nm magnetic beads

We present on-chip Brownian relaxation measurements on a logarithmic dilution series of 40 nm beads dispersed in water with bead concentrations between 16 μg/ml and 4000 μg/ml. The measurements are performed using a planar Hall effect bridge sensor at frequencies up to 1 MHz. No external fields are needed as the beads are magnetized by the field generated by the applied sensor bias current. We show that the Brownian relaxation frequency can be extracted from fitting the Cole-Cole model to measurements for bead concentrations of 64 μg/ml or higher and that the measured dynamic magnetic response is proportional to the bead concentration. For bead concentrations higher than or equal to 500 μg/ml, we extract a hydrodynamic diameter of 47(1) nm for the beads, which is close to the nominal bead size of 40 nm. Furthermore, we study the signal vs. bead concentration at a fixed frequency close to the Brownian relaxation peak and find that the signal from bead suspensions with concentrations down to 16 μg/ml can be...

Planar Hall effect bridge sensors with NiFe/Cu/IrMn stack optimized for self-field magnetic bead detection

The stack composition in trilayer Planar Hall effect bridge sensors is investigated experimentally to identify the optimal stack for magnetic bead detection using the sensor self-field. The sensors were fabricated using exchange-biased stacks Ni80Fe20(tFM)/Cu(tCu)/Mn80Ir20(10 nm) with tFM = 10, 20, and 30 nm, and 0 ≤ tCu ≤ 0.6 nm. The sensors were characterized by magnetic hysteresis measurements, by measurements of the sensor response vs. applied field, and by measurements of the sensor response to a suspension of magnetic beads magnetized by the sensor self-field due to the sensor bias current. The exchange bias field was found to decay exponentially with tCu and inversely with tFM. The reduced exchange field for larger values of tFM and tCu resulted in higher sensitivities to both magnetic fields and magnetic beads. We argue that the maximum magnetic bead signal is limited by Joule heating of the sensors and, thus, that the magnetic stacks should be compared at constant power consumption. For a fixed s...

Comment on “Planar Hall resistance ring sensor based on NiFe/Cu/IrMn trilayer structure” [J. Appl. Phys. 113, 063903 (2013)]

[J. Appl. Phys. 113, 063903 (2013)] DTU Orbit (26/01/2019) Comment on “Planar Hall resistance ring sensor based on NiFe/Cu/IrMn trilayer structure” [J. Appl. Phys. 113, 063903 (2013)] In a recent paper, Sinha et al. compared sensitivities of planar Hall effect sensors with different geometries that are all based on the anisotropic magnetoresistance of permalloy. They write that the sensitivity of a planar Hall effect sensor with a ring geometry is a factor of √2 larger than the sensitivity of the so-called planar Hall effect bridge (PHEB) sensor of equal size. Osterberg et al do not agree on the signal calculation for a ring sensor derived by Sinha et al. and claim that this adversely affects the results.