Planar Hall Effect Sensors for Biodetection

The work presented in this thesis contributes to the development of diagnostic tools also suited for use at the point-of-care. These devices may help to spread part of the biochemical analysis work from centralised laboratories to doctors' offices and in some cases to patients' homes. Point-of-care devices can effectively reduce the time for the analysis and the costs that are related to a delay in the diagnosis.

Many technologies are available for biosensing devices. In this work, we study and employ magnetic biosensing on magnetoresistive sensors. For magnetic biodetection magnetic beads are used as labels and planar Hall effect bridge (PHEB) magnetic field sensor as readout for the beads. The choice of magnetic beads as label is motivated by the lack of virtually any magnetic background from biological samples.

Moreover, magnetic beads can be manipulated via an external magnetic field and be employed for sample preparation in a lab-on-a-chip device. The PHEB sensors are formed by four magnetoresistive arms in a Wheatstone bridge geometry. In this thesis two different sensor geometries are used. In the first geometry (PHEB), the magnetic bead signals from the sensor arms are additive.

In the second geometry (dPHEB) half of the sensor is used as a local negative reference to subtract the background signal from magnetic beads in suspension. In all applications below, the magnetic beads are magnetised using the magnetic field due to the bias current passed through the sensor, i.e., no external magnetic fields are needed for the measurements.

The two sensor geometries are employed for two different types of biodetection. The PHEB senor is used for volume-based biodetection, where the effective hydrodynamic size of magnetic beads is increased upon binding to the analyte. The change affects the rotation response (Brownian relaxation) of the magnetic beads, which can be measured through magnetic AC susceptometry.

The dPHEB sensor is used for surface-based biodetection, where the analytes bind to capture probes immobilised on the sensor surface and allow for ligation of magnetic beads to the sensor surface. In this thesis, the theoretical PHEB and dPHEB sensor signals are derived. Also, the effects of shape anisotropy on the sensor output are considered.

We introduce Brownian relaxation of magnetic beads and chip-based magnetic susceptibility measurements using PHEB magnetic sensors. The effects of temperature upon the magnetic properties of the sensor stack and signal output are studied. A method is presented to discriminate the reversible and irreversible effects of temperature.

It is studied how a low-temperature annealing procedure can mitigate these effects. Experimental results show that it is impossible to avoid irreversible changes in the sensor, and thus we imply the need for a reference to correct for temperature effects in the sensor sensitivity. This reference is necessary whenever a biosensing experiment employs sizeable temperature variations.

PHEB sensors are used to for AC susceptometry measurements for volume-based bioassays. The setup allows to investigate the sensor responce as function of frequency in the range from DC to 5 MHz thus allowing for determining the hydrodynamic size of beads with nominal diameters from 10 nm to 250 nm and down to a concentration of 64 μg=mL (for 40 nm beads).

Beads with a diameter of 80 nm are found to be best suited for biodetection. Time-domain Brownian relaxation measurement are also demonstrated with PHEB sensors. The advantage of the time-domain technique is it lead to faster measurements, but at the cost reduced signal to noise. This method is compared to frequency-domain measurements in detection of biotin-conjugated bovine serum albumin (bBSA) and it is found that both techniques can detect bBSA in the nM range.

Furthermore, frequency-domain measurements are used for the detection of rolling circle amplification (RCA) products. RCA coils with a diameter of ≈ 1 μm are produced by RCA upon recognition of Vibrio cholerae and of Bacillus globigii spores and detected by incubation with properly functionalised magnetic beads.

Binding of the magnetic beads to the RCA coils slow their dynamics, which can be observed as an increase in the hydrodynamic diameter. The assay can detect down to 4 pM of DNA coils and the whole assay can detect down to 500 spores in the starting sample. These figures compare favourably to those obtained using a commercial susceptometer.

Differential PHEB sensors (dPHEBs) are used for a surface-based assay to detect DNA hybridisation in a sandwich assay. Biotinylated DNA target binds to surface tethered capture probes and allows for the ligation of 50 nm streptavidin coated magnetic beads to the sensor surface. The sensor can detect DNA to a concentration of 156 pM upon 60 min hybridisation.

The setup is capable of measuring DNA hybridisation in real-time, in a background of suspended magnetic beads. This characteristic is employed in single nucleotide polymorphism (SNP) genotyping, where the denaturation of DNA is monitored in real-time upon washing with a stringency buffer. The sensor setup includes temperature control and a fluidic system capable of generating both temperature and concentration gradients over the sensor surface.

The temperature and buffer concentration can be varied in order to perform denaturation analysis of the DNA hybrids. In this thesis, this kind assay is tested with temperature varying from 20ºC to 70ºC and Na+ ionic strength from 400 mM to 4 mM. This demonstrates the exibility of the presented sensing devices.