The use of magnetic particles in biosensing is advantageous for transport of target molecules in the device, for assay integration, and for labeled detection. The particles generally have a size between 100 nm and 3 ¿m and are of a superparamagnetic nature, being composed of thousands of iron oxide grains in a polymer matrix. In this thesis we describe a series of detailed microscopy studies of magnetic particles near to and coupled to a biosensor surface, in order to characterize their dynamic behavior and their magnetic properties. The first part of the thesis deals with the use of integrated microscopic current wires to study and manipulate unbound particles on a chip surface. The magnetic properties of individual particles are characterized in magnetic fields below 10 mT using on-chip magnetophoretic analysis and on-chip Brownian motion analysis. In magnetophoretic analysis, the volume susceptibility of 1 µm particles is determined by optically measuring the speed of particles moving between two current wires. The analysis reveals distinct differences in volume susceptibilities of particles with the same outer diameter. In addition to DC magnetic fields, also AC magnetic fields are applied, showing a decrease in particle susceptibility for increasing field frequencies. To reduce the hydrodynamic perturbation by the surface in on-chip magnetophoretic analysis, we present a chip design in which a particle can move back and forth in the channel between two large wires. In Brownian motion analysis, small particles of 150 to 450 nm are trapped in a tunable magnetic potential well above an integrated current wire. The histogram of two-dimensional particle positions reveals the strength of the particle magnetization. Using straight current wires, we demonstrate differences in bead susceptibility of an order of magnitude and differences in volume susceptibility of more than a factor of two. By using wires with surface barriers and wires with a tapered shape, the accuracy of the susceptibility determination is improved to better than 10%. We also show that combining a tapered wire with an external uniform field can give additional information on particle properties such as anisotropy or a small permanent magnetic moment. The second part of the thesis describes a study of the dynamics of particles that are biologically bound to a sensor surface. We show that an optical eva-nescent field can be used to study the thermal out-of-plane motion of bound particles with nanometer resolution, because the scattered light intensity of the particles depends on the height above the surface. By using a biological tether of known length (dsDNA of 290 bp/99 nm) we show that height variations can be quantitatively determined. We demonstrate that the accuracy of the height determination depends on the properties of the used particles, e.g. the shape, smoothness, and internal structure. Optimal results are found for polystyrene particles and magnetic particles that are smooth and spherical. Next, we show that the bond between a particle and a surface can be characterized by measuring the three-dimensional thermal mobility of the particle. As a model analyte we use four different lengths of dsDNA to bind the particle to the surface (590 bp/201 nm, 290 bp/99 nm, 141 bp/48 nm and 105 bp/36 nm). Plots of the minimum height, average height and maximum height as function of the in plane particle position reflect the differences in bond length, bond flexibility and bond orientation of the different DNA molecules. We also analyze ensembles of particles bound to the four DNA lengths and show that the height displacement is at maximum equal to the bond length, but large variations between particles are observed, which we attribute to non-specific interactions. The mobility of a bound particle can be influenced by external forces. We show that a magnetic force towards the surface brings bound particles on average closer to the surface. However, a magnetic force away from the surface does not always brings bound particles away from the surface, but can lead to medium or minimum heights. This can be explained by magnetic anisotropy in the particles, leading to particle alignment and subsequent height reduction. We describe a model that shows how particle alignment due to magnetic anisotropy brings bound particles into well-defined three-dimensional positions. Although particle alignment may interfere with the desired magnetic manipulation, it can also lead to additional information on for example rotational freedom of the bond and bond flexibility. Finally, we show that a bound particle can also be pulled away from the surface by an electrostatic force induced by replacing the buffer with a buffer of low ionic strength. We show that the height modulation is dependent on both the analyte length and the ionic strength, and describe a quantitative model to account for the measured height displacements for particles bound to four different lengths of DNA. The improved knowledge on the magnetic properties of individual particles and the mobility of individual particles bound to a biosensor surface, resulting from the experiments described in this thesis, may lead to improved detection limits and enhanced specificity in future biosensors.
|Qualification||Doctor of Philosophy|
|Award date||14 Jun 2010|
|Place of Publication||Eindhoven|
|Publication status||Published - 2010|