Magnetic particle actuation for functional biosensors

Molecular processes play a major role in the biology of the human body. As a consequence, molecular-level information can be very effctively used for medical diagnostics. In medical practice, samples of e.g. blood, urine, saliva, sputum, faeces or tissue are taken and investigated in specialized laboratories using a variety of biological tests. The tests can generally be separated into five process steps: (i) sample taking, (ii) sample preparation, (iii) specific recognition of the molecules of interest, (iv) transduction of the presence of the molecules into a measurable signal and (v) translation of the measured signal into a diagnostic parameter that can support the treatment of the patient. Particles with nanometer to micrometer sizes are widely used as carriers and labels in bio-analytical systems/assays. An important class of particles used in in-vitro diagnostics are so-called super-paramagnetic particles, which consist of magnetic nanoparticles embedded inside a non-magnetic matrix. The absence of magnetic material in biological samples allows a controlled application of magnetic fields. Super-paramagnetic particles are therefore powerful because they can be easily manipulated and reliably detected inside complex biological fluids. These properties are exploited in magnetic-label biosensors, which employ the magnetic particles as labels in order to measure the concentration of target molecules in a biological sample.In this thesis we investigate techniques for a novel generation of biosensors - called functional biosensors - in which the concentration as well as a functional property of biological molecules can be determined by controlled manipulation of the magnetic particles. We demonstrate real-time on-chip detection and manipulation of single super-paramagnetic particles in solution. The chip-based sensor contains micro fabricated on-chip current wires and giant magneto resistance (GMR) sensors. The current wires serve to apply force on the particles as well as to magnetize the particles for on-chip detection. By simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor, the sensitivity profile of the sensor was reconstructed and qualitatively understood from a single-dipole model. The manipulation of multiple particles in parallel combined with real-time detection of single particles opens the possibility to perform on-chip high-parallel assays with single-particle resolution. The drawback of on-chip magnetic actuation and detection is the limited amount of statistics since only a limited amount of particles (typically several dozens) can be used in a single experiment. To study a large number of particles (typically several hundreds) without hydrodynamic and magnetic particle-to-particle interactions, a magnetic tweezers setup is designed and built to apply translational pulling forces to magnetic particles. The magnetic tweezers setup is based on an electromagnet combined with an optical microscope for the detection of the particles. Using this setup the non-specific binding of protein coated particles to a glass substrate is measured for various buffer conditions. The increase of binding with increasing ionic strength is understood from the electrostatic interaction between the particles and the glass substrate. A complementary way to probe biological molecules or interactions is by applying a controlled torsion, i.e. a controlled rotation under well-defined torque. Particle based single-molecule experiments described in literature already indicate novel types of assays enabled by the application of rotation to biological molecules. Although the degree of rotation was known in these single-molecule experiments, the quantitative value of the applied torque was not controlled. In fact, it is a surprise that a torque can be applied because in an idealized super-paramagnetic particle, the angle difference between the induced magnetization and the applied magnetic field is zero and thus the torque should be zero as well. To answer the question which physical mechanism is responsible for torque generation, a rotating magnetic field was applied to single super-paramagnetic particles by on-chip current wires. We unraveled the mechanisms of torque generation by a comprehensive set of experiments at different field strengths and frequencies, including field frequencies many orders of magnitude higher than the particle rotation frequency. A quantitative model is developed which shows that at field frequencies below 10 Hz, the torque is due to a permanent magnetic moment in the particle of the order of 10¡15 Am2. At high frequencies (kHz - MHz), the torque results from a phase lag between the applied field and the induced magnetic moment, caused by the non-zero relaxation time of magnetic nanoparticles in the particle. A magnetic quadrupole setup is developed to upscale the rotation experiments to multiple particles in parallel. The advantage of the rotation experiments over the pulling experiments is that rotation experiments not only give information on dissociation but also on association processes. Using the quadrupole setup, the non-specific binding between protein coated particles and a glass substrate is measured for various buffer conditions. The increase of binding with increasing ionic strength and decreasing pH is understood from the electrostatic interaction between the particles and the glass substrate. When coating the glass substrate with bovine serum albumin (BSA), the non-specific binding of streptavidin coated particles is strongly reduced. Although the blocking effect of BSA is not fully understood, our measurements clearly show the feasibility of rotational excitation of particles to probe molecular interactions. Finally, we studied the feasibility of rotational actuation of magnetic particles to measure the torsional stiffness of a biological system with a length scale of several tens of nanometers. As a model system we used protein G on the particles that binds selectively to the crystallisable part of the IgG antibody that is physically adsorbed on a polystyrene substrate. The angular orientation of the particles that are bound to the substrate show an oscillating behavior upon applying a rotating magnetic field. The amplitude of this oscillation decreases with increasing anti-body concentration, which we attribute to the formation of multiple bonds between the particle and the substrate. By evaluating the details of the oscillatory behavior, we found a lower limit of the torsional modulus of the IgG-protein G complex of 6¢10¡26 Nm2. The torsional modulus is two orders of magnitude larger than typical values found in literature for DNA strands. A difference in torsional modulus is expected from the structural properties of the molecules i.e. DNA is a long and flexible chain-like molecule whereas the protein G and IgG molecules are more globular due to the folding of the molecule.