Rotational actuation of magnetic nanoparticle clusters for solution-based biosensing

Abstract

In-vitro diagnostics deals with the quantification of specific molecules or cells in a sample outside a living organism in order to highlight a particular physiological or pathological state. Samples are most easily taken from body fluids such as blood, saliva or urine. Detectable molecules with diagnostic value are called biomarkers. Examples of protein biomarkers are cardiac troponin (cTn) for the diagnosis of heart infarction or prostate specific antigen (PSA) for prostate cancer. Commonly antibody coated micro- or nano-particles are used to capture and extract biomarker molecules from a sample, because antibodies enable specific capture and because particles have a high surface to volume ratio. Magnetic particles have as additional advantage that they can be reliably manipulated by magnetic fields because biological materials are hardly magnetic. As a consequence magnetic particles are widely applied in in-vitro diagnostic assays. The detection of biomarkers in biological samples is complex due to the low concentrations of the biomarkers and the complicated nature of the sample matrix. Biosensing relies on specific interactions, but non-specific interactions can easily occur with abundantly available endogenous molecules. Traditionally, biological assays use series of different process steps in order to achieve good detection sensitivity and specificity. One of the aims of this dissertation is to explore the feasibility of a one-step technology based on the rotation of clusters of magnetic nanoparticles and to demonstrate rapid and sensitive detection of protein biomarkers directly in blood plasma. The first chapter of this dissertation provides an overview of the research project and describes the state of art in the field of magnetic clusterassays, which are based on biomarker-induced binding between magnetic particles. Our literature search highlights the lack of a background-free detection principle for magnetic cluster assays. This thesis presents a novel background-free detection principle based on frequency-controlled rotation. The rotational dynamics of magnetic particle clusters shows a critical frequency. The critical frequency is the maximum frequency at which the clusters can rotate synchronously with the external field. We model the rotational dynamics of individual two-particle clusters by deriving the equations of motion in an external rotating field (chapter 3). The theoretical model is based on the microscopic properties of the particles, in particular the magnetic susceptibility, a permanent magnetic moment of each cluster, and the size of the particles. We demonstrate that the rotational dynamics is most reproducible when the magnetic interaction is dominated by interactions between induced magnetic dipoles. In chapter 4 we describe how frequency-controlled rotation and optical scattering give a background-free detection principle. Clusters of magnetic particles have a uniaxial symmetry, in contrast to single particles which are essentially spherical. Rotating clusters expose an angle-dependent cross-section to an incoming light beam and thereby modulate the intensity of scattered light. Single particles hardly rotate and hardly generate a modulation of light. We demonstrate that the frequency-dependence of the optical modulation signal accurately reveals the number of clusters in solution and the value of the critical frequency. The method can be applied to an ensemble of nanoparticles and allows a measurement of cluster size and the magnetic susceptibility. The population of clusters can be modified by the addition of clusterinducing biological molecules. We have studied molecule-induced nanoparticle binding using biotinylated Bovine Serum Albumin (bBSA) and streptavidincoated nanoparticles, in buffer and in human plasma. Detection is performed by magnetic rotation and optical scattering, with an optical probing volume of approximately 1 nL. Using a two-step assay with a total assay time of less than 3 minutes, we demonstrate dose-response curves with a detection limit of 0.4 pM bBSA in buffer and 5 pM bBSA in human plasma. To achieve a one-step homogeneous assay format, the non-specific interactions need to be investigated and reduced (chapter 5). We analyze the effect of pulsed rotating magnetic fields on the amount of formed clusters, both specifically and non-specifically. We identify an optimum in the time between pulses and relate it to the diffusive properties of the nanoparticles. We hypothesize that the field-on period generates a concentration of particles, while during the field-off period the particles randomize their orientations which enhances the biomarker-induced binding, and the particles have a lower non-specific binding due to a reduced contact time. To further reduce the nonspecific interactions, we modify the surface molecular architecture of the nanoparticles by means of a double layer of polymer linkers. Antibodies are linked to the particles by a primary layer giving good molecular mobility. Using the antibody as anchoring point, we attach a secondary layer of linkers in order to further shield the particles without hindering the possibility for specific binding. We demonstrate a one-step homogeneous PSA immunoassay directly in human plasma, in a total assay time of less than 15 minutes. We obtain doseresponse curves with detection limit of 0.5 pM PSA. We quantitatively explain the dose-response curves with a model based on discrete binding of biomarker molecules onto the nanoparticles, which allows us to independently and quantitatively extract the reaction parameters for the binding of biomarker molecules onto the nanoparticles and for the biomarker-induced binding between nanoparticles. When performing an assay over several orders of magnitude in biomarker concentration, clusters of more than two particles appear. Chapter 6 presents an extension to the theoretical model presented in Chapter 3 and its experimental verification for clusters of different sizes. In conclusion, we have demonstrated a one-step homogeneous immunoassay by means of a new and versatile bionanotechnology based on rotating magnetic nanoparticles, which will enable a wide range of further studies in optics, magnetics, biophysics and biosensing.