Multivalent weak interactions enhance selectivity of interparticle binding.

Targeted drug delivery critically depends on the binding selectivity of cargo-transporting colloidal particles. Extensive theoretical work has shown that two factors are necessary to achieve high selectivity for a threshold receptor density: multivalency and weak interactions. Here, we study a model system of DNA-coated particles with multivalent and weak interactions that mimics ligand-receptor interactions between particles and cells. Using an optomagnetic cluster experiment, particle aggregation rates are measured as a function of ligand and receptor densities. The measured aggregation rates show that the binding becomes more selective for shorter DNA ligand-receptor pairs, proving that multivalent weak interactions lead to enhanced selectivity in interparticle binding. Simulations confirm the experimental findings and show the role of ligand-receptor dissociation in the selectivity of the weak multivalent binding.

Rate of Dimer Formation in Stable Colloidal Solutions Quantified Using an Attractive Interparticle Force.

We describe an optomagnetic cluster experiment to understand and control the interactions between particles over a wide range of time scales. Aggregation is studied by magnetically attracting particles into dimers and by quantifying the number of dimers that become chemically bound within a certain time interval. An optomagnetic readout based on light scattering of rotating clusters is used to measure dimer formation rates. Magnetic field settings, that is, field rotation frequency, field amplitude, and on- and off-times, have been optimized to independently measure both the magnetically induced dimers and chemically bound dimers. The chemical aggregation rate is quantified in solutions with different pH and ionic strengths. The measured rates are extrapolated to effective dimer formation rates in the absence of force, showing that aggregation rates can be quantified over several orders of magnitude, including conditions of very low chemical reactivity.

Single-Dimer Formation Rate Reveals Heterogeneous Particle Surface Reactivity.

Biofunctionalized micro- and nanoparticles are important for a wide range of applications, but methodologies to measure, modulate, and model interactions between individual particles are scarce. Here, we describe a technique to measure the aggregation rate of two particles to a single dimer, by recording the trajectory that a particle follows on the surface of another particle as a function of time. The trajectory and the interparticle potential are controlled by a magnetic field. Particles were studied with and without conjugated antibodies in a wide range of pH conditions. The data shows that the aggregation process strongly depends on the particle surface charge density and hardly on the antibody surface coverage. Furthermore, microscopy videos of single particle dimers reveal the presence of reactive patches and thus heterogeneity in the particle surface reactivity. The aggregation rates measured with the single-dimer experiment are compared to data from an ensemble aggregation experiment. Quantitative agreement is obtained using a model that includes the influence of surface heterogeneity on particle aggregation. This single-dimer experiment clarifies how heterogeneities in particle reactivity play a role in colloidal stability.

Interactions between protein coated particles and polymer surfaces studied with the rotating particles probe.

Nonspecific interactions between proteins and polymer surfaces have to be minimized in order to control the performance of biosensors based on immunoassays with particle labels. In this paper we investigate these nonspecific interactions by analyzing the response of protein coated magnetic particles to a rotating magnetic field while the particles are in nanometer vicinity to a polymer surface. We use the fraction of nonrotating (bound) particles as a probe for the interaction between the particles and the surface. As a model system, we study the interaction of myoglobin coated particles with oxidized polystyrene surfaces. We measure the interaction as a function of the ionic strength of the solution, varying the oxidation time of the polystyrene and the pH of the solution. To describe the data we propose a model in which particles bind to the polymer by crossing an energy barrier. The height of this barrier depends on the ionic strength of the solution and two interaction parameters. The fraction of nonrotating particles as a function of ionic strength shows a characteristic shape that can be explained with a normal distribution of energy barrier heights. This method to determine interaction parameters paves the way for further studies to quantify the roles of protein coated particles and polymers in their mutual nonspecific interactions in different matrixes.

Torsion stiffness of a protein pair determined by magnetic particles.

We demonstrate the ability to measure torsion stiffness of a protein complex by applying a controlled torque on a magnetic particle. As a model system we use protein G bound to an IgG antibody. The protein pair is held between a magnetic particle and a polystyrene substrate. The angular orientation of the magnetic particle shows an oscillating behavior upon application of a rotating magnetic field. The amplitude of the oscillation increases with a decreasing surface coverage of antibodies on the substrate and with an increasing magnitude of the applied field. For decreasing antibody coverage, the torsion spring constant converges to a minimum value of 1.5 × 10(3) pN·nm/rad that corresponds to a torsion modulus of 4.5 × 10(4) pN·nm(2). This torsion stiffness is an upper limit for the molecular bond between the particle and the surface that is tentatively assigned to a single protein G-IgG protein pair. This assignment is supported by interpreting the measured stiffness with a simple mechanical model that predicts a two orders of magnitude larger stiffness for the protein G-IgG complex than values found for micrometer length dsDNA. This we understand from the structural properties of the molecules, i.e., DNA is a long and flexible chain-like molecule, whereas the antibody-antigen couple is orders of magnitude smaller and more globular in shape due to the folding of the molecules.

Inter-particle biomolecular reactivity tuned by surface crowders.

The rate at which colloidal particles can form biomolecular bonds controls the kinetics of applications such as particle-based biosensing, targeted drug delivery and directed colloidal assembly. Here we study how the reactivity of the particle surface depends on its molecular composition, quantified by the inter-particle rate of aggregation in an optomagnetic cluster experiment. Particles were functionalized with DNA or with proteins for specific binding, and with polyethylene glycol as a passive surface crowder. The data show that the inter-particle binding kinetics are dominated by specific interactions, which surprisingly can be tuned by the passive crowder molecules for both the DNA and the protein system. The experimental results are interpreted using model simulations, which show that the crowder-induced decrease of the particle surface reactivity can be described as a reduced reactivity of the specific binder molecules on the particle surface.

Rotating magnetic particles for lab-on-chip applications - a comprehensive review.

Magnetic particles are widely used in lab-on-chip and biosensing applications, because they have a high surface-to-volume ratio, they can be actuated with magnetic fields and many biofunctionalization options are available. The most well-known actuation method is to apply a magnetic field gradient which generates a translational force on the particles and allows separation of the particles from a suspension. A more recently developed magnetic actuation method is to exert torque on magnetic particles by a rotating magnetic field. Rotational actuation can be achieved with a field that is uniform in space and it allows for a precise control of torque, orientation, and angular velocity of magnetic particles in lab-on-chip devices. A wide range of studies have been performed with rotating MPs, demonstrating fluid mixing, concentration determination of biological molecules in solution, and characterization of structure and function of biomolecules at the single-molecule level. In this paper we give a comprehensive review of the historical development of MP rotation studies, including configurations for field generation, physical model descriptions, and biological applications. We conclude by sketching the scientific and technological developments that can be expected in the future in the field of rotating magnetic particles for lab-on-chip applications.

On-chip manipulation and detection of magnetic particles for functional biosensors.

We demonstrate the real-time on-chip detection and manipulation of single 1 microm superparamagnetic particles in solution, with the aim to develop a biosensor that can give information on biological function. Our chip-based sensor consists of micro-fabricated 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. The sensitivity profile of the sensor was reconstructed by simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor. A single-dipole model reproduces the measured sensitivity curve for a 1 microm bead. For a 2.8 microm bead the model shows deviations, which we attribute to the fact that the particle size becomes comparable to the sensor width. In the range between 1 and 10 particles, we observed a linear relationship between the number of beads and the sensor signal. The real-time detection and manipulation of individual particles opens the possibility to perform on-chip high-parallel single-particle assays.

Surfactants modify the torsion properties of proteins: a single molecule study.

Surfactants are widely used in diagnostic assays to prevent protein aggregation and non-specific adsorption at surfaces. Here, a single molecule magnetic torque tweezers study is reported, aiming to quantify surfactant-induced changes in the torsional flexibility of a protein model system: protein-G-immunoglobulin G (IgG) attached to a glass surface. The influences of Sodium Dodecyl Sulphate (SDS) and Polysorbate 20 (Tween 20) on the protein pair have been investigated. The proteins were exposed to the surfactants at concentrations relative to the Critical Micelle Concentration (CMC), namely 0.1× CMC, 1× CMC and 10× CMC. Both surfactants increase the torsional flexibility of the protein-G-IgG complex. Tween 20 is most effective at increasing the torsional flexibility of the complex at the surface while SDS is more effective at dissociating the protein bonds. Tweezer data on the IgG-IgG protein pair show no influence of Tween 20 on the torsional flexibility. Furthermore, temperature dependent near-UV and far-UV Circular Dichroism (CD) data at 10× CMC show that Tween 20 does not significantly alter the secondary and tertiary structure of both protein-G and IgG while SDS does. These results provide evidence that both the mechanical properties of the protein structure and the interaction between proteins can alter the torsional rigidity measured with magnetic torque tweezers. This study shows for the first time the ability to use magnetic torque tweezers as a probe for surfactant-induced changes in proteins at a single molecule level.

Molecular interference in antibody-antigen interaction studied with magnetic force immunoassay.

Molecular interferences are an important challenge in biotechnologies based on antibody-antigen interactions, such as sandwich immunoassays. We report how a sandwich immunoassay with magnetic particles as label can be used to probe interference by surfactants. Surfactants are often used to improve the performance of immunoassays, however the surfactants can affect the involved proteins and the mechanism of action of surfactant molecules on the antibody-antigen system is mostly unknown. As an example, we investigated molecular interference by a nonionic surfactant (Pluronic F-127) in a cardiac troponin (cTn) sandwich immunoassay with two monoclonal antibodies. The influence of the surfactant below the critical micelle concentration (0.00-0.04%) on dissociation properties was quantified in a magnetic tweezers setup, where a force is applied to the molecules via magnetic particle labels. The force-dependent dissociation curves revealed the existence of two distinct cTn-dependent bond types, namely a weak bond attributable to non-specific binding of cTn, and a strong bond attributable to the specific binding of cTn. The dissociation rate constant of the strong bonds increased with the surfactant concentration by about a factor of two. Circular dichroism spectroscopy data showed that the nonionic surfactant influences the conformation of cTn while not noticeably affecting the two monoclonal antibodies. This suggests that the surfactant-induced increase of the dissociation rate of the specific sandwich-type cTn binding may be related to a conformational change of the antigen molecule. The described methodology is an effective tool to study the influence of surfactants and other interferences on assays based on protein interactions.

Ara h 1 protein-antibody dissociation study: evidence for binding inhomogeneities on a molecular scale.

The characterization of biomolecular interactions is essential when designing novel biosensors, since the interaction between the bioreceptor and the ligand determines important biosensing parameters such as sensitivity and selectivity. In this paper we study the interaction of the trimeric Ara h 1 protein with a monoclonal anti-Ara h 1 antibody by means of magnetic force-induced dissociation. The proteins were bound to magnetic particles and polystyrene surfaces by EDC/NHS reaction chemistry and by physisorption, respectively. Two different molecular configurations have been investigated, with either the Ara h 1 protein on the particles or the Ara h 1 protein on the polystyrene surface. A model with a Gaussian distribution of energy barriers for dissociation gives an adequate description for the measured multi-exponential decays. We hypothesize that distributions of molecular orientations as well as experimentally induced variations may underlay the observed distributions. The two molecular configurations show a different peak value of the energy distribution. Similarly, SPR experiments for two distinct configurations (either Ara h 1 protein on the surface, or anti-Ara h 1 antibody on the surface) also show clear differences in dissociation behavior. We hypothesize that the multivalency of the involved molecules leads to different modes of binding. The results of this work highlight the importance of molecular inhomogeneities when studying the interaction processes of biomolecular complexes.

In-target production of high specific radioactivity [15O]nitrous oxide by deuteron irradiation of nitrogen gas.

A simple and rapid production method for high specific radioactivity [15O]N2O has been developed based on the 14N(d,n)15O reaction on high-purity nitrogen gas in a flow-through target irradiated with a 0.5 microA beam of 7 MeV deuterons. The [15O]N2O formed during irradiation is selectively concentrated from the target effluent by adsorption on a zeolite during 150 s and subsequently released by rapid heating into a pulse with a full width at half maximum of 3.5 s. The radioactivity and specific radioactivity in the pulse amount to 4 MBq [15O]N2O and 4.5 x 10(13) Bq/mol respectively with a radiochemical purity >99.95%. A tenfold higher specific radioactivity may be feasible at larger beam currents. It was shown that stable N2O was also formed during irradiation. Based on responses to variations in various parameters during irradiation and on analyses performed on the products, an explanation is given on the mechanisms of in-target [15O]N2O and N2O formation, involving reaction of a particular excited state of O3 with N2.

Controlled torque on superparamagnetic beads for functional biosensors.

We demonstrate that a rotating magnetic field can be used to apply a controlled torque on superparamagnetic beads which leads to a tunable bead rotation frequency in fluid. Smooth rotation is obtained for field rotation frequencies many orders of magnitude higher than the bead rotation frequency. A quantitative model is developed, based on results from a comprehensive set of experiments at different field strengths and frequencies. At low frequencies (<10Hz), rotation is due to a small permanent magnetic moment in the bead. 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 bead. The control of torque and rotation will enable novel functional assays in bead-based biosensors.