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.

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.

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.

Distinct developmental changes in the distribution of calcium, phosphorus and sulphur during fetal growth-plate development.

Gradients in the concentrations of free phosphate (Pi) and calcium (Ca) exist in fully developed growth zones of long bones and ribs, with the highest concentrations closest to the site of mineralization. As high concentrations of Pi and Ca induce chondrocyte maturation and apoptosis, it has been hypothesized that Ca and Pi drive chondrocyte differentiation in growth plates. This study aimed to determine whether gradients in the important spectral elements phosphorus (P), Ca and sulphur (S) are already present in early stages of development, or whether they gradually develop with maturation of the growth zone. We quantified the concentration profiles of Ca, P, S, chloride and potassium at four different stages of early development of the distal growth plates of the porcine femurs, using particle-induced X-ray emission and forward- and backward-scattering spectrometry with a nuclear microprobe. A Ca concentration gradient towards the mineralized area and a stepwise increase in S was found to develop slowly with tissue maturation. The increase in S co-localizes with the onset of proliferation. A P gradient was not detected in the earliest developmental stages. High Ca levels, which may induce chondrocyte maturation, are present near the mineralization front. As total P concentrations do not correspond with former free Pi measurements, we hypothesize that the increase of free Pi towards the bone-forming site results from enzymatic cleavage of bound phosphate.