Daniel L. Graham

Biodetection using magnetically labeled biomolecules and arrays of spin valve sensors (invited)

On-chip spin-valve sensors (2×6 μm2) were used to detect the binding of streptavidin-functionalized superparamagnetic labels to sensor surface-bound biotin. Both micron-sized and nanometer-sized labels were studied. The detection of biomolecular recognition was demonstrated using 2 μm Micromer®-M and 250 nm Nanomag®-D labels, with signals ranging from ∼300 μV to ∼2 mV (8 mA sense current; ∼15 Oe in-plane magnetizing fields). For smaller labels, detection of biomolecular recognition was not achieved. The capability of detecting single labels was demonstrated for label moments down to 2×10−12 emu (2 μm labels), corresponding to signals of 100–400 μV. Although, theoretical calculations suggest that the minimum measurable moment is in the order of 6×10−15 emu, due to noise limitations of the present setup, this limit is in the order of 2×10−13 emu, corresponding to a single 250 nm label. On-chip tapered aluminum current line structures were used for placement of magnetic labels at sensor sites.

Single magnetic microsphere placement and detection on-chip using current line designs with integrated spin valve sensors: Biotechnological applications

Superparamagnetic labels, 400 nm dextran iron oxide particles and 2 μm polymer encapsulated iron oxide microspheres, with biomolecules immobilized on the surface, e.g., the enzyme horseradish peroxidase (20–40 molecules per label) were controllably placed on chip sites (5×15 μm2) using tapered Al current lines (10–20 mA current) and moved to and from adjacent spin valve sensors [2×6 μm,2, magnetoresistance (MR) ∼5%]. Average MR signals of 1.2 and 0.6 mV were obtained for the detection of bulk numbers of 400 nm and 2 μm labels respectively using an on-chip field of 15 Oe and a sense current of 5 mA. The moment per label was calculated at 5×10−13 emu for the 400 nm labels and 5×10−12 emu for the 2 μm labels, illustrating the higher density of the 400 nm particles. MR signals of ∼100 μV were obtained for single 2 μm labels positioned over the spin valve sensor using an on-chip field of 15 Oe and 8 mA sense current. The corresponding sensor saturation occurred at ∼1 mV, with a noise level of ∼10 μV. The estim...

Planar Hall effect sensor for magnetic micro- and nanobead detection

Magnetic bead sensors based on the planar Hall effect in thin films of exchange-biased permalloy have been fabricated and characterized. Typical sensitivities are 3 μV/Oe mA. The sensor response to an applied magnetic field has been measured without and with coatings of commercially available 2 μm and 250 nm magnetic beads used for bioapplications (Micromer-M and Nanomag-D, Micromod, Germany). Detection of both types of beads and single bead detection of 2 μm beads is demonstrated, i.e., the technique is feasible for magnetic biosensors. Single 2 μm beads yield 300 nV signals at 10 mA and 15 Oe applied field.

High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.

On-chip manipulation and magnetization assessment of magnetic bead ensembles by integrated spin-valve sensors

Manipulation and detection of magnetic beads on a semiconductor chip opens up new perspectives for analysis of magnetically labeled specimens in biomechanical micro-electromechanical systems for biological applications. Sensitive spin-valve sensors were integrated with magnetic field generating conductors to assess the behavior of ensembles of superparamagnetic nanoparticles 300 nm in diameter that contain 75%–80% magnetite. The spin-valve multilayer including a nanooxide layer achieves 8% magnetoresistance (MR) for an integrated device of 2×16 μm2. Motion of the magnetic particles towards and across the sensor is achieved by two tapered magnetic field generating current conductors. The spin-valve sensor detects the stray magnetic field that emanates from the ensemble of magnetic particles. We study the transients in the magnetic signal on the order of 1% MR. These results lead to a model that describes magnetization configurations of the cluster of beads.

Nitrile biotransformations using free and immobilized cells of a thermophilic Bacillus spp.

A thermophilic Bacillus spp. capable of transforming aliphatic nitriles, cyclic nitriles and dinitriles was used as a free cell suspension and immobilized in alginate beads to study the utilization of acetonitrile and acrylonitrile in a buffered biotransformation medium. The cells grew optimally at 65 degrees C and contained a nitrile hydratase-amidase enzyme system that transformed nitrile compounds stoichiometrically to the corresponding carboxylic acids. In the presence of urea or chloroacetone, amidase activity was inhibited and the amide intermediate was accumulated. Mass transfer limitation of nitrile utilization rates was observed with immobilized cells, but the alginate afforded the cells some degree of additional thermal stability and potential advantage in re-use. In vitro inhibition of the partially purified amidase was confirmed and the use of whole cells of this organism in a continuous bioreactor to generate amide products from nitrile substrates was demonstrated.

Rapid DNA hybridization based on ac field focusing of magnetically labeled target DNA

Rapid DNA-DNA hybridization between surface-bound probe DNA and magnetically labeled complementary target DNA was achieved using current carrying line structures and oscillating external magnetic fields. Magnetic particles of 250 nm in diameter were focused and manipulated over on-chip U-shaped current lines using dc currents of 40 mA and oscillating magnetic fields of 1.4kA∕mrms with frequencies ranging from 0.1 to 20 Hz. The focusing process was both time and frequency dependent and, consequently, hybridization degree varied with focusing efficiency. Extensive label binding was observed in 5–25 min at 0.1–20 Hz. This technique has strong potential in commercial DNA chip development.

Magnetoresistive DNA chips

Publisher Summary Magnetoresistance (MR) technology is being successfully applied to biomolecular recognition in different biological contexts. Micron-sized magnetic labels are already successfully used in biomolecular recognition experiments, but smaller magnetic labels that are non-remanent, non-clustering, with low anisotropy and high susceptibility are required. The existing magnetoresistive sensing technology allows the successful detection of single nanometer-sized magnetic labels. However, real biological recognition results with MR biochip prototypes done at INESC and elsewhere are successful only with micron-sized labels (INESC, NRL, U. Bielefeld) and with 250 nm labels (INESC). An important figure of merit when comparing biomolecular recognition detection platforms is the amount of target material that can be detected. The minimum target concentration that can be detected by MR biochip platforms depends intrinsically on label dimension, and the number of target biomolecules attached to the label that can hybridize. MR technology has shown the potential for single molecule process detection, a target not usually within the reach of most of the competing technologies.

Detection of cystic fibrosis related DNA targets using AC field focusing of magnetic labels and spin-valve sensors

Over the past few years the concept of using magnetic field sensors for biological applications in particular, the development of magnetoresistive biochips and biosensors, has generated increasing interest from laboratories and companies. A spin-valve sensor based biochip was used to detect cystic fibrosis related DNA targets for the purpose of developing an affordable diagnostic chip and detection system. The strategy is based on the AC magnetic field focusing technique. This method consists of the attraction, concentration and manipulation of magnetically-labelled target DNA within on-chip u-shaped current line regions surface functionalized with a cystic fibrosis-related DNA probe. Cystic fibrosis related probes were immobilized on the oxide surface and 250 nm diameter non-remanent magnetic particles were functionalized with cystic fibrosis related DNA targets complementary or non-complementary to the immobilized probes. The hybridization of the target is detected using a u-shaped spin-valve sensor fabricated within the line structure. The proximity of probe and target at the spin-valve sensor surface promotes the hybridization of complementary DNA strands. In this way, hybridization occurs in relatively short times, (5-25 minutes), in comparison with conventional hybridization approaches (3 to 12 hours), as limited by diffusion of the target DNA in solution. Magnetic labels bound to the sensor surface through the hybridization of complementary DNA strands have a magnetic stray field that changes the resistance of sensors enabling detection of the hybridization in real-time. Results show a discernable difference in sensor response after washing when using complementary or non-complementary DNA targets. The use of complementary target DNA resulted in distinct hybridization signals and the binding of the particles in the sensor area was verified by visual inspection. In addition, it was observed that hybridization signals decreased slightly after the more stringent wash indicating that non-specifically or weakly bound labels were washed away. The use of non-complementary target DNA resulted in negligible sensor response after washing and no particles were observed in the sensor area.

Effect of spin-valve sensor magnetostatic fields on nanobead detection for biochip applications

Spin valves are being used in biochip applications via the detection of biomolecular recognition using magnetic nanoparticles as labels. The magnetic moment of the labels and the sensor response depend on the magnetic fields involved. A calculation based on an external magnetizing field, incorporating the contribution from the magnetostatic fields created by the free and pinned layers of the sensor and the field due to the sensing current, showed that these fields are important. Experimental detection signals of high particle numbers agree with the model, showing that reasonable detection signals are possible even in the absence of an external field.