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Outline. Motivation Preparation of Biocompatible Magnetic Nanoparticles Dispersed in Water Investigation of Magnetic Nano-particles Applied to Immunoassay on Avidin Summary Future Work. Motivation. For example, to detect the antibody. Conventional Immunoassay

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  1. Outline • Motivation • Preparation of Biocompatible Magnetic Nanoparticles Dispersed in Water • Investigation of Magnetic Nano-particles Applied to Immunoassay on Avidin • Summary • Future Work

  2. Motivation For example, to detect the antibody • Conventional Immunoassay • - Enzyme-Linked Immunosorbent Assay(ELISA) On-chip bio-probe (anti-gen) Serum with Adding indicators Fluorescence/Isotopes Test chip Wash/separation The amount of is probed by detecting the intensities of fluorescence/isotopes

  3. To develop a convenient, high-sensitive, high-resolution, and reliable immunoassay • Disadvantages of Conventional Immunoassay • Complicated processes • - two pairs of anti-gen/anti-body: and • More uncertainties in the detected amount of antibody • - self-absorption/emission of fluorescence by bio-molecules

  4. Another promising candidate: Magnetically Labeled Immunoassay (MLI) A higher reliability may be expected because almost all anti-gens/anti-bodies are non-magnetic. Numerous novel methods have been actively developed: Surface Plasmas Resonance, Microfabricated transducers1 …… 1USA Naval Research Lab., http://stm2.nrl.navy.mil

  5. Magnetic properties of clusters are measured to detect the amount of bio-target (antibody). • Magnetic Labeling Use magnetic particles as an indicator. Magnetic particle Bio-probe Bio-target

  6. The biggest challenge in magnetically labeled immunoassay:  preparation of highly homogeneous magnetic nano-particles. For MLI, the sensitivity, resolution, and reliability deeply depend on the uniformity of the magnetic particles.

  7. Avidin is an antibody (glycoprotein) found in egg whites. Its conjugate antigen is biotin. • Goals • Preparation of Highly Homogeneous Biocompatible Magnetic Nano-particles Dispersed in Water • Investigation of Magnetic Nano-particles Applied to Immunoassay on Avidin

  8. centrifuge • Preparation of Biocompatible Magnetic Nano-particles Dispersed in Water • Preparation of Water-based Fe3O4 Magnetic Fluid FeCl2, FeCl3, H2O mixing dextran&CO(NH2)2 heating NH4OH coating Water dextrancoated Fe3O4 removing salt residue & large particles removing unbound dextran gel filtration chromatography Fe3O4 Dextran H.E. Horng et al., J. Magn. Magn. Mater., 283, 210 (2004) homogeneous water-basedFe3O4 magnetic fluid

  9. Particle Crystalline Water-based Fe3O4 magnetic fluid  No other detectable phase than Fe3O4

  10. Particle Size Distribution Water-based Fe3O4 magnetic fluid Average diameter = 25.6 nm S.D. = 5.0 nm (20 %) (cf. Commercial product: S.D. = 50 %)

  11. Hydrodynamic diameter (nm) Particle diameter (nm) • Controllable Particle Size Particle diameter Hydrodynamic diameter Urea decomposition time @ 90 oC (min.)

  12. Coating of Bio-probes on Magnetic Nano-particles Homogeneous water-based dextran-coated Fe3O4 magnetic fluid Example : Bio-target: avidin Bio-probe: biotin NaIO4 Oxidation dextran Water Biotin Biotin is bound to dextran Biotin Dialysis Fe3O4 removing unbound biotin Water-based biotin/detran-coated Fe3O4 magnetic fluid Dextran

  13. OD (a.u.) 3hr. 1Day 3Days 7Days 10Days 14Days • Toxicity Test (Fe3O4 MF + Human Osteoblast Cells)  Non-toxic Fe3O4 magnetic fluid with concentrations < 10-5 M J.S. Sun, YMH

  14. Bio-target: avidin Mixed & wait for 1 hour • Investigation of Magnetic Nano-particles Applied to Immunoassay on Avidin • Sample Preparation for Magnetic Measurement Biotin-dextran coated Fe3O4 water-based magnetic fluids: Volume = 1 c.c. Concentration = 0.07 emu/g Bitoin = 3, 5, 7 g

  15. With avidin Particle clusters: Mean diameter = 113.9 nm • Formation of Magnetic Clusters Associated with Bio-targets (avidin) Diameter distribution magnetic particles Without avidin Isolated particles: Mean diameter = 19.2 nm Using Laser Scattering Method

  16. Separation of Magnetic Clusters from Solution Filtrate the sample through a micro-filter possessing nano-sized holes of 50 nm in diameter. Mean diameter of particles = 28.4 nm Magnetic measurement Micro-filter (non-magnetic) Micro-filter (non-magnetic) Remove single particle

  17. In this work. Magnetic properties of the magnetic clusters detected in MLI: • Magnetic Relaxation1,2 • Mixed Frequency ac Magnetic Susceptibility3 • Magnetic Remanence4 • Saturated Magnetization 1R. Kütitz et al., JMMM, 194, 62(1999) 2J. Clarke et al., APL, 81, 3094(2002) 3Y. Zhang et al., Deutsche Patentanmeldung 10309132.7(2003) 4K. Enpuku et al., JJAP, 38, L1102(1999)

  18. To achieve a high-resolution in measuring saturated magnetization of clustered magnetic particles, an extremely sensitive detector is helpful. Superconductive QUantum Interference Devices (SQUIDs) The most sensitive detector of magnetic flux.

  19. Magnetic flux,  Magnetization of clusters Voltage signal Voltage, V • Superconductive QUantum Interference Devices (SQUIDs) Josephson junction Superconducting film (YBCO) V Bias current, Ib

  20. Earth field B (Tesla) Biomagnetic fields -4 -4 10 10 -5 10 mT -6 10 Urban noise -7 10 -8 10 Car @ 50 m nT Flux-gate magnetometer Lung particles -9 10 Human heart -10 10 Fetal heart -11 10 Transistor chip @ 2 m pT Human brain (a) -12 10 -13 Human brain (response) 10 -14 10 -15 10 Environmental fields Magnetic nano-particles High-Tc SQUID Low-Tc SQUID  SQUID is a sensitive detector to probe the magnetization of clustered magnetic particles.

  21. Magnetic Hysteresis of Magnetic Clusters Associated with Avidin Detected by the SQUID Magnetometer Saturated Magnetization, Ms

  22. Saturated Magnetization vs. Amount of Avidin High Resolution: ~ pg/ml High Sensitivity: ~pg/ml

  23. Summary • Magnetic Fe3O4 nano-particles: • highly homogeneous, • controllable size, • biocompatible, • non-toxic • Magnetically labeled immunoassay: • without secondary antibody, • high-resolution (~ pg/ml), • high-sensitivity (~ pg/ml)

  24. Front view Back view • Future Work Magnetic nano-particles coated with suitable bio-probes for interested bio-molecules will be synthesized. For example: C-Reactive Protein (CRP) CRP is composed of five identical, 21,500 MW subunits. CRP is released by the body in response to acute injury, infection, or other inflammatory stimuli.

  25. Protein A CRP Anti-CRP Protein A Fe3O4 Water Bio-probe: Anti-C-reactive protein     (Anti-CRP) Anti-CRP possesses a bio- functional group IgG, which can tightly bind with protein A IgG: Magnetic fluid: Other interested anti-bodies: VCAM-1,ICAM-1, MMP, VEGF…

  26. Applications of Magnetic Fluids to Photonic Devices Posters in Photonics West 2005, Jan. 25, 2005: • Tunable Photonic Band Gaps of Ordered Structures in Magnetic Fluid Films (5733-61) • Optical Logic Devices Based on Magnetic-fluid-coated Optical Fibers (5723-37) J. Appl. Phys., 81, 4275(1997) Appl. Phys. Lett., 75, 2196(1999) Appl. Phys. Lett., 79, 2360(2001) J. Appl. Phys., 94, 3849(2003) Appl. Phys. Lett., 84, 5204(2004) Opt. Lett., in press(2005) J. Appl. Phys., in press(2005)

  27. Thank you for your attention

  28. Measurement of amount of avidin: • Light Scattering Method •  mean diameter of single and clustered • magnetic particles • Magnetic Labeling •  saturated magnetization

  29. pg-order Mean Diameters of Magnetic Clusters Associated with Various Amounts of Bio-targets (avidin) g-order of magnitude  The more the amount of avidin, the larger the magnetic clusters. Sensitivity: 60 pg Resolution: 3 pg Dynamic range > 90 g H.E. Horng et al., IEEE Trans. Appl. Supercond., in press(2005)

  30. Magnetic Labeling vs. • Light Scattering Method

  31. Future Work • Material • Synthesis of magnetic nano-particles coated with suitable bio-probes for interested bio-molecules. • Interested bio-molecules (targets): • C-reactive protein, • Intracellular adhesion molecule-1(ICAM-1), • Vascular endothelial growth factor (VEGF), • Matrix metalloproteinase (MMP), (ICAM-1), • Vascular cell adhesion molecule-1 (VCAM-1)

  32. Instrumentation • Development of high-Tc SQUID measurement systems for various types of magnetic immunoassay. • Investigation of the specifications of the developed magnetic immunoassay measurement system. • Establishment of the magnetic immunoassay model in a noninvasive bio-molecular culture systems.

  33. Top view Side view Feeding liquid N2 Feeding liquid N2 Dewar Solenoid SQUID stage vertical translation controller SQUID stage (inside the dewar) 3-shell -metal magnetically shielded can Sample translation stage In our group, we design a high-Tc SQUID magnetometer/gradiometer system for magnetic immunoassay.

  34. Pathological diagnosis • Demonstration of the feasibility of noninvasive magnetic immunoassay in cell culture systems or in animals in vivo. • Parallel studies with existent diagnostic methods, such as • magnetic resonance imaging (MRI), • enzyme linked immunosorbent assay (ELISA), and • capillary electrophoresis (CE), etc.

  35. Configuration of Light Scattering for Detecting Particle Sizes 3 PORT SURFACE WAVEGUIDE LASER DIODE PHOTO DETECTOR SAMPLE CELL FFT - DSP HARDWARE COMPUTER ADC Microtrac, Nanotrac 150

  36. Great impacts to bio-medical academics and industry. Challenge: preparation of highly homogeneous magnetic nano-particles. • Motivation Applications of Magnetic Nano-particles Mechanical devices: vacuum seal, damper… Optical devices: modulator, switch, filter… Biomedicine: immunoassay, drug delivery…

  37. Distribution in moving velocity of particles Distribution in Doppler shift Velocity of Brownian Motion of particles depends on: • Particle size (Distribution) • Density of particle material (fixed value for a given particle material) • Viscosity of liquid (fixed value for a given liquid)

  38. Time Computation: Frequency Distribution to Particle Size Distribution Intensity Distribution Fast Fourier Transform Power Spectrum Photo Detector Power Intensity Intensity Percent Frequency Particle Size

  39. fscattered light freflected light due to Brownian Motion of particles  Doppler shift (Depending on the moving velocity of particles) • Light Scattering Method Working Principle From Laser Diode , 780 nm Reflected light 780 nm Scattered light To Photo Detector Waveguide Optical fiber Microtrac, Nanotrac 150

  40. Co-operation Groups： Prof. C.C. Wu(NTU Hospital) Prof. W.Y. Tseng(NTU Hospital) Dr. S.W. Chang(NTU Hospital) Dr. J.S. Sun(NTU Hospital) Prof. C.M. Liu(TMU) Co-operation Groups: Prof. H.C. Yang(NTU) Prof. S.Y. Yang(NTNU) Prof. Y. Zhang(Jülich Research Center, Germany) Co-operation Groups: Prof. C.-Y. Hong(DYU) Prof. W.Q. Jiang(USTC, China) Prof. H.C. Chang(CIT) • Three Areas of Research Involved in Magnetically Labeled Diagnosis Principal Investigator : Prof. Herng-Er Horng (NTNU) Bio-medicine High-Tc SQUIDs Magnetic Nano-particles

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