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Outline
MotivationPreparation of Biocompatible Magnetic Nanoparticles Dispersed in WaterInvestigation of Magnetic Nano-particles Applied to Immunoassay on AvidinSummaryFuture Work
Conventional Immunoassay - Enzyme-Linked Immunosorbent Assay (ELISA)
Serum with
Test chip
On-chip bio-probe (anti-gen) Adding
indicatorsFluorescence
/Isotopes Wash/separation
The amount of is probed by detecting the intensities of fluorescence/isotopes
For example, to detect the antibody
Motivation
Disadvantages of Conventional Immunoassay
More uncertainties in the detected amount of antibody - self-absorption/emission of fluorescence by bio-molecules
To develop a convenient, high-sensitive, high-resolution, and reliable immunoassay
Complicated processes - two pairs of anti-gen/anti-body: and
Numerous novel methods have been actively developed: Surface Plasmas Resonance, Microfabricated transducers1
……
1USA Naval Research Lab., http://stm2.nrl.navy.mil
A higher reliability may be expected because almost all anti-gens/anti-bodies are non-magnetic.
Another promising candidate: Magnetically Labeled Immunoassay (MLI)
Magnetic Labeling
Use magnetic particles as an indicator.
Magnetic particle
Bio-probe
Bio-target
Magnetic properties of clusters are measured to detect the amount of bio-target (antibody).
For MLI, the sensitivity, resolution, and reliability deeply depend on the uniformity of the magnetic particles.
The biggest challenge in magnetically labeled immunoassay:
preparation of highly homogeneous magnetic nano-particles.
Goals
Preparation of Highly Homogeneous Biocompatible Magnetic Nano-particles Dispersed in WaterInvestigation of Magnetic Nano-particles Applied to Immunoassay on Avidin
Avidin is an antibody (glycoprotein) found in egg whites.Its conjugate antigen is biotin.
Preparation of Water-based Fe3O4 Magnetic Fluid
Fe3O4 Dextran
Water
H.E. Horng et al., J. Magn. Magn. Mater., 283, 210 (2004)
mixing
NH4OH
removing salt residue & large particles
coating
centrifuge
gel filtration chromatograph
y homogeneous water-based
Fe3O4 magnetic fluid
FeCl2, FeCl3, H2O
heating
dextran coated Fe3O4
dextran & CO(NH2)2
removing unbound dextran
Preparation of Biocompatible Magnetic Nano-particles Dispersed in Water
Particle Crystalline
Water-based Fe3O4 magnetic fluid
20 30 40 50 60 702 (deg)
0
350
700
Inte
nsity
(a.
u.)
(220
)
(311
)
(400
)
(511
)
(440
)
Fe 3O 4
No other detectable phase than Fe3O4
Average diameter = 25.6 nm S.D. = 5.0 nm (20 %)(cf. Commercial product: S.D. = 50 %)
Water-based Fe3O4 magnetic fluid
Particle Size Distribution
Controllable Particle Size
Particle diameter
Hydrodynamic diameter
Urea decomposition time @ 90 oC (min.)0 10 20 30
U rea decom position tim e (m in)
0
5
10
15
20
25
30
Par
ticle
dia
met
er (
nm)
20
30
40
50
60
70
80
90
100
Hyd
rody
nam
ic d
iam
eter
(nm
)
Part
icle
dia
mete
r (n
m)
Hydro
dynam
ic d
iam
ete
r (n
m)
Coating of Bio-probes on Magnetic Nano-particles
NaIO4
Oxidation dextran
Biotin is bound to dextran
Homogeneous water-based dextran-coated Fe3O4 magnetic fluid
Biotin
Fe3O4
Dextran
Water
Biotin Dialysisremoving unbound biotin
Water-based biotin/detran-coated Fe3O4 magnetic fluid
Example :
Bio-target: avidin
Bio-probe: biotin
Toxicity Test (Fe3O4 MF + Human Osteoblast Cells)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
3 h 1 D 3 D 7 D 10 D 14 D
con (M)
1.09*10-11
1.09*10-10
1.09*10-9
1.09*10-8
1.09*10-7
1.09*10-6
1.09*10-5
3hr. 1Day 3Days 7Days 10Days 14Days
OD
(a.u
.)
Non-toxic Fe3O4 magnetic fluid with concentrations < 10-5 M J.S. Sun, YMH
Investigation of Magnetic Nano-particles Applied to Immunoassay on Avidin
Biotin-dextran coated Fe3O4 water-based magnetic fluids: Volume = 1 c.c. Concentration = 0.07 emu/g Bitoin = 3, 5, 7 g
Sample Preparation for Magnetic Measurement
Bio-target: avidin
Mixed & wait for 1 hour
Diameter distribution magnetic particles
Mean diameter =19.2 nm
Formation of Magnetic Clusters Associated with Bio-targets (avidin)
Isolated particles:Without avidin
With avidin
Mean diameter = 113.9 nm
Particle clusters:
Using Laser Scattering Method
Filtrate the sample through a micro-filter possessing nano-sized holes of 50 nm in diameter.
Micro-filter (non-magnetic)
Separation of Magnetic Clusters from Solution
Micro-filter (non-magnetic)
Magnetic measurement
Remove single particle
Mean diameter of particles = 28.4 nm
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)
In this work.
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.
Bias current, Ib
V
Josephson junction
Superconducting film (YBCO)
Superconductive QUantum Interference Devices (SQUIDs)
Magnetic flux,
M agnetic flux, /0
Vo
ltag
e,
V Magnetization of clusters Voltage signal
Volt
age, V
Environmental fields
Flux-gatemagnetometer
Magnetic nano-particles
Low-Tc SQUID
High-Tc SQUID
SQUID is a sensitive detector to probe the magnetization of clustered magnetic particles.
Biomagnetic fieldsUrban noise
Car @ 50 m
Transistorchip @ 2 m
Lung particles
Human heart
Fetal heart
Human brain ()
Human brain (response)
B (Tesla)10
-410
-4
10-5
10-6
10-7
10-8
10-9
10-10
10-11
10-12
10-13
10-14
10-15
T
nT
pT
Earth field
Magnetic Hysteresis of Magnetic Clusters Associated with Avidin Detected by the SQUID Magnetometer
Saturated Magnetization, Ms
-10000 -5000 0 5000 10000H (O e)
-600
-300
0
300
600
M (
x10-6
em
u/g)
Saturated Magnetization vs. Amount of Avidin
High Resolution: ~ pg/ml
High Sensitivity: ~pg/ml
0 100 200Avid in (pg/m l)
0
400
800
1200
Ms
(x10
-6 e
mu/
g)
0 3 6 9 12
Avid in (pM )
B iotin = 3 g
B iotin = 5 g
B iotin = 7 g
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)
Future Work
Magnetic nano-particles coated with suitable bio-probes for interested bio-molecules will be synthesized.
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.
Front view Back view
For example: C-Reactive Protein (CRP)
Bio-probe: Anti-C-reactive protein (Anti-CRP)
Anti-CRP possesses a bio- functional group IgG, which can tightly bind with protein A
IgG:
Protein A
Magnetic fluid:
Fe3O4
Protein A
Water
Anti-CRP
CRP
Other interested anti-bodies: VCAM-1,ICAM-1, MMP, VEGF…
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)
Light Scattering Method mean diameter of single and
clustered magnetic particles
Measurement of amount of avidin:
Magnetic Labeling saturated magnetization
Mean Diameters of Magnetic Clusters Associated with Various Amounts of Bio-targets (avidin)
g-order of magnitude
H.E. Horng et al., IEEE Trans. Appl. Supercond., in press(2005)
0 20 40 60 80 100Avid in (g)
40
60
80
100
Mea
n di
amet
er (
nm)
0 50 100Avid in (pg)
48
52
56
Mea
n di
amet
er (
nm)
The more the amount of avidin, the larger the magnetic clusters. Sensitivity: 60 pg Resolution: 3 pg Dynamic range > 90 g
pg-order
0 20 40 60 80 100Avid in (g)
40
60
80
100
Mea
n di
amet
er (
nm)
0 50 100Avid in (pg)
48
52
56
Mea
n di
amet
er (
nm)
Magnetic Labeling vs. Light Scattering Method
0 40 80 120Avid in (pg)
0
200
400
600
800
Ms
(x10
-6 e
mu/
g)
50
60
Mea
n di
amet
er (
nm)
0 2 4 6
Avidn (pM )
Biotin = 3 g
Future Work
Material Synthesis of magnetic nano-particles coated with suitable bio-pr
obes 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)
Instrumentation Development of high-Tc SQUID measurement systems for variou
s 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.
In our group, we design a high-Tc SQUID magnetometer/gradiometer system for magnetic immunoassay.
Feeding liquid N2
3-shell -metal magnetically shielded can
Dewar
Solenoid
SQUID stage (inside the dewar)
Sample translation stage
Side viewTop view
Feeding liquid N2
SQUID stage vertical translation controller
Pathological diagnosis Demonstration of the feasibility of noninvasive magnetic imm
unoassay 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.
LASER DIODE
PHOTO DETECTOR
3 PORT SURFACE WAVEGUIDE
SAMPLE CELL
ADCFFT - DSPHARDWARE COMPUTER
Configuration of Light Scattering for Detecting Particle Sizes
Microtrac, Nanotrac 150
Great impacts to bio-medical academics and industry.Challenge: preparation of highly homogeneous magnetic nano-particles.
Applications of Magnetic Nano-particles
Motivation
Mechanical devices: vacuum seal, damper…Optical devices: modulator, switch, filter…Biomedicine: immunoassay, drug delivery…
Velocity of Brownian Motion of particles depends on:
1. Particle size (Distribution)
2. Density of particle material (fixed value for a given particle material)
3. Viscosity of liquid (fixed value for a given liquid)
Distribution in moving velocity of particles
Distribution in Doppler shift
Computation: Frequency Distribution to Particle Size Distribution
Photo Detector Photo Detector
Po
wer
Frequency
PowerSpectrum
Fast FourierTransform
Particle Size
Inte
nsi
ty P
erce
nt
Intensity Distribution
Time
Inte
nsity
Light Scattering Method
From Laser Diode , 780 nmFrom Laser Diode , 780 nmFrom Laser Diode , 780 nmFrom Laser Diode , 780 nm
Reflected lightReflected light780 nm780 nm
Reflected lightReflected light780 nm780 nm
Scattered lightScattered light
Optical fiber WaveguideTo Photo DetectorTo Photo Detector
fscattered light freflected light due to Brownian Motion of particles
Doppler shift
(Depending on the moving velocity of particles)
Microtrac, Nanotrac 150
Working Principle
Three Areas of Research Involved in Magnetically Labeled Diagnosis
High-Tc SQUIDs Magnetic Nano-particles
Bio-medicine
Co-operation Groups:Prof. C.-Y. Hong(DYU)Prof. W.Q. Jiang(USTC, China)Prof. H.C. Chang(CIT)
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.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)
Principal Investigator : Prof. Herng-Er Horng (NTNU)