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)