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Markers for biomedical applications
Prof. Dr. Meinhard Schilling
Institute for Electrical Measurements and Foundations of Electrical Engineering
University of Technology, Braunschweig, Germany
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•
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Markers in Bioanalysis
Applications in Biochemistry and Medicine
Magnetic nanoparticles
„Fundamentals and Materials for Novel Sensors“
DAAD summer school
Rio de Janeiro 24.07.2009
Internat. graduate
school for metrology
metrolog
TU Braunschweig and PTB German National Metrology
Institute + Companies
p
20 foreign ph.d. students
+ 20 german ph.d.
h d students
t d t
Scholarships available
Application
pp
everytime
y
at
www.igsm.tu-bs.de
Nanotechnology - today
M k t
Market:
• Chiptechnology:
Chi t h l
I t l P ti
Intel-Pentium
• Colors and furnish, surface finishing
• New illumination and LED
LED-lamps
lamps
• Nanoparticles in biology and medicine
„Last technological revolution?“
Protagonists
P
t
it
and
d
• R. Feynman (QED)
• G.
G Binnig/H.
Binnig/H Rohrer (STM/AFM)
• R. Curl, H. Kroto, R. Smalley (C60)
• more...
Visionaries
Vi
i
i
• Erik Drexler
• Ray Kurzweil
• Vernor Vinge
• more...
Science Fiction: Nanorobots
www.
foresight.
org
Vision: Molecular Medicine
Id l IImmune system
Ideal:
t
On the human body live more bacteria than we have
cells in the body. Our bacteria amount to more than 1 kg.
But: In the blood of a healthy human one can find not a
single bacterium!
Our Immunesystem is our best protection.
How does the Immunesystem do this job?
Can y
you support
pp
the Immunesystem?
y
-> Molecular medicine
The needle in the haystack
A magnet can localize the needle!
Thus: Attach magnets to the molecules of interest
and find the molecules!
Why nanoscale markers?
Markers in bioanalysis
Antibodies
Prof. Dr. Stefan Dübel (TU-Braunschweig) „Antibody Factory“
Aims
Detection of biochemical activity (assay) without interaction
• in unchanged environment
• from large distance
• for longer duration
• selective for molecule
only small perturbation
radioactive, optical,
magnetic, electric
stable property
unique property
Solution:
Chemical linkage to marker, which is small and changes
the biochemical properties only marginal.
Assay Choices
Radioactive marker
radioactive elements
3H, 32P, 35S, 125I
Optical marker
luminescence, fluorescence
Magnetic marker
magn. nanoparticles, NMR
indirect electrical marker
conductivity
-> detection after separation:
filtering, centrifugation, electrophoresis, chromatography,
chemical selection (specif
(specif. linkage to antibodies or to
complementary DNA/RNA)
Binding of Marker
marker
probe
Specific linkage to DNA, protein, ... necessary:
di t
direct
probe
b coupled
l d tto marker
k
indirect
probe independent from marker,
marker attached later
Binding of markers by
antibodies
tib di
Radioactive Markers
Radioimmunoassay RIA
Determination of cencentration
of antigen by competitive radioimmunoassay (pg/ml):
1. prepare marked antigen
2. unknown concentration of
unmarked antigen reacts with
antibodies
3 wash
3.
4. remaining bonds are filled
with marked antigens
5. measurement of radioactivity
gives unknown concentration
Large signal at low concentration
Not Radioactive Markers
Fluorescence-Set-up
Lightsource with wide
spectrum,
fluorescence in all
directions !
Filters against excitation
li ht d
light
during
i d
detection
t ti
Monochromators admit
excitation- and
fluorescence-spectroscopyy
depending on the
change in wavelength in
M
Monochromator
h
t 1 or 2
-> microscopy !
Optical Active Transitions
red: light absorption
or emission
black: non-radiative
transitions
S singlet-states
T triplet-states
triplet states
Fluorescence wave
length larger than in
absorption,
phosporescence longer
observable than fluoresc.
Fluorescence Marker
When a chemical substance is excited by light of short
wavelength (high energy) and light of longer wavelength is
emitted this observation called fluorescence.
emitted,
fluorescence
Excitation and emission-spectra
p
are influenced byy
environmental conditions.
iinternall flfluorescence (autofluorescence)
(
fl
):
z.B. proteins: tryptophan (280nm ->350nm)
externe fluorescence:
additional g
groups
p at molecule: fluorophores
p
additional molecules / nanoparticles as marker
fluorescein, rhodamin, coumarin
Quantum Dots
Fluorescence markers with tunable wavelength are
realized by semiconductor nanoparticles:
Many markers can be used simultaneously!
ELISA/EIA-Tests
amplification
Enzymimmunoy
Enzymlinked
y
Enzymimmunoy
assay, competitive, Immunosorbentassay homogen
heterogenous
assay, heterogenous
HIV-Antibody Test
Zur Anzeige wird der QuickTime™
Dekompressor „Animation“
„Animation
benötigt.
HIV positive
Zur Anzeige wird der QuickTime™
Dekompressor „Animation“
„Animation
benötigt.
HIV negative
www.biology.arizona.edu/immunology
Marker only binds, if antibodies are attached.
Dye only stains, if antibodies with enzyme are bound.
Biochips
All gene segments of DNA are
labeled. The counterparts are bound
to the chip. After reaction and
washing of unbound, not matching
material fluorescence is measured.
This is done twice: red/green for
comparison of normal and changed
cell state
state.
Marker based bioanalysis
Magnetic nanoparticles
Magnetic Nanoparticles MNP
Properties
• diameter > 10 nm
(Hemoglobin Molecule 6 nm)
(Hemoglobin-Molecule
• superparamagnetic
• magnetit
g
Fe3O4 biocompatible
p
Function
• shell for chem. stabilization
• functionalization on shell
• rotate (field), move (gradient)
25 nm
Statistics in Suspension
• shell diameter distribution (Präp.)
• shell functionalization distr.
distr (Präp.)
(Präp )
• thermal distribution (Langevin)
Image of magnetic nanoparticles
from scanning electron microscope
Markers for protein detection
Solid phase - immunoassay
MARIA: MAgnetic Relaxation ImmunoAssay
ELISA: Enzyme linked immunosorbent Assay (AIDS)
Liquid phaseimmunoassay
marker, i.e. magn. nanoparticle
detecting antibody
antigen
fixing antibody
substrate
sandwichimmunoassay
direct immunoassay
aggregation-assay
Immobilization and purification
Antigene
MNP
Magnets
Tube
N
N
N
S
S
S
Contrast agent for
magnetic resonance imaging
Diagnosis
„Resovist“ Fe3O4-contrast agent for MRI from Bayer-Schering
Future: Specific markers by use of antibodies
Hyperthermia = „overheating“
Heating to 40 - 44 °C can kill cancer cells by apoptosis,
also higher effect of radiation and chemotherapy,
chemotherapy
better circulation, rate for damage repair in cells decreases
To much heat kills all cells in environment: blood stain
IInterstitial
t titi l Hyperthermia:
H
th
i LLocall application
li ti
tto overheat
h t
tumor from within
-> www.magforce.de (A.Jordan)
Hyperthermia with magnetic
nanoparticles
ti l
motion
generates
t heat
h t
S
N
100 kHz
Tumor cell incorporates MNP
Drug delivery - „trojan horse“
S
N
Tumor
Further steps
p
1. Magnetic concentrate
2. Magnetic
g
diagnose
g
3. Find disease by themselves
Drug
D
Body
Applications of magnetic markers
Conclusions
Marker binding by proteins (antibodies!)
Measurement of
- Radioactive Markers
RIA
- Optical/enzyme Markers
EIA/ELISA
- Magnetic Markers
MARIA
- Electrically active/enzyme Markers
- Applications of magnetic nanoparticles
Download from www.emg.ing.tu-bs.de/PUC-RIO
Conclusion and Outlook
Magnetic
M
ti nanoparticles
ti l ffor bi
biology
l
and
d medicine
di i
are a dynamic, interdisciplinary research field
•
Magnetic nanoparticles can be employed for
analysis, diagnosis and therapy
•
By functionalization with antibodies they bind
specifically to the target molecules
•
Magnetorelaxation MRX can be used in laboratory
for concentration measurement and for
nanoparticle-characterization / quality control
Acknowledgement
Institute staff
Dr. Frank Ludwig
Dr. Wenzhong Liu
E ik H
Erik
Heim
i
Rainer Piel
Kai Kuchenbrandt
Kerstin Franke
Björn Kuhn
Garnet Alps
p
Fama Ghaffari
Kai Pöhlig
Dennis Rühmer
Philip Martens
Sascha Mäuselein
Thilo Wawrzik
research partners
K. Enpuku, Kyushu Univ.
TU Braunschweig
S 578
SFB
8
PTB: Braunschweig and
Berlin
HZI Braunschweig (GbF)
Magnicon GbR
Chemicell
GlycoThera
Lionex
Tantec
IST-FHG
Klinikum BS
Uni Tübingen
Tübingen, Anatomie
Thank you for your attention