Fluorogenic Polydiacetylene Nanosomes:
Application to Label-Free Nanobio Sensors
Dong June Ahn
[email protected], +82-2-3290-3301
Department of Chemical & Biological Engineering
College of Engineering, Korea University
Seoul, Korea
April 17-19, 2008
D. J. Ahn, Korea University
Needs for Sensors
New & Mutated Infectious Diseases
Diagnoses & Drug Discovery
SARS
Anthrax
Mutated
Cholera,
etc.
Astronomical cost
spent each year
worldwide !
Need for Disease Monitoring
Bio Chips & Chemical Chips
Safety for Processed Food
D. J. Ahn, Korea University
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Outline
¾ Polydiacetylene Supramolecules
¾ Detection Examples
¾ Nanosome-Based Chips
¾ Nanosome Strips and Fibers
¾ Summary
D. J. Ahn, Korea University
Current Concept for Sensing Systems and Chips
• Sensor Systems
Target Substance
A
B
C
C
B B
Recognizing Materials
A
• Diagnostic Array Chips
Human Judgment
A
A
A
B
C
A
A
C
Interface I
Target / Sensor Matrix
Interface II
Sensor Matrix / Human
Affinity
Shape
Size
Signal Transducer
Transistors / Optics
Electrochemistry
Mechanics / Nanowires
Labels: Cy5, Cy3,
nanoparticles, radio tracers
T
C
A
A
G
T
T
C
A
Pre-treatments
DNA Chip
For Labeling Analytes
Protein Chip
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Why Conjugated Systems?
• Conventional Chemosensor
T
target molecule
S-T
S
Sensitivity depends on the equilibrium constant.
Keq = [S-T] / [S] [T]
• π-Conjugated Polymer Sensor
target molecule
Signal amplification can be achieved.
D. J. Ahn, Korea University
π-Conjugated Polymers
• π-Conjugated Polymers
O
O
O
O
O
O
O
O
R
R
O
R
O
R
S
R
NH HN
S
R
n
n
Polythiophene
Polypyrrole
Poly(phenyleneethylene)
R
N
R
R
N
R
Combination (PPE + Bipyridil)
R
R
Polydiacetylene
• Electrical and/or optical properties
Easily dispersed into aqueous media !
D. J. Ahn, Korea University
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Polydiacetylene Supramolecules
• Synthetic Diacetylene Supramolecules
• Natural Cell Membranes
O
Hydrophobic tail
Ligand
diacetylene unit
inner aqueous compartment
Hydrophilic head
molecular recognition
self-assembly
Langmuir-Blodgett
Langmuir-Schaefer
dispersion by
sonication
solid-supported films
X
X
X
O
O
O
hydrophobic
tails
X
X
X
O
O
O
lipid bilayers
hydrophilic
head groups
nanosome (vesicle) solution
oligosaccharide
hydrophobic
α-helix
glycolipid
O
X
O
X
O
O
O
X
X
O
X
X
AFM
SEM
phospholipid
integral
protein
cholesterol
molecular spacing : 4.8±0.2Å
Cytosol
size : 50 nm
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Optical Characteristics
Red
Blue
Colorless
Stimuli
hν
X
X
X
O
O
O
O
O
X
O
X
O
X
O
X
X
O
X
O
X
O
X
O
X
O
X
O
X
O
X
O
X
X
O
X
O
Real-Time
Observation
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Polydiacetylene-Based Sensor Systems
target
color change
conjugated p-orbital
twisted p-orbital
blue
red
(Increased response)
Blue phase
(Non-fluorescent)
Red phase
(Fluorescent)
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Detection Examples
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Detection of Influenza Virus A
Charych et al., Science, 261, 585 (1993); J. Am. Chem. Soc., 117, 829 (1995).
increase of virus concentration
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Detection Examples
• Influenza Virus A: Charych (1993, 1995)
sialic acid
• Antibody: Jelinek (2001)
OH
HO
OH
O
COOH
O
AcHN
HO
NH
O
O
peptide
O
O
OH
O
NH
O
OH
5
5
6
5
6
O
O
R1
N
H
H
N
O
R3
N
H
R2
6
• Small Molecule (Cyclodextrin):
Ahn & Kim (2003, 2004)
• DNA: Wang & Ma (2005)
pore match
NH
O
NH
O
NH
O
5
5
6
6
5
6
D. J. Ahn, Korea University
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O
OH
O
OH
O
“Conceptual Evolution”
OH
5
5
6
6
5
6
Nanosome Solutions
Films on Substrates
Method: Langmuir-Schaefer
Use: Film-type Sensor
Low Signal
One Binding Event per Film
New
Concept
integration
Method: Sonication
Use: Solution-type Sensor
Medium-High Signal
One Binding Event per Solution
NH 2
H 2N
NH 2
H 2N
NH 2
H 2N
NH 2
H 2N
N
H
N
H
200㎛
Method: Surface Chemistry
Embedded
Nanosomes
Advanced Materials, 15, 1118 (2003).
Method: Polymer Blending
Patterned Nanosomes
“Nanosome Strips”
“Nanosome Fibers”
Surface Immobilized Nanosomes
Method: Microcontact Printing
Use: Sensor
One Binding Event per Patterned Nanosome Surface
Method: Molding & Spinning
Use: Chemical & Bio Sensor
Blue
“Nanosome Chips”
Blue
Method: Inkjet Spotting
Use: Chemical & Biological Chip
Simultaneous Screening & Detection
Blue
Purple
Pink
Self-Emitting Fluorescence
“Label-Free System”
Red
J. Am. Chem. Soc., 127, 17580 (2005).
Advanced Functional Materials,
16, 2103 (2006).
Advanced Materials, 19, 521 (2007).
D. J. Ahn, Korea University
Nanosome-Based Chips
D. J. Ahn, Korea University
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Nanosome Array Chips
Inkjet (non-contact), single tip
< Ink-jet Microarrayer >
AP
S
gl
as
s
< Microarray of Nanosomes >
1 spot (4 nL), width: 200 ㎛
Pre-spot Nanosome Library
Solutions in 96 or 384 well plates
•
•
•
•
Conventional sol’n analysis: 0.1 mL
25,000-fold saving
Tiny amount of analytes
Simultaneous multiple screening
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Microarray-Based Fluorescence Profiles
α-CD (30mM )
γ-CD (30mM)
γ-CD → Heating
Linear Carbohydrate :
Maltoheptaose(30mM)
Maltoheptaose → Heating
Polyacrylic acid (30mM)
Ahn et. al., J. Am. Chem. Soc., 127, 17580 (2005).
D. J. Ahn, Korea University
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A Label-Free Detection of Protein-Protein Interaction
initial
after exposure to anti-rabbit
secondary antibodies
when primary after heated
antibody is absent
after heated
after exposure to anti-mouse
secondary antibodies
after heated
Advanced Functional Materials (2007); Macromolecular Research (2006).
D. J. Ahn, Korea University
A Label-Free Detection of Bacteria
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Detection of E. Coli
PCDA-Aniline + PCDA-EDEA-s-NHS
PCDA + PCDA-EDEA-s-NHS
when primary
antibody is absent
Submitted to Angew. Chem. Int. Ed. (2007).
D. J. Ahn, Korea University
DNA Detection Sensitivity
Spotting of vesicle-probe DNA complex
3` - GAT GGA TGC CCA TTG - 5`-NH2
Polymerization
5` - CTA CCT ACG GGT AAC - 3`
E. coli lethal factor sequence 15 mer.
200 ㎛
target DNA
250
Intensity (a.u.)
200
150
100 nM
1 nM
10 nM
10 pM
100 pM
100
50
0
100 nM 10 nM 1 nM 100 pM 10 pM 1 pM 100 fM 10 fM
Target DNA conc.
1 fM
--
1 pM
100 fM
10 fM
1 fM
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DNA Detection Selectivity:
Perfect Match vs. Single-Base Mismatch
Vesicle
Vesicle & probe DNA
Pre spot Solution
NH2 – 5` - GTT ACC CGT AGG TAG - 3`
3` - CAA TGG GCA TCC ATC - 5`
TAG
A
G
T
200 ㎛
Single strand
Polymerization
Immobilization
[
Si O Si O Si O Si O Si O Si O
O
O
Si
Si
O
O
O
Si
Si
O
Si
]
n
Si
Double strand
target DNA
Mismatched DNA
Si O Si O Si O Si O Si O Si O
TAG
G
A
O
O
O
O
O
O
Si
Si
Si
Si
Si
Si
]
n
T
Filter 3.
(green)
D. J. Ahn, Korea University
Nanosome Strips and Fibers
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Nanosome-Embedded Strip Films
PCDA liposomes in PVA
Drying
Thermal Response
Confocal Fluorescence
Microscopic Image
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“Litmus-Type” Strip Sensors for Chemicals
O
OH
O
OH
O
OH
O
OH
5
5
7
7
5
5
7
7
control
α-CD
β-CD
γ-CD
strip
solution
Chemistry Letters, 35, 560 (2006).
D. J. Ahn, Korea University
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Image Display Using Nanosome-Embedded Films
Advanced Functional Materials, 16, 2103 (2006).
after
photopolymerization
thru a photomask
after heating
after
photopolymerization
of the remaining area
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Electrospun Polydiacetylene Fibers
R2
R1
R1
R1
R2
R2
R2
R1
R2
R1
Randomly-distributed DA
electrospinning
R2
R1
R1
R2
R1
R2
R2
R1
Self-assembled DA
hv
R2
R2
R2
R2
R1
R1
R1
R1
PDA-embedded fiber
Advanced Materials, 19, 521 (2007).
D. J. Ahn, Korea University
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Visual Detection of Food Transmutation
beef, pork
pH=8.8
hairtail, croaker, skate, etc
chicken
pH=11.0 pH=12.3
Gas Analytes
Liquid Analytes
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Visual Alarm System
(Example Case: Formaldehyde)
HCHO
detection
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Summary of Sensor Research
Blue
Blue
Red
100㎛
+ Target
+ Target
Blue
Blue
+ Target
Purple
- Target
Pink
Red
100㎛
Self-Emitting Fluorescence
“Label-Free Chip System”
Color-Changing
“Label-Free Strip System”
Label-Free Chips and Strip Sensors
No Fluorescence
Fluorescence
Reusable or Continuous-Monitoring System
D. J. Ahn, Korea University
Acknowledgments
My Dear Students
Home Coming Day
Dr. D. H. Charych (Lawrence Berkeley Nat’l Lab. / Chiron Corp. / Five Prime Corp.)
Prof. Jong-Man Kim (Hanyang University)
Research Funds from Korean Government (KISTEP, KRF, KOSEF, MOH) and Private Sectors
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