Transcript Slide 1

Polydiacetylene Vesicles: Direct
Biosensors with a Colorimetric
Response
Margaret A. Schmitt
Samuel H. Gellman Group
University of Wisconsin, Madison
February 20, 2003
Outline

Biosensors



Definition and Introduction
Direct and Indirect
Polydiacetylenes




Polymerization Reaction
Chromic Response to Environmental Changes
Harnessing Chromic Response in a Useful Biosensor
Construct
Variables Associated with Designing an Appropriate
PDA Biosensor for a Variety of Systems
Biosensors




Device incorporating a biological
sensing element directly connected
to a signal transducer
Biosensor design research attempts
to couple Nature’s “lock-and-key”
interactions with cleverly
engineered signal transduction
mechanisms
Molecular recognition assumes
many forms: enzyme-substrate,
antibody-antigen, and receptorligand interactions
Two-fold utility:


Basic science level: develops an
understanding of complex biological
processes
Applied science level: broad
applicability in industrial and medicinal
settings
Analyte
“recognition”
Signal
Biologically Sensitive
Element
X
No interaction
No signal
Importance of Biosensing Techniques

Glucose level monitoring in
individuals with diabetes
1
3
2
4
http://www.healthchecksystems.com/bioscanner.htm

Rapid detection of toxins
and other biological warfare
agents
Response
Designing Usable Biosensors
Response
Conc. Analyte
Conc. Analyte


Signal must have a direct relationship to quantity of material being
analyzed
Sensor must demonstrate specificity and selectivity in recognizing a
single compound or group of compounds in a varied mixture
Biosensor Detection Ranges
-1

Detection limit of sensor
must be within a relevant
range
Sensor must have a
reasonable response
time
Glucose
Cholesterol
Metabolites:
mM range
-3
Log Concentration (1/mol)

-2
-4
Iron
-5
-6
Antibodies/Antigens:
nM - pM range
-7
-8
Syphilis
Rubella
-9
Rh Antigen
-10
Hepatitis
-11
-12
Indirect vs. Direct Biosensors
Indirect: Relies on detection of a
labeled ligand after a binding event
has occurred.
Direct: Binding event is directly linked to
a signal transduction event for detection
in real-time.
Indirect Biosensor: ELISA
REPORTER
SUBSTRATE
REPORTER
REPORTER
Substrate turnover
and signal detection
ANTIBODY
BINDER
Wash
Wash
ANTIBODY
Incubate
ANTIBODY
Incubate
BINDER
BINDER
TARGET



BINDER
TARGET
BINDER
TARGET
Based upon tight binding between an antigen and antibody
Labeling agents for ligands include fluorescent probes and radioisotopes
Most commonly used reporter enzyme horseradish peroxidase (HRP), which
upon reaction with substrate produces a bright green color
Direct Biosensor: SPR

Optical detection method for
studying interactions between
a soluble analyte and
immobilized ligand

Binding of the analyte
molecule changes the
refractive index in a way that is
approximately proportional to
the mass of the molecules
which have entered the
interface

Stoichiometry of binding can
be examined
http://www.astbury.leeds.ac.uk/Facil/spr.htm
Advantages and Disadvantages of Indirect
and Direct Biosensors

Indirect
Advantages





Amount of material required
Can detect virtually any material
(ELISA)
Sensitivity
Signal amplification
Disadvantages


Labeled ligands or secondary
reagents required
Background problems – washing is
necessary
Direct

Advantages



Binding event results in signal
transduction
Signal measures only the desired
interaction
Disadvantages



Specialized machinery is often
required
Signals more difficult to amplify
Time-consuming
“Ideal” Biosensors



Response is directly coupled to recognition event: Direct
Signal readily detectable without the use of expensive or
large instrumentation
Adaptable to detect many types of analytes
Outline

Biosensors



Definition and Introduction
Direct and Indirect
Polydiacetylenes




Polymerization Reaction
Chromic Response to Environmental Changes
Harnessing Chromic Response in a Useful Biosensor
Construct
Variables Associated with Designing an Appropriate
PDA Biosensor for a Variety of Systems
Diacetylene Polymerization

Topochemical polymerization reaction

Reaction is very sensitive to the surrounding environment and packing
of substituents

Reacting carbon atoms must be less than 4 Å away from each other or
polymerization is not likely to occur
R1
+
R2
R1
R1
+
R2
R2
R2
R1
R1
R2
R2
R1
Mechanism of Polymerization Reaction
R1
R1
+
R2
R1
R1
hν
+
R2
R1
C
C
R2
R2
R1
C
R1
C
R2
C
R2
R1
R1
R2
R2
R1
R2
C
R2
R2
R1
R1
C
R2
R2
C
R2
C
R2
R1
R2
R1
R2
C
R1
R1
R1
PDA Response to Environmental Changes
R2
R1
R1
R2
R2
R1
R1
R1
R1
R1
R2
R2
R2
R1
R1
R1
C C
R2
R2
R2
R2
n
R2
R1
C C
R2
R1
C C
R2
Blue Phase (ppm)
131.6
107.4
C
C
R2
n
No butatrienic structure indicated in either blue or red form as indicated by
Carbon
>C=
−C≡
R1
R1
13C
NMR
Red Phase (ppm)
132.0
103.6
Tanaka, H.; et. al. Macromolecules 1989, 22, 1208.
Effect of Side Chain Conformation on
Chromatic Response
Carbon
δ-CH2
α-CH2
ε-CH2
β,γ-CH2
Blue (ppm)
66.6
37.3
32.9
24.5
Red (ppm)
65.5
37.8
32.6
26.4
R1
R1
R2


Only β,γ-carbons show significant
shift between 2 phases

Conformational change around
backbone single bonds is minimal
as α-carbon chemical shifts to not
change significantly
R1

O
O
N
H
R2
Tanaka, H.; et. al. Macromolecules 1989, 22, 1208.
Polydiacetylenes as Biosensors




R1
R1
R1
R2
R2
R2
R1
R1
R2
R2
n
Incorporation into vesicles
Methodology of assay
Physical changes in vesicles and relationship to color change
Variables associated with appropriate biosensor design


Position of diacetylenic functionality
Incorporation of recognition element
?
Two Supramolecular Approaches for
Utilizing Polydiacetylenes in Sensors
Immobilization of polymer as a
thin film on a solid glass support
HO
O
HO
O
HO
O
HO
O
HO
O
HO
O
Solution-based sensors incorporating
PDA vesicles (liposomes)
HO
O
HO
O
HO
O
O
HO
OH
O
O
HO
OH
O
O
HO
OH
O
O
OH
O
OH
O
OH
Advantages of Vesicles




Liposomes can be made more simply and reproducibly
Vesicle assays and analysis can be done in a 96-well plate format
Liposomes mimic the cell membrane more closely than thin films
Ability to immobilize and remain functional on a surface
HO
O
HO
O
HO
O
HO
O
HO
O
HO
O
Vesicle Immobilization onto Au Films

Use lipid with disulfide
containing headgroup to
immobilize vesicles on gold

Disulfide remains oxidized,
reducing vesicle aggregation

Vesicles remain highly
monodisperse over periods of
3 days in buffer
Stanush, I.; Santos, J.; Singh A. J. Am. Chem. Soc. 2001, 123, 1008.
PDA Vesicle Stability

Stable at 4ºC in solution for
months

Can be made stable to
lyophilization and resuspension

Not sensitive to white light

Show no evidence of fusion to
form large aggregates

Not destroyed osmotically by
salts
PDA Vesicles: Synthesis

Diacetylenic monomer molecules self-assemble into an ordered
array by the same driving forces which occur in the formation of
biological membranes

Vesicle formation is encouraged by sonication

Monitor polymerization reaction by appearance of a deep blue color
Design of Colorimetric Assay




Analyze peptide-membrane interactions
Utilize well-characterized antimicrobial peptides and related mutants to
examine interactions at vesicle surface and colorimetric response (CR)
Amphiphilic peptides severely disrupt membrane surface and may insert
into membrane and form pores
Vesicles contained 6:4 mole ratio of TRCDA and phospholipid (e.g. DMPC)
OH
O
(
)m
O
O
O
O
O
P
O O
O-
+
N
DMPC
M = 6; N = 7
TRCDA
(
)n
Kolusheva, S.; Boyer, L.; Jelinek, R. Nature Biotechnology 2000, 18, 225.
Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 15851.
Colorimetric Assay

Vesicle solutions buffered with Tris to pH 8.5

Incubate peptide and vesicles for 30 min at 27ºC and measure CR
Control: no peptide
Cells containing amphiphilic peptides
Kolusheva, S.; Boyer, L.; Jelinek, R. Nature Biotechnology 2000, 18, 225.
Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 15851.
Colorimetric Response (CR)
 ABlue   ABlue 



 ABlue  ARed 0  ABlue  ARed  f100
 ABlue 


 ABlue  ARed 
0

Calculation of quantitative value
for extent of color transition from
initial blue state to final red state

A: absorbance at the “blue”
(~640nm) or “red” (~500nm)

Depending upon background
levels and non-specific
interactions, interactions can be
detected with as little as 5-7% CR
Kolusheva, S.; Boyer, L.; Jelinek, R. Nature Biotechnology 2000, 18, 225.
Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 15851.
Non-Specific PDA-Analyte Interactions

Measure CR with pure PDA
vesicles to determine changes
due to interactions between
analyte and negatively charged
PDA portion of vesicles

Even at μM concentrations,
melittin can be detected above
background
Kolusheva, S.; Boyer, L.; Jelinek, R. Nature Biotechnology 2000, 18, 225.
Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 15851.
Negative Controls



Expose vesicles to mismatched analyte to rule out CR resulting from non-specific
interactions with the recognition element
Examine response due to presence of peptides not expected to be membrane
active (e.g. neuropeptides)
Peptide-membrane interactions are non-specific; use to ensure CR is due only to
membrane interactions and disruption and not presence of other analytes
No peptide
Antimicrobial Peptides
Neuropeptide
(no membrane interaction)
Kolusheva, S.; Boyer, L.; Jelinek, R. Nature Biotechnology 2000, 18, 225.
Kolusheva, S.; Shahal, T.; Jelinek, R. Biochemistry 2000, 39, 15851.
Polydiacetylenes as Biosensors




R1
R1
R1
R2
R2
R2
R1
R1
R2
R2
n
Incorporation into vesicles
Methodology of assay
Physical changes in vesicles and relationship to color change
Variables associated with appropriate biosensor design


Position of diacetylenic functionality
Incorporation of recognition element
?
Mechanisms of Biochromatic Response
Effective length of conjugation in the polymer shortens
as a result of desired interaction resulting in a strong
blue-red color transition

Insertion of viral membrane or
toxin hydrophobic domains
into the PDA bilayer

Multipoint interactions of the
receptor at the PDA-vesicle
surface changing packing of
lipid headgroups near surface
Observation of Physical Changes in
Vesicles in Conjunction with CR
OH

O
Detection of antibody-epitope
recognition
(
)m
O
O
O

HA peptide-epitope is
presented at the N-terminus of
a hydrophobic -helix
designed to span lipid bilayers
O
O
P
O -O
O
+
N
DMPC
m = 6; n = 7
TRCDA
(
)n
Kolusheva, S.; et. al. J. Am. Chem. Soc. 2001, 123, 417.
Antibody-Epitope Interaction Results
in a Physical Change
Prior to addition of antibody
Vesicles Blue
Incubated with HA antibody
Vesicles Red
Incubated with incorrect antibody
Vesicles Blue
Kolusheva, S.; et. al. J. Am. Chem. Soc. 2001, 123, 417.
Varied Mechanisms of Membrane Interaction



Evidence of phospholipase activity : PLA2, PLC, and PLD
Enzymes which hydrolyze cell membrane phospholipids
Each enzyme cleaves PC in a different location, but activity of each
results in a similar colorimetric response
PLA2 PLC
O
O
O
O
P
O -O
O
PLD
+
N
O
Jelinek, R.; et. al. Chem. Biol. 1998, 5, 619.
Okada, S.; Jelinek, R.; Charych, D. Angew. Chem. Int. Ed. 1999, 38, 655.
Cleavage Products Disrupt Membrane
O

OH
+
O
OH
O

O
O
P
O
N
O
-

+
O
OH
+
O
HO
O
O
P
- O
O
N
+
PLC (phosphodiesterase)

Cleavage product is 1,2-diacylglycerol

Lipid chains spread apart and expose
hydrocarbon core to aqueous surface
PLC

PLD (phosphodiesterase)

O

O
O
O
O
Cleavage products leave membrane matrix
Forms “pits” in membrane surface, resulting
in changes in lipid packing
PLA2

O
PLA2 (acyl hydrolase)
O
P
HO O
+
HO
N
+

Cleavage product is phosphatidic acid (PA)
PA has affinity for Ca2+ ions in buffer
Interaction with cations results in vesicle
condensation
PLD
Jelinek, R.; et. al. Chem. Biol. 1998, 5, 619.
Okada, S.; Jelinek, R.; Charych, D. Angew. Chem. Int. Ed. 1999, 38, 655.
Polydiacetylenes as Biosensors




R1
R1
R1
R2
R2
R2
R1
R1
R2
R2
n
Incorporation into vesicles
Methodology of assay
Physical changes in vesicles and relationship to color change
Variables associated with appropriate biosensor design


Position of diacetylenic functionality
Incorporation of recognition element
?
Location of Polymerization Group

10,12-PDAs have a much more
rigid hydrophobic chain prior to
the diacetylene moiety
OH
O
(


Strong connection between
conformation of alkyl chain and
polymer electronic properties
5,7-PDAs are expected to be
more responsive to environmental
changes
)m
m = 0 or 6
m = 0: TCDA and DCDA
m = 6: TRCDA and PCDA
(
)n
Thermochromism of 5,7- and 10,12-PDAs

Examine thermochromism in
response to incubation at 50ºC
as a function of time

Vesicles composed of 5,7-PDAs
express an enhanced response
compared to 10,12-PDAs

Drawback of this enhanced
response is that 5,7-PDAs are
more readily affected by
properties of their solution: salt
content, pH, etc
Okada, S.; et. al. Acc. Chem. Res. 1998, 31, 229.
Location of Polymer Backbone and
Effective Biochromic Response


Positive response to cholera toxin with
5,7-PDA vesicles (and ganglioside
receptor)
No response to cholera toxin with 10,12PDA vesicle

Positive response to E. coli with 2,4-PDA
vesicles (and sialic acid receptor)

No response to E. coli with 10,12-PDA
vesicles
OH
O
OH
O
OH
O
O
OH
vs.
vs.
(
)n
(
)n
(
)n
(
)n
Pan, J.; Charych, D. Langmuir 1997, 13, 1365.
Ma, Z.; et. al. J. Am. Chem. Soc. 1998, 120, 12678.
Incorporation of Recognition Element

Incorporated on separate membranespanning peptide in antibody-epitope
studies

Synthetically attach recognition
element to lipid containing diacetylene
moiety
HO
OH
HO
COOH
AcHN
O
O
HO
O

N
H
O
O
O
N
H
Incorporate recognition element
through a lipid in the system which
does not contain a diacetylene moiety,
and therefore cannot be polymerized
Synthetic Attachment of Recognition
Element


Bifunctional molecule incorporates both the recognition element (sialic
acid) and the reporter diacetylene moiety
Surface lectin of influenza virus (hemagglutinin) binds terminal glycosides (sialic acid residues) on cell surface glycoproteins and
glycolipids
HO
OH
HO
COOH
AcHN
O
O
HO
O
N
H
O
O
O
N
H
O
10,12-pentacosadiynoic acid (PCDA)
HO
Reichert, A.; et. al. J. Am. Chem. Soc. 1995, 117, 829.
PDA Vesicle Detection of Influenza Virus

HA binds cell surface
sialic acid residues and
initiates viral infection
Detection of as little as 11
HAUs of virus particle
(~11 x 107 virus particles)
90
Colorimetric Response [%]

80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
Amount of Virus [HAU]
Reichert, A.; et. al. J. Am. Chem. Soc. 1995, 117, 829.
Incorporation of Recognition Element on
a Non-Polymerizable Lipid



Useful when receptor of interest is already lipid linked or when
attaching receptor to diacetylenic lipid may be synthetically
challenging
Gangliosides are lipid molecules that reside on the surface of the
cell membrane and display carbohydrate recognition groups
Cholera toxin recognizes GM1 ganglioside
5,7-docosadiynoic acid (DCDA)
Pan, J.; Charych, D. Langmuir 1997, 13, 1365.
Detection of Cholera Toxin


Detection of slightly less than
100 μg/ml cholera toxin
Response is slightly
sigmoidal


Binding cooperativity –
binding one ligand makes the
vesicle more accessible for
others
Polymer side chain
conformations – once the
effective conjugated length of
the vesicle is perturbed as the
result of toxin binding,
subsequent perterbation is
more favorable
Pan, J.; Charych, D. Langmuir 1997, 13, 1365.
Screening a Library with PDA Vesicles
NH2



Examine structure-activity
relationships in a library of
amphiphilic co-polypeptides
Relationship between
polypeptide -amino acid
composition and interaction
with phospholipids found in
cell membranes
Suggest important factors for
designing new antimicrobial
peptides
N
H
O
Lys
N
H
O
N
H
O
N
H
Ala
N
H
Ile
O
Phe
N
H
O
Leu
O
Val
Wyrsta, M.; Cogen, A.; Deming, T. J. Am. Chem. Soc. 2001, 123, 12919.
Detection of Peptide-Membrane Interactions



Most membrane-active
peptides are of intermediate
chain length and high
hydrophobic content
Peptides containing -helix
favoring amino acids interact
with vesicles and produce a
colorimetric response
Peptides containing β-sheet
favoring amino acids do not
produce any colorimetric
response
Ala, Phe, and Leu: α-helix favoring
Ile and Val: β-sheet favoring
B) Lys/Ala peptides
C) Lys/Phe peptides
D) Lys/Leu peptides
E) Lys/Ile peptides
F) Lys/Val peptides
Blue = negative
Red/Orange = positive
Wyrsta, M.; Cogen, A.; Deming, T. J. Am. Chem. Soc. 2001, 123, 12919.
Future Directions




Continue to examine the mechanism of PDA biochromic
response
Apply vesicle methodology in evaluation of compounds with
unknown activity (e.g. potential antimicrobial peptides or enzyme
inhibitors)
Correlate colorimetric response with desired biological
interaction
Examine biochromic responses in new constructs and
immobilized vesicles
?
Conclusions

Polydiacetylene vesicles mimic the properties of cell signaling by
directly coupling a bio-recognition event to signal transduction

Recognition events in a PDA vesicle result in a visible colorimetric
signal which changes from blue to red

PDAs are able to detect peptide-membrane interactions, antibodyepitope recognition, enzyme binding and catalysis, and virus and
toxin molecule recognition

If a relationship between the colorimetric response of PDAs and
desired bio-recognition events can be shown, PDA vesicles could
become a useful sensing technique with a wide variety of
applications
Acknowledgements
Gellman Group
Nick Fisk
Terra Potocky
Tim Peelen
Jon Lai
Marissa Rosen
Erin Carlson