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Transcript of Polydiacetylene Vesicles: Direct Biosensors with a Colorimetric Response Margaret A. Schmitt Samuel...
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 receptor-ligand interactions
Two-fold utility: Basic science level: develops an
understanding of complex biological processes
Applied science level: broad applicability in industrial and medicinal settings
Biologically SensitiveElement
Analyte“recognition” Signal
X No interactionNo signal
Importance of Biosensing Techniques
Glucose level monitoring in individuals with diabetes
Rapid detection of toxins and other biological warfare agents
1 2
3 4
http://www.healthchecksystems.com/bioscanner.htm
Designing Usable Biosensors
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
Conc. Analyte
Conc. AnalyteR
esp
onse
Re
spo
nse
Biosensor Detection Ranges
Detection limit of sensor must be within a relevant range
Sensor must have a reasonable response time
-1
-2
-3
-4
-5
-7
-8
-9
-10
-11
-12
-6
GlucoseCholesterol
Iron
SyphilisRubella
Rh Antigen
Hepatitis
Lo
g C
on
cen
tra
tio
n (
1/m
ol)
Metabolites: mM range
Antibodies/Antigens: nM - pM range
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
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
TARGET
ANTIBODY
REPORTER
Substrate turnoverand signal detection
SUBSTRATE
TARGET TARGET
BINDER
BINDER
BINDER
BINDER BINDER
REPORTER
REPORTER
ANTIBODY
ANTIBODY
Wash
Incubate
Wash
Incubate
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
R2
R1
R2
R1
R2
R1
R1
R2
+ +
R2
Mechanism of Polymerization Reaction
hν+
R1
R2
R1
R2
R1
R2
R1
R2
CC
R1
R2
R1
R2
R1
R2
R1
R2
CC
CC
R1
R2 R1
R2
CC
R1
R2
R1
R2 R1
R2 R1
R2
R1
R2
R1
R2
CC
R1
R2
+
PDA Response to Environmental Changes
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
R1
R2
C C C C C C
R1
R2
R1
R2
R1
R2
CC
R1
R2n
R2
R1
n
No butatrienic structure indicated in either blue or red form as indicated by 13C NMR
Carbon Blue Phase (ppm) Red Phase (ppm) >C= 131.6 132.0−C≡ 107.4 103.6
Tanaka, H.; et. al. Macromolecules 1989, 22, 1208.
Effect of Side Chain Conformation on Chromatic Response
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
R2R1
R1
R2
O NH
O
Carbon Blue (ppm) Red (ppm)
δ-CH2 66.6 65.5
α-CH2 37.3 37.8
ε-CH2 32.9 32.6
β,γ-CH2 24.5 26.4
Tanaka, H.; et. al. Macromolecules 1989, 22, 1208.
Polydiacetylenes as Biosensors
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
R1
R2
R1
R2
R1
R2R2
R1
R2
R1
n
?
Two Supramolecular Approaches for Utilizing Polydiacetylenes in Sensors
Immobilization of polymer as a thin film on a solid glass support
Solution-based sensors incorporating PDA vesicles (liposomes)
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OHO
OOH
OOH
OOH
OOH
OOH
OOH
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
OHO
OHO
OHO
OHO
OHO
OHO
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)
OOH
O
OO
PO
OO
N
O
ODMPC
( ) m
( ) n
M = 6; N = 7 TRCDA
-+
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)
100
RedBlue
Blue
RedBlue
Blue
RedBlue
Blue
AAA
AAA
AAA
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
0 f
0
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
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
R1
R2
R1
R2
R1
R2R2
R1
R2
R1
n
?
Mechanisms of Biochromatic Response
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
Effective length of conjugation in the polymer shortens as a result of desired interaction resulting in a strong blue-red color transition
Observation of Physical Changes in Vesicles in Conjunction with CR
Detection of antibody-epitope recognition
HA peptide-epitope is presented at the N-terminus of a hydrophobic -helix designed to span lipid bilayers
OOH
O
OO
PO
OO
N
O
ODMPC
( ) m
( ) n
m = 6; n = 7 TRCDA
-
+
Kolusheva, S.; et. al. J. Am. Chem. Soc. 2001, 123, 417.
Antibody-Epitope Interaction Results in a Physical Change
Prior to addition of antibody Incubated with HA antibody Incubated with incorrect antibody
Kolusheva, S.; et. al. J. Am. Chem. Soc. 2001, 123, 417.
Vesicles Blue Vesicles Red Vesicles Blue
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
O
OO
PO
OO
N
O
O
-
+
PLA2 PLC PLD
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
OP
O
OO
NO
O
-
+
O
O
OH
P
O
OHON
O
O
+
O
O
OP
O
HOO N
O
O
+
+
+
PLA2
PLC
PLD
OH +
O-
HO-
PLA2 (acyl hydrolase) Cleavage products leave membrane matrix Forms “pits” in membrane surface, resulting
in changes in lipid packing
PLC (phosphodiesterase) Cleavage product is 1,2-diacylglycerol Lipid chains spread apart and expose
hydrocarbon core to aqueous surface
PLD (phosphodiesterase) Cleavage product is phosphatidic acid (PA) PA has affinity for Ca2+ ions in buffer Interaction with cations results in vesicle
condensation
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
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
R1
R2
R1
R2
R1
R2R2
R1
R2
R1
n
?
Location of Polymerization Group
10,12-PDAs have a much more rigid hydrophobic chain prior to the diacetylene moiety
Strong connection between conformation of alkyl chain and polymer electronic properties
5,7-PDAs are expected to be more responsive to environmental changes
OOH
( ) m
( ) n
m = 0 or 6
m = 0: TCDA and DCDAm = 6: TRCDA and PCDA
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 E. coli with 2,4-PDA vesicles (and sialic acid receptor)
No response to E. coli with 10,12-PDA vesicles
Positive response to cholera toxin with 5,7-PDA vesicles (and ganglioside receptor)
No response to cholera toxin with 10,12-PDA vesicle
OOH
( ) n
OOH
( ) nPan, J.; Charych, D. Langmuir 1997, 13, 1365.Ma, Z.; et. al. J. Am. Chem. Soc. 1998, 120, 12678.
OOH
( ) n
OOH
( ) n
vs.vs.
Incorporation of Recognition Element
Incorporated on separate membrane-spanning peptide in antibody-epitope studies
Synthetically attach recognition element to lipid containing diacetylene moiety
Incorporate recognition element through a lipid in the system which does not contain a diacetylene moiety, and therefore cannot be polymerized
O
NH
O
OCOOH
HOAcHN
HO
HOOH
OO
ONH
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
O
NH
O
OCOOH
HOAcHN
HO
HOOH
OO
ONH
O
HO10,12-pentacosadiynoic acid (PCDA)
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)
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40 50 60 70
Amount of Virus [HAU]
Co
lori
me
tric
Re
sp
on
se
[%
]
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
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
NH O
NH2
Lys
NH OAla
NH O
Phe
NH O
Leu
NH O
Ile
NH OVal
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 B) Lys/Ala peptides E) Lys/Ile peptides
C) Lys/Phe peptides F) Lys/Val peptidesD) Lys/Leu peptides
Wyrsta, M.; Cogen, A.; Deming, T. J. Am. Chem. Soc. 2001, 123, 12919.
Ala, Phe, and Leu: α-helix favoringIle and Val: β-sheet favoring
Blue = negativeRed/Orange = positive
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, antibody-epitope 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