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


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


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


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)





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.




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

polydiacetylene vesicles: direct biosensors with a colorimetric response margaret a. schmitt samuel...

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