Sirigen Confidential

1

Abstract

Improving Photophysical Properties of Brilliant Violet™ Polymers via

Structural Modifications

Sirigen/BDB

Lan Tran

May 2013

Professional Masters Degree Program

Cal State University, San Marcos

Department of Product Development; Sirigen Inc., San Diego, Ca

Sirigen’s HSF™ dyes are conjugated polymers which are designed to be brighter than

conventional dyes for detection in diagnostics and research applications. The properties

of these fluorophore-enriched polymers are engineered through modifications of core

monomers, followed by selective polymerization techniques. The goal of this research

was to improve the photophysical properties of existing Brilliant Violet™ polymers via

structural modification where discrete polyethylene glycol (dPEG) is coupled to the

internal linker monomer. Polymers were generated via Yamamoto polymerization

technique using the modified internal linker monomer and comonomer. This technique

uses nickel complex as catalyst, and allows symmetric coupling of monofunctional

comonomers. Generated polymers were conjugated to acceptor dyes at 570nm, 650nm,

and 785nm emission bands and solubility, FRET efficiencies, and photostability were

evaluated and compared to existing Brilliant Violet™ products. The introduction of

dPEG to the internal monomer linkers did not result in a significant improvement in

initial energy transfer of donor to acceptor emission; however, addition of varying

dPEG-spacer lengths to lengthen the internal linker shown tremendous improvement on

photostability of the polymer-dye complexes. The fluorescence measurements shown

modified polymers with longer internal linkers resulted in less dye quenching and

reduced photodegradation compare to current Brilliant Violet™ product.

Improving Photophysical Properties of Brilliant Violet™ Polymers

via Structural Modifications

By: Lan Tran

April 2013

1

t display bright distinguish colors

Sirigen’s High Sensitivity Fluorescence Technology: background

Technology originated at UCSB and developed by Sirigen (founded in 2005)

High Sensitivity fluorescence (HSF)

HSF generated from conjugated polymers (materials tha

under UV light detection)

Signal amplification process

Key advantages:

Offer broad range of colors for multiplex detection

High brightness for increased sensitivity and accuracy

Improved alternative to existing small molecule dyes (conventional fluorophores)

Slide 2

HSF delivers enhanced signals due to light harvesting capability of polymer

HSF materials deliver enhanced signals = lowering limits of detection and improve diagnosis

Light harvesting capability

Collection of optical segments

Slide 3

Energy “collected” by the polymer transfers to a localized accepter dye

Slide 4

Excitation results in electronic delocalization along the chain

High Sensitivity Fluorescence

Slide 5

Light harvesting effect allow standard fluorescent labels to be amplified

Current Brilliant Violet™ Polymers Application: Flow cytometry allows for detection of

multiple target cells simultaneously in a single assay

Slide 6

Improve detection methods : early and accurate detection in cells via Flow Cytometry

n

n

liquid flow

detectors

Research Goal: Improve Current Brilliant Violet™ polymers

Slide 7

Impro ve solubility, energy transfer, and photostability

Polymer generation

Key is to make well defined monomers These are then polymerized to make the polymer of interest

Tailoring the monomer allows one to “tune” the properties of the final materials

= +

Monomer building blocks

￭ Organic synthesis

Monomer Selection &

Polymer Design

￭ Based on literature &

experience (Sirigen core)

Polymer Generation

￭ Metal catalyzed coupling

￭ GPC, FPLC, UV-VIS

characterizations

Materials & Methods: Multistep synthesis to generate 3 base polymers

Slide 8

R0 R1

+

Modified internal linker monomer

comonomer

CH3

n

R0

R1

Site of Dye Attachment

3 modified base polymers differ in internal linker subsituents per repeat

unit

Slide 9

n

R0

R1

CH3

L1-spacer L2-spacer CH3

LX-spacer

(dye) (dye) (dye)

Site of Dye Attachment

Materials & Methods: Conjugation to small molecule dyes to yield 9 products across

3 emission bands

Slide 10

=

a b c

POL1a-c : a = 570nm, b = 650nm, c = 785 nm

POL2a-c : a = 570nm, b = 650nm, c = 785 nm

POLXa-c : a = 570nm, b = 650nm, c = 785 nm

@ 570nm, 650nm, 785 nm emission bands

n

R0

R1

n

R0

Results & Discussions: Characterizations using GPC and FPLC

Slide 11

Gel Permeation Chromatography (GPC)

Also known as SEC; equipped with UV-VIS detector Separates dissolved molecules on the basis of their size

Organic solvent as the mobile phase

Mn, Mp, and PDI are all within specifications of current product

Determination of the Percent incorporation of modified internal linker monomer in the polymer

Fast Performance Liquid chromatography (FPLC) with 3-wavelength UV-detector

Separation of polymer-dye complex from free dye via size exclusion chromatography

Calculations using dye and polymer Absorption peak ratios

Confirmed desired results aligned with the controlled ratio of co-monomer content in

Yamamoto coupling.

Dye wavelength peak

Polymer wavelength peak

Results & Discussions: Energy Transfer

Slide 12

Energy transfer measurements via fluorescence spectroscopy

Polymer (donor) emission peak & dye (acceptor)

emission peak

POL1 and POL2 are comparable to current products

Initial D/A Ratio

Polymers

λem POL1 POL2 POLX

Brilliant

Violet™

Comparisons

570 35% 32% 43% 18-31% Slightly worse

650 12% 14% 36% 9-21% Within range

785 18% 22% 34% 18-30%

Within range

0

0.2

0.4

0.6

0.8

1

1.2

400 450 500 550 600 650 700 750 800

Brilliant Violet(TM) Emission

Emission

Rela

tive S

ign

al

In

ten

sit

y

Wavelength, nm

Results & Discussions: Improved photostability of polymer-dye complexes

across all three emission bands

Slide 13

Fluorescent signal of polymers was measured over 24 hours

Monitored the emission of the donor and acceptors

POL1 and POL2 with linker-spacers retains photostability property across at 570nm, 650nm,

and 785nm emission bands.

−POL1-D/A Ratio

−POL2-D/A Ratio

−POLX-D/A Ratio

−Brilliant Violet™-D/A Ratio

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25

650 D/A Ratio

Rela

tive S

ign

al

In

ten

sit

y

Time, hr

0

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25

570 D/A Ratio

Rela

tive S

ign

al

In

ten

sit

y

Time, hr

0

0.5

1

1.5

2

0 5 10 15 20 25

785 D/A Ratio

Rela

tive S

ign

al

In

ten

sit

y

Time, hr

Conclusions

Slide 14

Three structurally modified base polymers were generated, characterized and studied

9 total polymer-dye complexes were generated, characterized and studied

Yamamoto coupling yield the polymers within the acceptable molecular weights, and percent internal linker monomer incorporation.

Modification of internal linker (same attachment site or extended linker-spacer) did not

conclusively show an improvement on initial energy transfer

Improvement on photostability compared to current Brilliant Violet™ polymers

Reduced photobleaching of dyes across three distinct emissions

Application: with continuous excitation/illuminations in biological detection processes, we

can have products that retain its fluorescence property

Confidence in the results that our products generate

Tim

e

Before After

Retains fluorescence intensity under continuous illumination (depiction)

Future Research

Slide 15

Optimization of internal monomer linker lengths to confirm photostability and energy transfer

Introducing longer internal linker-spacers

Various substituents at the site of dye attachment

Solubility study beyond concentrations and solvents of base polymer; study on

improvement of solubility for antibody conjugation

Understanding/study polymer-dye complexes interaction to further improve

Sirigen’s technology

Acknowledgements

Slide 16

Sirigen & BDB BD Biosciences

Glenn Bartholomew Jim Waters

Brent Gaylord

Janice Hong

Frank Uckert

Barry Leonard

All my coworkers at Sirigen

Cal State San Marcos

Dr. John Drewe

Dr. Betsy Read

Jill Litschewski

References

Slide 17

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Alt man, R., Terry, D. S., Zhou, Z., Zheng, Q., Geggier, P., & Kolster, R. (2012). Cyanine fluorophore derivatives with enhanced

photo stability. Nature Methods, 9, 68-71. Retrieved from

Gaylord, S. Brent (2012) Patent No.8,158,444 B2. Santa Barbara, CA: U.S.

Patil, A. V., & Bidkar, S. G. (2009, September 23). Characterization of Polymer. Scribd. Retrieved October 2, 2012, from

Schmidt J., Werner M., Thomas A. (2009). Conjugated Microporous Polymer Networks via Yamamoto Polymerization. Marcomolecules, 42(13), 4426-4429.

Sirigen : High Sensitivity Fluorescence. (n.d.). Sirigen : Light harvesting materials delivering High Sensitivity Fluorescence in diagnostic

and Life Science applications. Retrieved September 4, 2012, from http://www.sirigen.com/high_sensitivity_fluorescence.html

What is a dPEG®. (n.d.). Quanta BioDesign. Retrieved March 5, 2013, from http://www.quantabiodesign.com/what-is-dpeg.html

Zhao, W.,Chen, C., Li X., Zhao J. 2002.“Photodegradation of Sulforhodamine-B Dye in Plantinized Titania Dispersions under Visible Light

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