Metal ion burst: Examining metal ion diffusion using ultrafast fluorescence spectroscopy in reverse...

METAL ION BURST:

EXAMINING METAL ION

DIFFUSION USING

ULTRAFAST FLUORESCENCE

SPECTROSCOPY IN REVERSE

MICELLES

CHELSEY CROSSE

ORALS PRESENTATION

APRIL 29, 2014

to develop methods

to measure the

impact of metal ions

on a wide range of

timescales.

1

RESEARCH FOCUS:

Example: Proposed work with Dr. Crans

observing interaction of Cu2+ and β-amyloid. All

current work is directed in this capacity.

Illustration courtesy of Alzheimer’s Disease Research, a program of the American Health Assistant Foundation

A STEP BACK:

TIME RESOLVED SPECTROSCOPY

2

Initialization

t0

tChanges Equilibrium

…

0

1

2

3

0 5 10 15 20

Ab

so

rba

nc

e

(~6

20

nm

)

Time

Example Kinetic Trace at Single Wavelength

0

0.2

0.4

0.6

0.8

1

400 500 600 700

Ab

so

rba

nc

e

(t~

8)

Wavelength (nm)

Example Spectrum at Single Time

• Rate of observation

• Rate of initialization

3

LIMITS OF TIME RESOLVED

SPECTROSCOPY

t01s

Pouring

Human Eye

Photolysis Release

Optical Gating

Highest Possible Time Resolution

1ps

(approximate temporal orders)

Micro Channel Plate (electronic limit)…

pH jump

• Photoacids excited by femtosecond pulses

• Releases H+

• Regenerates

• Well characterized release dynamics

Caged-metal organic complexes

• Cages photolysed by femtosecond pulses

• Releases metal ion

• Does not regenerate

• Release dynamics not characterized on fs scale to our knowledge

4

PHOTOLYSIS RELEASE

TECHNIQUES

HA hn¾ ®¾ A- +H+ time¾ ®¾ HA

AMB hn¾ ®¾ A+M +B

M2+ M2+

hn

H+

hnH+

Donten, M. L., Hamm, P., & VandeVondele, J. (2011). A Consistent Picture of the Proton Release Mechanism of oNBA in

Water by Ultrafast Spectroscopy and Ab Initio Molecular Dynamics. The Journal of Physical Chemistry B , 115, 1075-1083.

First Step

5

METAL ION DETECTION BY

FLUORESCENT PROBE

1. Photolysis

2. Diffusion

3. Interaction

1. Photolysis

2. Diffusion

3. Interaction

1. Photolysis

2. Diffusion

3. Interaction

1. Photolysis

2. Diffusion

3. Interaction

(1) hnM2+(3) Interaction

M2+

Probe

(2) DiffusionM2+

M2+

6

Requirements:

• Ultrafast probe technique

• Well characterized probe

• Limited diffusion distance

(1) hnM2+

M2+

ProbeM2+

M2+


FLUORESCENT PROBE

• Energy of electronic

states depends on:

• Molecule geometry

• Interactions with

environment

• Changes can be

observed as:

• Changes in fluorescence

intensity

• Shifts in fluorescence

spectra

7

TIME RESOLVED FLUORESCENCE

ON MOLECULAR TIMESCALE

System Coordinate

t

t

Flu

ore

scen

ce

In

ten

sity

Detection of fluorescence

signal using non-linear

optical gating.

8

FLUORESCENCE

UPCONVERSION

wgated =w fl +wgate

Non-Linear Crystal

Gated

Fluorescence Signal (ωgated)

Portion of fluorescence

signal sampled can be

changed by:

• Changing gate arrival

time temporal region

• Changing crystal angle

spectral region

9

FLUORESCENCE UPCONVERSION:

TEMPORAL RESOLUTION

Non-Linear Crystal

Gated

Fluorescence SignalIn

tensity

Crystal Out

Crystal In

• Each frequency requires

realignment of crystal

angle

• Kinetic traces can be

used to reconstruct

temporal fluorescence

spectra

10


EXAMPLE DATA

Zhang, X. -X.; Würth, C.; Zhao, L.; Resch-Genger, U.; Ernsting, N. P.; Sajadi, M. Femtosecond broadband fluorescence

upconversion spectroscopy: Improved setup and protometric correction. Review of Scientific Instruments 2011, 82,

063108.

t

Flu

ore

scen

ce

In

ten

sity

11


TEMPORAL RESOLUTION

BBO

Sample

Nd:Vanadate

Pump Laser

Mode-locked

Ti:Sapphire Laser

Optical

Delay Stage

BBO

Elliptical

Mirror

Mo

no

chro

mato

r

PM

T

LabVIEW VI

Used successfully by

Levinger group to

examine water

environments

I have rebuilt most of this

system, repairs &

replacements have

prevented completion

Gate

Ex

citation

Gated

Signal

I have experience aligning

necessary optical systems:

• Assembled &

successfully mode-locked

Ti:Sapphire oscillator

system

• Used & adjusted FROG

system

• Designed & built other

non-linear system

12


PERSONAL EXPERIENCE

13

RELATIVE

TIMESCALES

• Fluorescence lifetime: ~10-9 s

• Diffusion of metal ion: 10-13-10-6s

(1) hnM2+

M2+

ProbeM2+

M2+

14

Requirements:

Ultrafast probe technique

• Well characterized probe



M2+

Probe

(2) DiffusionM2+

M2+


FLUORESCENT PROBE

Ideal fluorophore:

1. Inert

2. Selective & sensitive

3. Detectable by our system, currently:

• Absorption: 400-450nm

• Emission: 450-500nm

• Detectable intensity

15

FLUOROPHORE

SELECTION

Initial characterization:

• Steady State

Fluorescence

• Fluorescence Lifetime

(TCSPC)

Have been able to observe

quenching by Cu2+.

16

COUMARIN 343 (C343)

Previously used successfully by Levinger group in

fluorescence upconversion studies.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

450 460 470 480 490 500 510 520 530 540 550

Flu

ore

scen

ce I

nte

nsit

y [

co

un

ts]

Wavelength (nm)

Steady State Fluorescence Spectra for Solutions of C343 excited by 372nm NanoLED with Varying Concentrations of CuSO4

0.0E+00

5.5E-06

1.1E-05

2.2E-05

4.4E-05

1.1E-04

2.2E-04

Concentration CuSO4 (M)

17

C343: STEADY STATE

FLUORESCENCE

0

1

2

3

4

5

0.0E+00 1.0E-04 2.0E-04

Th

ou

sa

nd

s

Concentration CuSO4 [M]

Peak Fluorescence Intensity

18

C343: FLUORESCENCE

LIFETIME

Fluorescence Lifetime Measurements of 2.2E-5 [M] C343

with Varying Concentration of CuSO4

Used to determine complex

stoichiometry.

1. Measure absorbance of

mixture solution at

different mole fractions

2. Subtract the absorbance

of the pure compounds

in solution at the

concentration measured

19

JOB PLOT

Harris Quantitative Chemical Analysis Fig. 19-8

A(PX, x) = A(solution, x)-A(P,[P])-A(X,[X])

C343: BEER’S LAW

20

In cooperation with Angela Warner.

A =ebc

y = 0.253xR² = 0.9636

0

0.1

0.2

0.3

0.4

0.5

0.6

0 0.5 1 1.5 2 2.5

Absorb

ance

Concentration of C343 (10-5M)

Beer's Law Plot C343 at 409 nm

y = 0.3417xR² = 0.9997

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.5 1 1.5 2 2.5

Pe

ak

Ab

so

rba

nc

e

Concentration (10-5M)

Beer's Law Plot of Tryptophan at 212 nm

C343:

R² = 0.96364

JOB PLOT:

TRYPTOPHAN

21

0

0.005

0.01

0.015

0.02

0.025

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Co

rre

cte

d A

bso

rba

nce

Mole Fraction of Cu2+

Job Plot of Tryptophan with CuSO4

Corrected Abs@204nm

Corrected Abs@212nm

Corrected Abs@219nm

Corrected Abs@230nm

Corrected Abs@246nm

Corrected Abs@279nm

Corrected Abs@300nm

In cooperation with Angela Warner.

0

0.2

0.4

0.6

0.8

200 250 300 350

Absorb

ance

Wavelength (nm)

Absorbance Spectrum of Tryptophan(2.2 × 10-5 M)

22

Requirements:


Well characterized probe



M2+

Probe

(2) DiffusionM2+

M2+


FLUORESCENT PROBE

• Open solution:

• Sensor-ion interaction

determined by Brownian

diffusion

• Large probability distribution

• Limited measurement

resolution

• Confined environment:

• Sensor-ion interaction

probability enhanced by

proximity

• Increased reaction times have

been shown previously

• Choose reverse micelles

(RMs)

Levinger group has

expertise in characterization

& preparation.

23

CHARACTERIZATION OF

METAL ION RELEASE

M2+

Probe

M2+

Probe

24


• Fluorescence upconversion

• Currently being reassembled

Well characterized probe

• Analytical characterization techniques have been validated

• Still in the process of testing candidates

Limited diffusion distance

• Confinement in reverse micelles

• Relying on expertise of Levinger group


FLUORESCENT PROBE

Immediate:

• Identify fluorophore

• Build upconversion

experiment

• Characterize full

fluorophore system using

fluorescence

upconversion

Future:

• Extend upconversion to

metal ion burst

• Measure metal ion

dynamics in interesting

systems

25

GOALS

M

Probe

Dr. Nancy Levinger

Dr. Debbie Crans

Angela Warner

Ben Wiebenga-Sanford

All the Levinger & Crans

group folks

Barisas (use of the IBH)

CIF

Jenee Cyran & Laura Tvedke

26

ACKNOWLEDGEMENTS

27

ANATOMY OF A

REVERSE MICELLE

• Organic solvent

• Aqueous suspension

• Surfactant molecules

28

REVERSE MICELLE: TERNARY

PHASE DIAGRAM

• Size

• Described in number of water

molecules (w0) in each RM

• Easily adjusted by changing

proportions of solution

• Well documented

characteristics

• Rate enhancements

observed in confined

reaction systems

Magalhaes – Phase diagram of a lyotropic mixtrue sodium bis (2 ethylexyl) sulfosuccinate-codecanol-water:…(1998)

Confinement papers from Levinger (78-80 PRF)

• Photolytic release of

metal ions into solution

• Complex dissociation has

been observed to be

faster than 10 s

• Variety of compounds

with different optical

absorbances

• Diffusion time of metal

ion has yet to be

characterized

29

CAGED METAL

ORGANIC COMPLEXES

H. M. D. Bandara, D. P. Kennedy, E. Akin, C. D. Incarvito, and S. C. Burdette, "Photoinduced Release of Zn2+ with ZinCleav-1: a Nitrobenzyl-Based Caged Complex",

Inorganic Chemistry 48 (17), 8445-8455 (2009).

H. M. D. Bandara, T. P. Walsh, and S. C. Burdette, "A Second-Generation Photocage for Zn2+ Inspired by TPEN: Characterization and Insight into the Uncaging Quantum Yields

of ZinCleav Chelators", Chemistry-a European Journal 17 (14), 3932-3941 (2011).

K. L. Ciesienski and K. J. Franz, "Keys for Unlocking Photolabile Metal-Containing Cages", Angewandte Chemie-International Edition 50 (4), 814-824 (2011).

K. L. Ciesienski, K. L. Haas, and K. J. Franz, "Development of next-generation photolabile copper cages with improved copper binding properties", Dalton Transactions 39 (40), 9538-9546 (2010).

Because we are observing quenching effects, we will be working in lower intensity regions.

It may be necessary to increase instrument sensitivity.

OPAGE would gate signal in an OPA crystal to account for this

It has been tried before, but have been some challenges

30

OPAGE

31

C343 CHARACTERIZATION:

QUALITATIVE FLUORESCENCE

0

200000

400000

600000

800000

1000000

1200000

1400000

440 460 480 500 520 540

Inte

nsit

y

Emission [nm]

0 drops

1 drop 0.01 M

2 drops 0.01 M

1 drop 0.1M

2 drops 0.1M

3 drops 0.1M

1 drops 1M

2 drops 1M

3 drops 1M

Fluorescence Measurements of 2.2E-5 M C343 with Varying

Concentrations of CuSO4 Excited at 400 [nm]

Amount of CuSO4

added

JOB PLOT:

TRYPTOPHAN

32

0

0.005

0.01

0.015

0.02

0.025

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Ab

so

rba

nce

Mole Fraction of Cu2+

Job Plot of Tryptophan with CuSO4

Corrected Abs@204nm

Corrected Abs@212nm

Corrected Abs@219nm

Corrected Abs@230nm

Corrected Abs@246nm

Corrected Abs@279nm

Corrected Abs@300nm

EVERYONE LOOK HOW AWESOME ANGELA IS!!!

33

C343

CHARACTERIZATION:

ABSORPTION

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

300 350 400 450 500 550

Ab

so

rban

ce

Wavelength [nm]

Absorbance of 2.2E-5 [M] C343 Solutions with Varying CuSO4 Concentrations

0.0E+00

1.7E-04

3.4E-04

5.0E-04

6.7E-04

8.4E-04

1.0E-03

1.2E-03

Concentration*

of CuSO4 [M]

*Assumed molarity of a drop of CuSO4 solution in 2 [mL] of C343 = 1.7E-4

Isobestic Point?

FWHM: ~25 nm

34

EXAMPLE NANOLED

SPECTRUM

0

1000

2000

3000

4000

5000

6000

7000

250 300 350 400 450 500 550 600 650 700 750 800

Co

un

ts

Wavelength (nm)

372nm NanoLED Spectrum

FLUORESCENCE

LIFETIME FITS OF C343

AND CU2+

35

C343

2.5E-5M

0.01M CuSO4

3 drops

3.35E-3M

0.1M CuSO4

1 drop

1.12E-2M

0.1M CuSO4

2 drops

2.24E-2M

1M CuSO4

1 drop

0.112M

Single

exp.

Double

exp.

Single

exp.

Double

exp.

Single

exp.

Double

exp.

Single

exp.

Double

exp.

1 [s] 4.14E-9 4.19E-9 2.18E-9 4.00E-9 2.12E-10 3.84E-9 2.45E-10 3.55E-9 2.16E-10

2 [s] --- --- 4.24E-9 --- 4.03E-9 --- 3.89E-9 --- 3.64E-9

A -0.502 -0.199 -0.282 -0.152 -0.209 0.133 4.82E-2 0.202 0.103

B1 0.118 0.119 5.77E-3 0.117 6.75E-3 0.117 3.97E-2 0.115 0.139

B2 --- --- 0.115 --- 0.114 --- 0.112 --- 0.107

2 1.385 1.380 1.358 1.398 1.120 1.687 1.210 2.935 0.984

Point at which two chemical

species have the same molar

absorptivity, or are linearly

related.

Can be used to tune

absorbance for total reaction

because it remains constant

over the entire reaction.

36

ISOSBESTIC POINT

Wikipedia. http://en.wikipedia.org/wiki/File:Bromocresol_green_spectrum.png Created 03/02/2006, Accessed 04/17/14

http://en.wikipedia.org/wiki/File:Bromocresol_green_spectrum.png

37

PH JUMP EXAMPLE

Donten, M. L., Hamm, P., & VandeVondele, J. (2011). A Consistent Picture of the Proton Release Mechanism of oNBA in

Water by Ultrafast Spectroscopy and Ab Initio Molecular Dynamics. The Journal of Physical Chemistry B , 115, 1075-1083.

38

TIMESCALES OF

CHEMICAL PROCESSES

Optical component

measured delay times:

• SHG crystal: 0.36 mm

(108 fs)

• Microscope slide: 0.7 mm

(208.8 fs)

• Sapphire window: 2.39

mm (717 fs)

• Lens set 1: 21 mm

(6301.2 fs)

• Lens set 2: 3.74 mm

(1122 fs)

39

AUTOCORRELATOR & OPTICAL

DELAY MEASUREMENTS

40

R928 SPECTRAL

RESPONSE

Metal ion burst: Examining metal ion diffusion using ultrafast fluorescence spectroscopy in reverse...

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