9/25/08

Quantum Dot Systems for

Specific Biosensing Applications

Jay L. Nadeau Department of Biomedical Engineering, McGill

University

Adjunct Member, The Jackson Laboratory

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Energy- and electron-transfer processes can be

exploited to quench and enhance QD

fluorescence in response to specific cellular events

1. Introduction: synthesis and properties of particles

2. QD-dopamine conjugates and the flow of electrons a. Evaluation of quality using the OPA assay

b. Electron paramagnetic resonance spectroscopy

c. Time-resolved spectroscopy

d. Blinking statistics

3. FRET constructs for voltage sensing a. Hydrophobic QDs in membranes

b. Genetically-encoded sensors

Introduction and outline

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CdSe, CdS,

ZnS,CdTe,

etc

•Emission wavelength is related to the size

of the crystal

•Slow to photobleach and radiation

resistant

•Emission can be quenched/modulated by

attaching electron donors or acceptors to

the surface

•Can be suspended in aqueous and non-

aqueous environments

•Many colors obtained with a single UV

excitation source

•Surface can be conjugated to chemically

and biologically important molecules

Why QDs?

450 500 550 600 650 700

0

1

Norm

aliz

ed inte

nsitie

s

(nm)

Absorption Emission

3 to 10 nm

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QD Synthesis/Solubilization

CdSe/ZnS core-shell

Synthesis via a two-step, single flask method.

Injection of Selenium precursor into hot coordinating solvent (octadecane) containing the cadmium precursor, CdO.

Leads to nucleation and growth of particles

Injection of Zn and S solutions arrests growth, forms cap around

particles.

Water solubilization was done by oleic acid cap exchange with thiol

mercaptosuccinic acid (MSA) or mercaptoacetic acid (MAA)

Reflux in methanol for 6 hours

Yields water-soluble particles

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3 nm

Conjugation to primary amines Solubilized QDs at a 1 M concentration are added to 2-10mM of

conjugate and 1mg/mL of EDC

EDC forms amide bond between amino and carboxyl groups

Excess reagents removed with ultrafiltration or dialysis.

O H

O S

O H O

S

O H

O

S

O H

O

S

O H O

S

N H 2 R

N

O S

R H N

O

S

R

H

O H

O

S

O H

O

S

N O

S

R

H

(CdSe)MAA

+

EDC ethyl-dimethylaminopropyl carbodiimide hydrochloride

primary amine

containing molecule

amide bond formation

CdSe CdSe 1-2 hours in dark

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QD-dopamine: a redox sensor

Charge separation is a classic feature of semiconductors that has been used in nonfluorescent

nanocrystals such as TiO2

CB

h VB

h O

R

O, R

Energy

Dopamine is an excellent electron donor

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Evaluation of ligand number with OPA

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Observation of DA radical/ quinone

DA+

Using EPR spectroscopy Oxidized dopamine shows blue fluorescence

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Time-resolved emission

At least 4 components with different lifetimes are seen

Total area under the curve ~ steady-state fluorescence output

Most importantly, quenching correlates with uptake by cells

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Model for electron flow: normal conditions

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Reducing conditions

The addition of an antioxidant (e.g., beta-mercaptoethanol) eliminates the appearance of the oxidized DA

without preventing electron transfer

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Successful uptake of quenched QDs

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With antioxidants

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Partitioning/redox dependence

QD only Redox Red

Overlay

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Make cells more oxidizing

Addition of the

glutathione synthesis

inhibitor BSO (10 mM) affects the intracellular

redox potential without

altering that of the

medium

10 μμ

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Or more reducing

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Photooxidation

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Blinking statistics

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Open questions

Does electron transfer correlate with greater bioavailability?

If so, is this a property of the QDs or of the the cells?

What is the relationship between conjugate and phototoxicity?

Do blinking statistics correlate with the number of dopamines per particle? Could this be use for tracing in cells?

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Quantum dot-FRET systems for

imaging of neuronal action potentials

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Specific disease model

motor neuron astrocyte

The image cannot be displayed. Your computer may not have enough memory to open the image, or the

presynaptic

terminal NMDA R

AMPA R

kainate R

glutamate

GLT-1 transporter

other transporters mGluR

Gln

Ca++ Ca++

?

Ca++

CaBP CaBP ER ER Mito Mito

VGCC

Ca++ Na+

Na+-Ca++ exchanger

Na+

Ionotropic

Na+ channels

Na+,

Ca++

Ca++

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Imaging needs in neuroscience

Many different systems have a need for

optical recording from ensembles of neurons

Voltage-sensitive dyes have very low signal

to noise and high toxicity

A genetically-encoded probe would really be

ideal!

To visualize neuronal action potentials,

minimum resolution is 100 mV in 10 ms

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Quantum dots and membranes

A quantum dot is a fluorescent

semiconductor nanocrystal of a

diameter comparable to that of a

biological membrane…

…or, alternatively, a

QD is roughly the

size of streptavidin

4 nm

Streptavidin (MW = 52, 000)

cell membrane

with transmembrane

proteins

Protein

(pores up to 7 nm)

Lipid

(5 nm)

QD

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Model system

Internal solution contains 1 mM KCl

External solution contains 150 mM KCl

Equilibrium occurs when G = 0

G = RTln([Kin]/[Kout]) + zFE =0

E = RT ln [K]out

.........zF.....[K]in

Valinomycin makes vesicle permeable to K

Lipid vesicle with QDs embedded in the membrane

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QDs in vesicle membranes

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FRET with ANEPPS amplifies effects of

voltage and K ions

As they are, QDs are not voltage-sensitive

enough

0

100

200

300

400

500

600

500 550 600 650 700 750Emission (nm)

1/1

150/1

150/150

1/150

no Val

Spectra suggest that QDs are acting as FRET acceptors (J.

Phys. Chem. B 2004 108: 17042)

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Delivery to living cells

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QDs are capped with pyridine and

delivered by Pluronic

Pluronic is a non-ionic surfactant

MW ~12 kD

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The results are intriguing…

400 450 500 550 600 650 700

Em

issio

n (

arb

. u

nits)

Wavelength (nm)

RH421+QD+glut

RH421+QD

RH421QD

autofluorescence

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Toxicity is a problem

Delivery by other methods? (liposome

fusion)

Capping by other groups (amines)

Use of different materials (ZnS)

Take QD out of the membrane!

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Taking the QD out of the membrane

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Initial tests

Channel construct with 6His leads to

cell agglomeration and death with

certain cell lines (NIH3T3, not HEK293)

Sequence verified

Tests underway with channel blockers

(nifedipine)

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Expression in HEK293 cells

Green: QDs; Red: Red fluorescent protein co-expressed

with 6His channel; yellow: overlay

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Questions

Do QD-fluorescent protein systems

show any voltage sensitivity?

Could we alter the QDs (size and

shape) to enhance voltage sensitivity?

Can we use the toxicity to our

advantage?

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Conclusions

There is a great need for voltage-sensitive probes in neurons

No existing technology works well enough

QDs show promise but have problems

The same is true for genetically-encoded sensors

FRET is a general principle that can be used to create specific probes

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Acknowledgements

Students: Samuel Clarke, Daniel

Bahcheli, Rafael Khatchadourian

Postdoc: Annette Hollmann

Collaborators: Netta Cohen, Stephen

Bradforth, Diana Suffern

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Acknowledgements

Lab Members

Samuel Clarke

Annette Hollmann

Daniel Bahcheli

Rafael Khatchadourian

Collaborators

Trish Holden

Steven Bradforth

Diana Suffern

Nada Dimitrijevic

Paul Wiseman

Alexia Bachir