Nanoparticles 2

Biological applications and non-semiconductor materials

Biological applications of fluorescent QDs

God made the bulk and the devil the surfaces (Pauli)

The surface is 50% of atoms

Problems are weak fluorescence or loss of fluorescence, cap decay, aggregation, toxicity

Dozens of suggestions have been proposed; none is perfect

Properties of QDs

Fluorescence (photoluminescence, PL) results from trapped or free carrier recombination

Radiative recombination of the free exciton is desired; traps red-shift or eliminate fluorescence

The surface is not round and smooth, so the optical properties are highly dependent upon its features!

TOPO provides “passivation” c.b.

v.b.

shallow trap states

deep trap states

radiative

radiative &

non-radiative

non-radiative

500 550 600 650 700 7500

20

40

60

80

100F

luore

scence (

arb

. units)

Emission (nm)

Trap states arise from defects and un-coordinated atoms on the nanocrystal surface.

CdSe CdSe

Coating the surface with a higherband gap material or a polymerincreases the fluorescence.

Weller, 1993

Add an excess of thiol to the organic solvent; reflux for 2- 24

hours; add aqueous buffer at basic pH; QDs will partition into

the aqueous phase and can be removed and further purified by centrifugation and washing

The original solubilization method

“Cap exchange” using mercaptoacetic acid to replace

trioctylphosphine oxide

P

R R

RO

P

RR

RO

PR

R R

O

PR

RR

O

PR

RR

O

SHOH

O

OH

OS

OH

O

S

OH

O

S

OH

O

S

OHO

S

(CdSe)TOPOin CH2Cl2

+CdSeprecipitates from CH2Cl2

shake 1 hour in dark

MAAmercaptoacetic acid

(CdSe)MAA,rinsed and stored in H2O

CdSe

Chan and Nie 1998

Other chain lengths can be used

In general, all this will change is the dissociation constant and

the degree of hydrophilicity

Mercaptoundecanoic acid

Probably the most popular

Mercaptohexadecanoic acid

See e.g.

Huge problem: loss of fluorescence and instability!

CdSe CdSe

+ O2

Occurs over days to weeks,depending on solvent, lightexposure, temperature, etc...

oxidizedsurface layer

SeO2

oxidized non-oxidized

500 550 600 650 700 750

0

500

1000

1500

2000

2500

3000

3500

Flu

ore

scence (

arb

. units)

Emission (nm)

after 3 days light exposure

fresh prep.

(CdSe)MAA

Overall “cap decay” scheme Dabbousi et al. 1997

Aldana et al. 2001

What can be done?

Problem: the degree of fluorescence quenching is even

greater than with mono-dentate thiols! CdSe solubilized by

dihydrolipoic acid is nonfluorescent

Another classic

Bruchez et al Science 25 September 1998

Problem: difficult to perform; leads to a great reduction in

fluorescence

Oligomeric phosphines

Kim and Bawendi, J. Am. Chem. Soc., 125 (48), 14652 -14653, 2003

Triblock copolymers

Problem:

requires

elaborate synthesis;

greatly

increases size

of the particle

Gao et al

Nature

Biotechnology

22, 969 - 976 (2004)

Phospholipid micelles

Dubertret et al, Science 29 November 2002:

Vol. 298. no. 5599, pp. 1759 - 1762

An even worse problem for biologists!

QDs that do get into cells just sit in endosomes

Suggested solution: hyperbranched copolymer

Duan and Nie, J. Am. Chem. Soc., 129 (11), 3333 -3338, 2007

And the latest

Liu et al, J. Am. Chem. Soc., Web Release Date: November 6, 2007

•CdSe(ZnCdS)

•Very small

•Cleared by kidneys (rat)

Sub-conclusions

Everyone recognizes the problem

Many people have tried to fix it

The majority of researchers

continue to use MAA, MUA, or DHLA because these methods are easy and generally acceptable

Toxicity

“Quantum dots are nontoxic” is a lie

The most obvious (but not most common) source of toxicity is Cd ions

The most common source is reactive oxygen species (ROS) OR direct oxidation (unclear which!)

Hepatocytes hate Cd

Derfus et al

Nano Letters, 4

(1), 11 -18, 2004.

Some, but not all QDs (might) make ROS

Expose to UV/blue light

Measure direct singlet oxygen luminescence (1271 nm) or use

terephthalate

CdS, CdSe yes

CdSe/ZnS no

Ipe et al, Small Volume 1, Issue 7, Pages

706-709

ROS leads to lipid peroxidation and loss of mitochondrial membrane

potential

mitochondrial

membrane lipid

peroxidation

Caspase 8

FADD

NAC

NAC

plasma membrane

lipid peroxidation

metabolic

activity CELL DEATH

cytochrome c caspase

cascade

Fas

ROS

M

QD

cardiolipin

peroxidation

Proposed mechanism

(D. Maysinger, Pharmacology)

Simple molecules can photosensitize QDs

Clarke et al. NATURE MATERIALS 5 (5): 409-417 MAY 2006; and Clarke et al in progress

Really ROS or just oxidation?

False positive results with oxidation-based dyes using

fullerenes (Lyon et al, Nano Letters 8(5):1539)

Using a reduction-based dye (XTT), no ROS was detected

Nevertheless, oxidative damage to cells can occur

Apart from oxidation, charge matters

Rasmussen et al, Journal of Investigative Dermatology (2007) 127, 143-153

Is there a solution?

Toxicity can be used to kill unwanted cells

In vitro, QDs must be passivated to prevent toxicity; antioxidants also help

It’s very unlikely that Cd-containing particles will be used in vivo; other types are needed!

The ideal particle

Made of non-toxic materials

Small

Multifunctional (e.g. fluorescent and provides MR contrast)

Easy to make

Many types of nanoparticles fit one or more of these criteria: ZnS, Au, Ag, TiO2, …

Fluorescent but nontoxic

ZnO

ZnS

Problems:not a wide range of

fluorescence; very blue, excite with UV

Nonfluorescent but interesting

TiO2

Ag

Au

FeO3

etc

Ag is antimicrobial

Sondi Journal of Colloid and Interface Science Volume 275, Issue 1, 1 July 2004, Pages 177-182

Au has many interesting properties It all started in 1994: Brust et al, JOURNAL OF THE CHEMICAL

SOCIETY-CHEMICAL COMMUNICATIONS (7): 801-802 APR 7 1994

Colloids stabilized with alkanethiols as in CdSe

Au-S bond is very stable

Simple synthesis: reduction of AuCl4- by sodium borohydride in the presence of the thiol

All the “particle in a box” quantum mechanics discussed for QDs still applies

All of the solubilization/encapsulation methods also apply!

Daniel and Astruc, Chem. Rev., 104 (1), 293 -346, 2004

The surface plasmon

Au colloids are deep red due to surface plasmon band absorbance

Collective oscillations of 6s electrons in conduction band in response to EM field of visible light

Varies with shape and especially size: for particles of 9, 15, 22, 48, and 99 nm, the SPB maximum max was observed at 517, 520, 521, 533, and 575 nm. Absent in bulk gold and in particles < 2 nm

Some remarkable features

Aggregation causes colour change

This can be used as a basis for

sensing

Electron transfer to semiconductors

Enhancement of CdSe

Hsieh et al, Nanotechnology 18 (2007) 415707

Au toxicity

Pan et al, Small Volume 3, Issue 11 , Pages 1941 - 1949

CT contrast agents

Usually iodine-based (high X-ray absorption coefficient)

Problems of toxicity (kidneys)

Fast clearance--> short imaging times

Alternatives of nontoxic materials include bismuth and gold

PEG-SH coated Au

CT images of rat hepatoma

using Au-PEG

nanoparticles injected by tail vein as a contrast agent Kim et al, J. Am. Chem. Soc., 129 (24), 7661

-7665, 2007

Multifunctional QDs

a,b, Quantum dots having

different molecules for target-specific interaction,

and, attached to the surface, paramagnetic lipids

(a) and chelators (b) for

nuclear-spin labelling. (c) The silica sphere has QDs

and paramagnetic nanoparticles inside and

target-specific groups

attached to the outside. (d) The structure of a

multimodal QD probe, based on silica-shelled

single-QD micelles

Bakalova et al, Nature

Photonics 1, 487 - 489 (2007)

Iron oxide FDA approved as MR contrast agent “SPIO”

Superparamagnetic

Unlike ferromagnetic materials, paramagnetic

materials do not retain magnetization when B = 0.

Superparamagnetism is the ability to exhibit paramagnetism below the Curie temperature. In

nanoparticles, thermal energy is sufficient to change

the direction of all atoms in the crystallite.

A quick synthesis method

0.2 mL of Fe(CO)5 to 10mL octyl ether and 478 μL oleic acid at 100 ºC, reflux at 280 ºC for 1 h; should turn black.

Let cool, add 0.34 g of trimethyl-N-oxide, heat to 125 ºC for 2 h under nitrogen and then reflux at 280 ºC for 1 h.

Micelle encapsulation: dilute original solution tenfold, add 100 μL to 900 μL chloroform and 1 mg mPEG-2000 PE, 0.25 mg of DPPC, 34 μL of a 5.4 mM DiI solution. Allow solvents to evaporate, resuspend in 1 mL H2O.

Appearance

Usefulness in MR and fluorescence imaging

0

20

40

60

80

100

120

0 200 400 600 800

Conc (uM)

1/T

2 (

se

c-1

)

1

T 2=

1

T 2H2O

+ R2 Fe[ ]

MR imaging

Conclusions Quantum confinement not restricted to semiconductors

Metals and insulators show interesting properties at the nanoscale that are not observed in bulk materials

Wet-chemical synthesis methods are similar to those for QDs

Methods of solubilization, encapsulation, etc are also similar, aided by the Au-S bond strength for Au

Multifunctional particles can permit several different applications at once

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