Nanoscale Biosensors Based on Luminescent Quantum DotsHedi Mattoussi and Ellen R. Goldman

Optical Sciences Division and Center for Bio/Molecular Science and Engineering (CBMSE)

To develop QD multianalyte biosensors

Sensors capable of long-term environmental monitoring

Objectives

QD

Q

Exc

A

analyte

hνPrototype Concept

QD

Q

Exc

quencherFRET

Add analyte

Regenerate

Unexploded Ordnance Detection:Continuous monitoring for underwater detection of explosives

Biological Screening of Toxins (Force Protection):Sensing systems for monitoring of drugs, toxins, pathogens, and environmental contaminants

Payoffs

Different color QDs

Design and implement new syntheses of highly luminescent QDs

Utilize QDs with bioreceptors to assemble multi-analyte biosensors for long-term environmental monitoring

Assembly of biomolecular receptors onto luminescent QDs

Control FRET between a QD and dye-labeled receptor upon interaction with target analyte

Approaches

P=O:P:Caps:

TOP TOPO

• Synthesis of CdSe QDs: Murray et al., J. Am. Chem. Soc. 115, 8706 (1993) • Synthesis of CdSe-ZnS QDs: (1) Hines et al., J. Phys. Chem. 100, 468 (1996)

(2) Dabbousi et al., J. Phys. Chem. B 101, 9463 (1997)

Unique features of Quantum Dot fluorophores

UV source at λ ≅ 365 nm

R = 12Å 14Å 18Å 22Å 25Å 30Å

(Dabbousi et al., J. Phys. Chem. B 101, 9463 (1997))

organic capsZnS shell

4eV 2eV CdSe coreR0~10-60Å

Tunable photoemission:

Maintain the same composition but vary the size across the range of accessible wavelengths

sizechoice of group II or VI elements

Binary CdSe QDs

Wavelength (nm)450 500 550 600 650

Size

: R (Å

)

Photoluminescence varies with QD size

12

20

40

Homogeneous core formationTunable photoemission:

Maintain uniform size across wide wavelength domain

% compositionchoice of group II or VI elementssize

Design of Alloyed QDs

Wavelength (nm)300 400 500 600 700 800 900

CdSe

CdSexTe1-x

ZnxCd1-xSe

Composition

Photoluminescence domain varies for a fixed QD size

4. FRET-based sensor concept

dye

5-his

QDExcitation

QD emission

• FRET Involves the non-radiative exciton transfer from a donor to an acceptor

• Transfer efficiency varies as ~ (1 / r 6)

• Proximity driven

• It is a powerful sensing tool, used to detect binding events

• Requires spectral (or energy) overlap

QD emission

FRET

Dyeemission

Design of QD nanoscale TNT sensor based on Fluorescence Resonance Energy Transfer (FRET)

475 500 525 550 575 600 625 650 675 7000

5e+5

1e+6

2e+6

2e+6

3e+6

3e+6

0:1 MBP-Cy3/QD1:1 MBP-Cy3/QD2:1 MBP-Cy3/QD3:1 MBP-Cy3/QD4:1 MBP-Cy3/QD5:1 MBP-Cy3/QD7:1 MBP-Cy3/QD10:1 MBP-Cy3/QD

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0

555 QDsMBP-Cy3E (QD PL quenching)

λ (nm)

PL (a

.u.)

Rel

ativ

e PL

; effi

cien

cy

ratio MBP-Cy3/QD

555 nm emitting QDs titrated with MBP-Cy3 Integrated PL vs. acceptor ratio

7

System predisposed for multiplexing (multiple analytes can be targeted simultaneously)

QD

Cy3MBP

QD

Cy3MBP

• J(λ) = 8.91×10-13 cm3/M => R0 = 58.6 Å

• Tunable QD excitation => Choose a minimum for the dye

I. Medintz et al., Nature Materials 2, 630 (2003) and A. Clapp et al., J. Am. Chem. Soc., In press

2. Advantages of QDs in FRET

5. TNT monitoring system

Recognition elements: withstand nonstandard matrixes (seawater)Robust reporters: photo- and chemically stable QDsEasily identifiable: ‘On’ or ‘Off’ luminescence

3. Illustration of energy transfer : Time-resolved fluorescence

t = 0.5 ns t = 2.5 ns t = 4.5 ns t = 6.5 ns

QD/10MBP

QD/3MBP-Cy3

Cy3 only

510 570 510 570 510 570 510 570

1. FRET concept

FRET

Exc

FRET

Exc

QD

‘On’ State: Strong luminescence

QSY-7

QD

substrate

Exc

TNT

QDQD

‘On’ State: Strong luminescence

QSY-7QSY-7QSY-7

QD

substratesubstrate

ExcExc

TNTTNT

FlexibleLinker

‘Off’ State: No emission

Antibodyfragment

TNT analog

Quencher dye

QSY-7

substrate

QD FlexibleLinker

‘Off’ State: No emission

Antibodyfragment

TNT analog

Quencher dye

QSY-7QSY-7QSY-7

substratesubstrate

QDQD

TIME

SIG

NA

L: P

L

QDQDQDQDQD hν

QAnalyte

Analyte in recognition site. QD emits

Exc

hν

QDQDQDQDQDQDQDQDQDQD hν

QQAnalyte

Analyte in recognition site. QD emits

ExcExc

hν

QQ

Receptor

No analyteNo QD emission

ExcExc

Analog-labeledquencher

QDQD

FRETFRET

ExcExc

FRETFRET FRETFRET

QDQDQDQD

QQ

No analyteBaseline signal

RESET

ExcExc

Properties: • Robust, real time • Able to regenerate • Reset by flushing with buffer

Maltose Concentration uM10-2 10-1 100 101 102 103

Fluo

resc

ence

at 6

00 n

m

200

400

600

800

1000

1200

1400

kd~ 55.2

Cy3.5

6 carbon spacer

15 carbon space

Flexible hybridized DNA linker

Biotin for attachment to NeutrAvidin β-cyclodextrin

Cy3.5

6 carbon spacer

15 carbon space

Flexible hybridized DNA linker

Biotin for attachment to NeutrAvidin β-cyclodextrin

Titration of P1-Cy3.5-B-CD on NeutrAvidin plate with MBP-95C-QSY7 added versus Maltose concentration

MBPQ

QSY7

NeutrAvidin

Already constructed and tested: a prototype of a fixed-tether assay

Substrate

Flexible hybridized DNA linker

FRET

Biotin

β-CD-Cy3.5

• Assay is based on competition displacement• Employs reverse quenching as the

transduction signal

Exc

Add Maltose MBPQ

QSY7

QSY9-MBP Displaced

Direct Emission

NeutrAvidin

Substrate

Flexible hybridized DNA linker

Biotin

β-CD-Cy3.5

Exc

Tether structure

Key research milestones

Design and develop new QDs with broad emission wavelengths

Develop QD-surface ligands for water-compatibility and covalent conjugation

Optimization of QD-acceptor FRET spectral overlap and separation distances

Sensor optimization by linker and donor-acceptor pairs

Design and optimization of multireceptor QD-bioconjugates

Develop sensors that target analytes of interest to the Navy– TNT– RDX – Toxins– Pathogens– Environmental contaminant

Collaboration with:• Professor M.G. Bawendi and his group at MIT• Professor S. Simon and his group at Rockefeller UniversityFunding from ONR, Keith Ward program

Publications• 14 refereed publications, 1 patent, 9 proceedings, and 3 book

chapters

Work highlighted in Science, Nature Biotechnology, and Small Time magazine

Seminal papers:• H. Mattoussi, J.M. Mauro, E.R. Goldman, G.P. Anderson, V.C. Sundar, F.V.

Mikulec and M.G. Bawendi, J. Am. Chem. Soc. 122, 12142-12150 (2000).• E.R. Goldman, G.P. Anderson, P.T. Tran, H. Mattoussi, P.T. Charles, and J. M.

Mauro, Analytical Chemistry 74, 841-847 (2002).• J.K. Jaiswal, H. Mattoussi, J.M. Mauro, and S.M. Simon, Nature Biotechnology 21,

47-51 (2003).• I.L. Medintz, A.R. Clapp, H. Mattoussi, E.R. Goldman, and J.M. Mauro, Nature

Materials 2, 630-638 (2003).

Acknowledgments: