Nanogold &Quantum Dot

as

Novel Biosensors

Amornpun Sereemaspun, MD. PhD.E-mail : amornpun.s@gmail.com

Nanobiomedicine Laboratory

Department of Anatomy

Faculty of Medicine

Chulalongkorn University

Gold Nanoparticle as Biosensor

• Nano gold (Colloidal Gold)

– Nanometer-sized particles of gold in a fluid

– Size 1-100 nm.

– Intense red or yellowish color

www.nanopartz.com/Gold_Nanorods.htm

Why Gold Nanoparticles ?

• Easy to synthesis

• Protocol have been approved (J. Turkevich et al. 1951)

• Stable in room temperature

• Red color ;easy to monitor or detect

• Biocompatibility

• Can conjugate with nucleic acid or protein

From; http://www.nature.com/nprot/journal/v3/n2/fig_tab/nprot.2008.1_F2.html

Au3++ Au0 Au0

reduction stabilization

Gold Nanoparticles Synthesis

Gold Nanoparticles and

Biomolecules

• Nanogold size is similar to many cellular objects

• Gold surface can be coated by various biomolecules

Optical Properties of Nanogold

• The optical properties of gold nanoparticles can be

tuned carefully by controlling their size and shape

webexhibits.org www.azonano.com

Basic optical properties of

nanoparticlesOptical Properties of AuNPs

8

NanoGold As Products

From http://microgravity.hq.nasa.gov/general_info/homeplanet_lite.html

Lateral flow strip test

NanoGold As Products

• A worldwide common zoonosis in mammalian

• Spirochete-born disease

• Empirical diagnosis-based

• Staining – Gram unstainable

– Silver stain OK

• Culture – special media,

Take times

Leptospirosis

Leptospirosis

control 10 102 104 105 106 CFU

AuNP

106

5 ×105

105

5 ×104

104

Dot-Blot ELISA

CFU

Urine Pregnancy Test

Nanogold Comparision with comercial kit

Rojanathanes R. et al. 2008200820082008, Taiwan OB-GYN

Fluorescence-based detection of

protein kinase

Kim, Y.-P., et al., Biosens. Bioelectron. (2007),

Mirkin et al. (Science 1997 ) reported DNA sandwich hybridization

assay using DNA-nanogold conjugate.

16

Nanogold and DNA Detection

Kiley et al.(Nanomedicine. 2008)

Lateral Flow Strip Test

Microchromatographic-based

http://www.rapid-diagnostics.org/index.htm

Test lineTest lineControl lineControl line

Sample padSample padConjugate probeConjugate probe

Conjugate probe test line probe Control line probe

Xun et al. (Anal. Chem. 2009) applied nucleic acid biosensor based on the oligonucleotide functionalized Au-NPs and lateral flow for the detection of human genomic DNA directly with a detection limit of 2.5 µg/mL (1.25 fM)

Lateral flow nucleic acid test strips

Ioannis et al. (Anal. Chem. 2007)

reported the first dry-reagent

dipstick assay for SNP

genotyping by primer extension

Lateral flow nucleic acid test strips

20

(Zhao et al., PNAS,2004)(Wang et al., Bioconjugate Chem, 2007)

(Rosi et al., Science, 2006)

What Are Quantum Dots?

• Crystalline fluorophores• CdSe semiconductor core/ ZnS Shell• Unique Spectral properties

– Broad absorption

– Narrow emission– Wavelength depends on size

3 nm

QDs vs. Other Fluorescence

Quantum dots conjugate - redAlexa 488 conjugate - green

Wu et al. Nature; 2003

• Photostability (quantum dots do not photobleach)

QDs vs. Other Fluorescence

Jaiswal & Simon 2004

• Broader excitation spectrum and narrower emission spectrum

• No spectral overlap between dots of different size

Conjugating quantum dots to biomolecules

• Avidin or protein-G with positively charged tail conjugated to negatively charged DHLA coat of quantum dots

AvidinAvidin

protein G

Quantum dots

Summary

• Gold Nanoparticle are key components of numerous

assays for biologically analytes, including proteins,

nucleic acids, small molecules and metal ions.

• Colorimetric assays provide a sensitive test

• Gold nanoparticle improve the performance of

many conventional assays.

Future Outlook

• Development of QD lasers at communication wavelengths

• Gain and stimulated emission from QDs in polymers

– Polymeric optoelectronic devices?

• Probe fundamental physics

• Quantum computing schemes (exciton states as qubits)

– Basis for solid-state quantum computing?

• Biological applications

• Material engineering

– How to make QDs cheaply and easily with good control?

• Let’s not forget the electronic applications too!

• Lots to do!

C. Seydel. Quantum dots get wet. Science, 300, p. 80-81, Apr 2003.

Thank you

T C

MT

G

UTG

Methylation probe

Probe Sequence

AuNP-Probe Met 5’-thiol-TTTTTTTTTTACCTTACCCGCTCCATCGCG -3’

Test line (T) Met’ 5’-TCACTAACCGCTCCTCAAACAAATACG-TEG-biotin-3’

Control (C) Met Com 5’-biotin- TTTTTTTTTTCGCGATGGAGCG GGTAAGGT-3’

Methylation probe

AuNPs-Probe: Methylation-probe 15 µL Test line(T): 1/10 Streptavidin-Biotin-Probe (Methylation)Control line(C): 1/10 Streptavidin-Biotin-Probe (Control)Hybridization buffer: 6XSSC, 0.5% SDS, 50% Formamide

T C

MTG=meth

ylation

MT

G

Probe µl Sequence

AuNP-Probe Unmet 15 5�-thiol-TT TTT TTT TTC ACA ACT AAC CTT ACC CAC TCC ATC ACA -3�

Test line (T) 1/10 Unmet� 1 5�-CAT CAA ACA TCT CCA ACA ACC ACT CCA C-TEG-biotin-3�

Control (C) 1/10 Unmet 1 5�-biotin-TTTTTTTTTTTGTGATGGAGTGGGTAAGGTTAGTTGTG-3�

Hybridization buffer 1 6×SSC, 1%BSA, 0.01% SDS, 0.2% Tween-20,

Hybridization buffer 2 6XSSC, 1% BSA, 0.01% SDS, 0.2% tween 20, 50% Formamide

Condition adjustment of new unmethylation biotin-probe

MT

G

UTG

UTG

Hybridization

buffer 1

Hybridization

buffer 2

0.1 µM Synthetic target

(Met or Unmet) 10 µl

Add 90 µl

Hybridization buffer

Apply mixture to

sample pad

Result: Buffer 2 can reduce non specific hybridization

MTG=methylation target, UTG=unmethylation target

T C MT

G

Probe µl Sequence

AuNP-Probe Met 15 5�-thiol-TTT TTT TTT TAC CTT ACC CGC TCC ATC GCG -3�

Test line (T) 1/10 Met� 1 5�-CGT CAA ACA TCT CCG ACG ACC GC-TEG-biotin-3�

Control (C) 1/10 Met 1 5�-biotin- T TTT TTT TTT CGC GAT GGA GCG GG TAA GGT-3�

Hybridization buffer 2 6XSSC, 1% BSA, 0.01% SDS, 0.2% tween 20, 50% Formamide

Condition adjustment of new methylation biotin-probe

MT

G

UTG

0.1 µM Synthetic target

(Met or Unmet) 10 µl

Add 90 µl

Hybridization buffer

Apply mixture to

sample pad

MT

G UTG

MTG=methylation target, UTG=unmethylation target

T C MT

G

Condition adjustment of strip test with genomic DNA

MT

G

UTG MT

G UTG

DNA 5 µl

(treat bisulfite)

Denature

at 100oC, 5 min

Chill in ice, 15 min

Apply DNA to

sample pad

Apply buffer to

sample pad

Probe µl Sequence

AuNP-Probe Unmet

Met

15

15

5�-thiol-TT TTT TTT TTC ACA ACT AAC CTT ACC CAC TCC ATC ACA -3�

5�-thiol-TTT TTT TTT TAC CTT ACC CGC TCC ATC GCG -3�

Test line (T)

1/10

Unmet

Met

1

1

5�-CAT CAA ACA TCT CCA ACA ACC ACT CCA C-TEG-biotin-3�

5�-CGT CAA ACA TCT CCG ACG ACC GC-TEG-biotin-3�

Control (C)

1/10

Unmet

Met

1

1

5�-biotin-TTTTTTTTTTTGTGATGGAGTGGGTAAGGTTAGTTGTG-3�

5�-biotin- T TTT TTT TTT CGC GAT GGA GCG GG TAA GGT-3�

Hybridization

buffer

6×SSC, 1%BSA, 0.2% Tween-20, 0.01% SDS

Met

Unmet

B=Bisulfite treatment DNA, N=No treatment DNA

B

B

N

N

1 µg DNA(Male)

Denature

at 100oC, 5 min

Chill in ice, 10 min

Apply DNA to

sample pad

Apply buffer to

sample pad

ZP3 SRY

Probe µl Sequence

AuNP-Probe SRY

ZP3

10

10

5�-thiol-T TTT TTT TTT GAT GAT TAC AGT CCA GCT GTG CAA G-3�

5�-thiol-TTT TTT TTT TAG CCA TCC TGA GAC GTC CGT ACA-3�

Test line (T)

1/10

SRY

ZP3

1

1

5�-GAA TAT TCC CGC TCT CCG GAG AAG TTT TTT TTT T-biotin-3�

5�-GCC CGT ACT GGT GGA GTG TCA TTT TTT TTT T-biotin-3�

Control (C)

1/10

SRY

ZP3

1

1

5�-biotin-TT TTT TTT TTC TTG CAC AGC TGG ACT GTA ATC ATC-3�

5�-biotin-TTT TTT TTT TTG TAC GGA CGT CTC AGG ATG GCT-3�

Hybridization

buffer

6×SSC, 1%BSA, 0.2% Tween-20, 0.01% SDS

T C

Result: SRY test line appear red band

Condition adjustment of SRY strip test