Nanogold & Quantum Dot as Novel Biosensors

1. Gold Nanoparticle as Biosensor
Nanogold &Quantum Dot
as
Novel Biosensors
www.nanopartz.com/Gold_Nanorods.htm
Amornpun Sereemaspun, MD. PhD.
E-mail : amornpun.s@gmail.com
Nanobiomedicine Laboratory
• Nano gold (Colloidal Gold)
Department of Anatomy – Nanometer-sized particles of gold in a fluid
Faculty of Medicine – Size 1-100 nm.
Chulalongkorn University
– Intense red or yellowish color
Why Gold Nanoparticles ? Gold Nanoparticles Synthesis
• Easy to synthesis reduction stabilization
• Protocol have been approved (J. Turkevich et al. 1951) Au3+
+
Au0 Au0
• 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
Gold Nanoparticles and Optical Properties of Nanogold
Biomolecules
webexhibits.org www.azonano.com
• Nanogold size is similar to many cellular objects
• Gold surface can be coated by various biomolecules • The optical properties of gold nanoparticles can be
tuned carefully by controlling their size and shape

2. Basic optical properties of Optical Properties of AuNPs
nanoparticles
8
NanoGold As Products NanoGold As Products
Lateral flow strip test
From http://microgravity.hq.nasa.gov/general_info/homeplanet_lite.html
Leptospirosis
• A worldwide common
zoonosis in mammalian
• Spirochete-born disease
• Empirical diagnosis-based
• Staining – Gram unstainable
– Silver stain OK
• Culture – special media,
Take times

3. CFU
Leptospirosis 106 Urine Pregnancy Test
5 ×105
Nanogold Comparision with comercial kit
105
Dot-Blot ELISA
5 ×104
104
AuNP
control 10 102 104 105 106 CFU
Rojanathanes R. et al. 2008 Taiwan OB-GYN
2008,
Fluorescence-based detection of Nanogold and DNA Detection
protein kinase
Kiley et al.(Nanomedicine. 2008)
Mirkin et al. (Science 1997 ) reported DNA sandwich hybridization
assay using DNA-nanogold conjugate.
Kim, Y.-P., et al., Biosens. Bioelectron. (2007), 16
Lateral Flow Strip Test
Microchromatographic-based
Conjugate probe test line probe Control line probe
http://www.rapid-diagnostics.org/index.htm
Control line
Control line
Test line
Test line
Sample pad
Sample pad Conjugate probe
Conjugate probe

4. Lateral flow nucleic acid test strips Lateral flow nucleic acid test strips
Xun et al. (Anal. Chem. 2009) Ioannis et al. (Anal. Chem. 2007)
applied nucleic acid biosensor reported the first dry-reagent
based on the oligonucleotide dipstick assay for SNP
functionalized Au-NPs and genotyping by primer extension
lateral flow for the detection of
human genomic DNA directly
with a detection limit of 2.5
µg/mL (1.25 fM)
20
(Zhao et al., PNAS,2004)
(Wang et al., Bioconjugate Chem, 2007)
What Are Quantum Dots?
• Crystalline fluorophores
• CdSe semiconductor core/ ZnS Shell
• Unique Spectral properties
– Broad absorption
– Narrow emission
– Wavelength depends on size
3 nm
(Rosi et al., Science, 2006)

5. QDs vs. Other Fluorescence QDs vs. Other Fluorescence
• Photostability (quantum dots do not photobleach) • Broader excitation spectrum and
narrower emission spectrum
• No spectral overlap between dots
of different size
Quantum dots conjugate - red
Alexa 488 conjugate - green
Wu et al. Nature; 2003
Jaiswal & Simon 2004
Conjugating quantum dots to biomolecules Quantum dots
Avidin
• Avidin or protein-G with positively
charged tail conjugated to negatively
charged DHLA coat of quantum dots
protein G
Summary Future Outlook
• Gold Nanoparticle are key components of numerous • Development of QD lasers at communication wavelengths
• Gain and stimulated emission from QDs in polymers
assays for biologically analytes, including proteins,
– Polymeric optoelectronic devices?
nucleic acids, small molecules and metal ions. • Probe fundamental physics
• Colorimetric assays provide a sensitive test • Quantum computing schemes (exciton states as qubits)
– Basis for solid-state quantum computing?
• Gold nanoparticle improve the performance of • Biological applications
many conventional assays. • 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.

6. Methylation probe
T C
MT
G
UTG
Thank you 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’
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
Condition adjustment of new unmethylation biotin-probe Condition adjustment of new methylation biotin-probe
0.1 µM Synthetic target T C 0.1 µM Synthetic target
(Met or Unmet) 10 µl MT (Met or Unmet) 10 µl MT T C
Hybridization
buffer 1 G MTG
UTG UTG
Add 90 µl Add 90 µl G
Hybridization buffer Hybridization MT Hybridization buffer MT
UTG
buffer 2
G
UTG G
MTG=methylation target, UTG=unmethylation target MTG=methylation target, UTG=unmethylation target
Apply mixture to Apply mixture to
sample pad Probe µl MTG=methSequence sample pad Probe µl Sequence
AuNP-Probe Unmet 15 ylation
5 -thiol-TT TTT TTT TTC ACA ACT AAC CTT ACC CAC TCC ATC ACA -3 AuNP-Probe Met 15 5 -thiol-TTT TTT TTT TAC CTT ACC CGC TCC ATC GCG -3
Test line (T) 1/10 Unmet 1 5 -CAT CAA ACA TCT CCA ACA ACC ACT CCA C-TEG-biotin-3 Test line (T) 1/10 Met 1 5 -CGT CAA ACA TCT CCG ACG ACC GC-TEG-biotin-3
Control (C) 1/10 Unmet 1 5 -biotin-TTTTTTTTTTTGTGATGGAGTGGGTAAGGTTAGTTGTG-3 Control (C) 1/10 Met 1 5 -biotin- T TTT TTT TTT CGC GAT GGA GCG GG TAA GGT-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
Hybridization buffer 2 6XSSC, 1% BSA, 0.01% SDS, 0.2% tween 20, 50% Formamide
Result: Buffer 2 can reduce non specific hybridization
Condition adjustment of strip test with genomic DNA Condition adjustment of SRY strip test
DNA 5 µl
(treat bisulfite) T C 1 µg DNA(Male) T C
B
MT
Denature G Met Denature ZP3 SRY
at 100oC, 5 min N MTUTG at 100oC, 5 min
G
B MT
Chill in ice, 15 min UTG Unmet Chill in ice, 10 min
N G Apply DNA to
Apply DNA to
B=Bisulfite treatment DNA, N=No treatment DNA
sample pad sample pad Probe µl Sequence
AuNP-Probe SRY 10 5 -thiol-T TTT TTT TTT GAT GAT TAC AGT CCA GCT GTG CAA G-3
Probe µl Sequence 5 -thiol-TTT TTT TTT TAG CCA TCC TGA GAC GTC CGT ACA-3
ZP3 10
AuNP-Probe Unmet 15 5 -thiol-TT TTT TTT TTC ACA ACT AAC CTT ACC CAC TCC ATC ACA -3
Test line (T) SRY 1 5 -GAA TAT TCC CGC TCT CCG GAG AAG TTT TTT TTT T-biotin-3
Apply buffer to Met 15 5 -thiol-TTT TTT TTT TAC CTT ACC CGC TCC ATC GCG -3
Apply buffer to 1/10 ZP3 1 5 -GCC CGT ACT GGT GGA GTG TCA TTT TTT TTT T-biotin-3
Test line (T) Unmet 1 5 -CAT CAA ACA TCT CCA ACA ACC ACT CCA C-TEG-biotin-3
sample pad 1/10 Met 1 5 -CGT CAA ACA TCT CCG ACG ACC GC-TEG-biotin-3
sample pad Control (C) SRY 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
1/10 ZP3 1
Control (C) Unmet 1 5 -biotin-TTTTTTTTTTTGTGATGGAGTGGGTAAGGTTAGTTGTG-3
5 -biotin- T TTT TTT TTT CGC GAT GGA GCG GG TAA GGT-3 Hybridization 6×SSC, 1%BSA, 0.2% Tween-20, 0.01% SDS
1/10 Met 1 buffer
Hybridization 6×SSC, 1%BSA, 0.2% Tween-20, 0.01% SDS
buffer
Result: SRY test line appear red band