nanogold & quantum dot as novel biosensors
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Nanogold & Quantum Dot as Novel BiosensorsTRANSCRIPT
Nanogold &Quantum Dot
as
Novel Biosensors
Amornpun Sereemaspun, MD. PhD.E-mail : [email protected]
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