1. Miss. Siriporn Tontipattananon ID 565020075-3
Department of Biochemistry, Faculty of Science, Khon Kaen University
Gold-based optical biosensor for single mismatched DNA
detection using salt-induced hybridization
Zongrui Zhan, Xingyi Ma, Cuong Cao, Sang Jun Simb
Biosensors and Bioelectronics 32 (2012) 127–132

2. 2
Statistic of cancer
(http://www.chulacancer.net)

3. BRCA1 gene located
Breast Cancer
BRCA1 -
 The BRCA1 protein is classified as
a tumor suppressor
3
Breast Cancer

4. Method of breast cancer detection
 mammography
 magnetic resonance imaging (MRI)
 Biopsy
4

5.  polymerase chain reaction (PCR)
 Biosensor
Early detection
http://www.biomed.in.th/biosensor/ 5

6. Previous of study
6

7. (http://www.parkafm.com/jp/gallery_list.php?id=167)
The atomic force microscope (AFM) of gold nanoparticles
Gold-based optical biosensor
gold nanoparticle (Au-NP)
 stable metal particles
 conduct electricity, magnetic and
optical properties
7

8. The diameter of gold nanoparticles determines the wavelengths of light
absorbed and the colors in this diagram illustrate this effect.
8
gold nanoparticle (Au-NP)

9. Main steps of sandwich ELISA assay
(http://www.epitomics.com/products/product_info/1257)
sandwich ELISA assay
9

10. DNA hybridization
Scheme of DNA hybridization
(http://www.ncbi.nlm.nih.gov/books/NBK26837/figure/A1569/?report=objectonly) 10

11. Objectives
To investigate in various concentrated salt
solutions discriminating single-mismatched DNA
in a homogeneous solution.
To evaluate the relative importance of the salt
concentration that contributes to hybridization
efficiency.
11

12. Scope of study
12
1.Preparation of the gold colloid
2.Preparation of gold nanoprobes
3.Preparation of PMP capture probes
4.DNA hybridization procedure
5.Experimental procedures
for BRCA-1 detection

13. Materials and methods
Preparation of the gold colloid
Sodium citrate
(reducing agent)
HAuCl4
e-
gold colloid
(colorless red)
Au+3
(aquoues)
+
Auo
(solid)
3e-
13

14. Scope of study
14
1.Preparation of the gold colloid
2.Preparation of gold nanoprobes
3.Preparation of PMP capture probes
4.DNA hybridization procedure
5.Experimental procedures
for BRCA-1 detection

15. centrifuged and washed
14,000 rpm,
30 min, 4oC
Preparation of OEG-stabilized gold nanoprobes
Materials and methods
Absolute
ethanol
EG6-COOH/EG3-OH
1:9 molar
Au-NPs
solution
9 mL
1 mL
overnight
incubation
supernatant
(decant)
pellet
Added NHS/EDC
solution
suspended in
PBS buffer
solution
*5’-Amino-(C6)-
add detection probe*
15
OEG : Oligo ethylene glycol

16. Fig. 2 Scheme of the nanoprobe preparation and comparison of UV–vis spectra
for colloidal gold nanoparticles in aqueous solution, OEG-functionalized AuNPs,
and nanoprobes in PBS solution. The inset image shows the size of colloidal gold
nanoparticles.
Results and
discussions
 Characterization of gold nanoprobes
16

17. Scope of study
17
Preparation of the gold colloid
Preparation of gold nanoprobes
preparation of PMP capture probes
DNA hybridization procedure
Experimental procedures
for BRCA-1 detection

18. preparation of PMP capture probes
Materials and methods
PMPs
Incubated 30min at room temp.
and wash 0.01M PBS solution
conjugated with biotinylated
capture DNA
wash
stored in a 0.5 mL stock solution
(0.01 M PBS solution contained 1% BSA)
streptavidin/capture probe*
*Capture probe 5’-GAAACCCTATGTATGCTCTTTTTTTTTT-Biotin-3’
(Streptavidin coated
MagnetSphere Para-
Magnetic Particles)
18

19. Scope of study
19
1.Preparation of the gold colloid
2.Preparation of gold nanoprobes
3.Preparation of PMP capture probes
4.DNA hybridization procedure
5.Experimental procedures
for BRCA-1 detection

20. DNA hybridization procedure
Materials and methods
wash with
hybridization buffer
DNA solutions
(non-cognate DNA
and target DNA*)
*Detection probe 5-Amino-(C6)-TTTTTTTTTTTTTTTGTATGAATTATAATCAAA-3
Non-cognate DNA 5-ACACGCTTGGTAGACTTTTTTTTTTAGCATCGATAACGTT-
3
Incubate
1h at 37oC
wash
+ Au-NPs
+ Buffer**
Incubate
wash
** 0.1-0.5M Hybridization buffer 50 uL 20

21. Materials and methods
Experimental procedures for BRCA-1 detection
Au 5’-Amino-(C6)-(T)15GTATGAATTATAATCAAA-3’
3’-CATACTTAATATTAGTTT∙∙∙CTTTGGGATACATACGAG-5’
3’-CATACTTAXTATTAGTTT∙∙∙CTTTGGGATACATACGAG-5’
5’-GAAACCCTATGTATGCTC(T)10-Biotin-3’
detection probe
Capture probe
PM
P
Fig. 1 Schematic illustration for
the entire experimental strategy
for BRCA-1 detection.
Target DNA
Single-mismatched DNA
21

22. Fig. 3 (A) Absorbance spectra of gold nanoprobe in various concentrations of salt.
(Experimental condition: nanoprobes 50 µL; Na+ concentrations of HB range from
0.1 to 0.5 M; hybridization 1 h at 37 ◦C.)
Results and discussion
 Effect of salt concentration
Absorbance
Wavelength (nm)
A
22
527 nm

23. MMT : single-mismatched DNA
PMT : perfectly matched DNA
Fig. 3 (B) Effect of salt concentration on DNA hybridization efficiency.
Results and
discussions
 Effect of salt concentration
0.25 M 0.4 M
23
0 M

24. Fig. 4 (A) Nanoprobe-based colorimetric detection for single-base
mismatches.(Experimental condition: target DNA 10 pM; Na+ concentrations of
HB 0.25 M; hybridization 1 h at 37 ◦C.)
Results and
discussions
 Discrimination of single mismatches
24
Target DNA
5’-GAGCATACA··· ATTATAATTCATAC-3’
Single-mismatched DNA
5’-GAGCATACA··· ATTATXATTCATAC-3’ (“X” stands for T, C, G)

25. Fig. 4 (B) Absorbance intensity in colorimetric detection of target DNA spanning a 0.1
fM to 100 pM concentration range.
(Experimental condition: target DNA 100 aM to 100 pM; Na+ concentrations of HB 0.25
M; hybridization 1 h at 37 ◦C.)
Results and
discussions
25
 Hybridization signal intensity versus DNA concentration

26. Fig. 5. Detection of 10 pM DNA target (black bars) and 10 nM non-cognate DNA
(gray bars) in buffer, 1/5 diluted serum, and undiluted serum.
(Experimental condition: target DNA 10 pM; Non-cognate DNA 10 nM;
Na+ concentrations of HB 0.25 M; hybridization 1 h at 37 ◦C.)
Results and
discussions
 Hybridization signal intensity versus DNA concentration
26

27. Conclusions
The absorption peak of detection probe (Au-OEG-DNA)
centered at 527 nm was assigned to 15 nm
NaCl concentration also caused the AuNP finally to aggregate
as a result of decreased electrostatic repulsion between AuNPs
27

28. Conclusions
Increasing salt concentration from 0.1 to 0.5 M,
optical absorbance of MMT and PMT increased and
A maximum difference appeared at 0.25 M
28
selectively identify DNA targets concentration of
0.1 fM ; Na+ concentrations of HB 0.25 M

29. Thank you!

30. Previous studies have shown factors of hybridization
 Steric crowding
 steric hindrance
Results and
discussions
Electrostatic repulsive
►functionalized group
►spacer

31. Au
C6H5Na3O7
Au
Results and
discussions

32. centrifuged
14,000 rpm,
30 min, 4oC
Preparation of OEG-stabilized gold nanoprobes
Materials and method
Absolute
ethanol
EG6-COOH/EG3-OH
1:9 molar
Au-NPs
solution
9 mL
1 mL
overnight
incubation
supernatant
(decant)
pellet
HS(CH2)11(OCH2CH2)6OCH2COOH(EG6-COOH)
and HS(CH2)11(OCH2CH2)3OH (EG3-OH)

33. DNA hybridization procedure
Materials and method

34. Capture probe 5’-GAAACCCTATGTATGCTCTTTTTTTTTT-Biotin-3’
Detection probe 5’-Amino-(C6)-
TTTTTTTTTTTTTTTGTATGAATTATAATCAAA-3’
Target DNA
5’-GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-3’
Single-mismatched DNA
5’-GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATXATTCATAC-3’
(“X” stands for T, C, G)
Non-cognate DNA 5’-
ACACGCTTGGTAGACTTTTTTTTTTAGCATCGATAACGTT-3’

35. HS(CH2)11(OCH2CH2)6OCH2COOH (EG6-COOH)
HS(CH2)11(OCH2CH2)3OH (EG3-OH)

36. N-(3-dimethylaminopropyl)- N’-ethylcarbodiimide (EDC)
and N-hydroxysuccinimide(NHS)
• Covalent
• non-cytotoxic in vitro
• Crosslinking agents introduce &zero length' amide-
crosslinks between carboxylic acid groups from
aspartic and glutamic acid residues, and amino
groups from (hydroxy-) lysine residues

38. BRCA1 founder mutations
c.181T>G (BIC: 300T>G), c.4035delA (BIC: 4154delA)
and c.5266dupC (BIC: 5382insC) and found negative earlier.
BRCA1 mutations
(Breast Cancer Information Core : BIC database)

40. Phosphate buffered saline : PBS buffer
One of the common composition of PBS
Salt Concentration Concentration
(mmol/L) (g/L)
NaCl 137 8.01
KCl 2.7 0.20
Na2HPO4 • 2 H2O 10 1.78
KH2PO4 2.0 0.27
pH 7.4

43. Au—ester----NHS/EDC---peptide bond---DNA