Nanoscale-Controlled Surface Materials, Bioanalysis, and Commercialization - NANO KOREA

Transcription

1 Nanoscale-Controlled Surface Materials, Bioanalysis, and Commercialization - NAN KREA Aug 28, 2007 Joon Won Park NanoSurface Biosciences PSTECH Pohang University of Science and Technology

2 Biomolecules on the Surface in Various Applications Biosensors Biochips (DNA Chip, Protein Chip) In-situ Kinetic Study of Biomolecules Biomolecules on the Surface Affinity Chromatography High Throughput Screening Immunoassay 1

3 Biomolecules Require Mesospacing for Comfortable Anchoring on the Surface 23.7 A B-DNA 45 A Lysozyme 139 A ATP synthase 2

4 Peptide Folding on the Spacing Controlled Gold Surface Whitesell et al. Science 261, 73 (1993) 3

5 An ptimized Dendron H H H N H H N NH H H H H H H 4

6 Inert Base Layer and Covalent Bonding H H H H H H H H H H H H H H H H H H H H H H H H H NH NH NH H NH H H H H H H H H H H H H H H H H H H H 5

7 Immobilization of Gold Nanoparticles at the Dendron Surface d: 1.4 nm Park et al. Langmuir 4257 (2005) 6

8 Spacing between Dendron Molecules on Surface SEM Image Distance between Dendron Molecules Population Particle number / area : 130 ea / 50 x 50 nm 2 Density: ea/nm Distance (nm) Average distance: 3.2 nm Standard deviation: 0.4 nm 7

9 8 Various Spacing up to 10 nm H H H H H H NH H H H N H N H N H H H H H H H H H H NH NH H H H H H H H H H H N H H H H H H H H H H H H H H H NH H H H NH NH H HH NH H H H NH H H H [9]-acid [18]-acid [27]-acid, [81]-acid ~ 3 nm ~ 10 nm Spacing

10 Dendron-Coated Surface Improves gnificantly SNP Discrimination Efficiency and Detection Limit of Gene Expression Profiling Joon Won Park NSB PSTECH Pohang University of Science and Technology KREA 9

11 Fabrication of DNA Microarray on Dendron-Modified Surface Dendron Substrate ne base mismatch site 100 : < 1 PM site MM site US Patent Application (2004) 10

12 Probe & Target ligonucleotides Probe oligonucleotide Probe 1: 5 -NH 2 -C 6 -CAT TCC GXG TGT CCA-3 Probe 2: 5 -NH 2 -C 6 -(T) 30 -CAT TCC GXG TGT CCA-3 X = A (complementary), T, G, C (mismatched) Target oligonucleotide Target 1: 5 -Cy3-TGG ACA CTC GGA ATG-3 Target 2: 5 -Cy3-CCT ACG AAA TCT ACT GGA ACG AAA TCT ACT TGG ACA CTC GGA ATG-3 11

13 Fluorescence Image of the Microarrays (I) 15-Base oligonucleotide (Probe 1) & 15-Base oligonucleotide (Target 1) PM IMM(TT) IMM(GT) IMM(CT) PM(AT) IMM(TT) IMM(GT) IMM(CT) Normalized gnal Ratio Hybridization condition: 1 nm target at 50 o C for 1 h 12

14 Molecular Beacon showed 100: 1 Ratio in Solution Molecular beacon in solution: 100: ~1 SNP discrimination ratio (Wang, H. et al. Nucleic Acids Res. 2002, 30, e61.) (MM/PM) in solution: 1 % (MM/PM) on agarose film: 19 % (MM/PM) on glass slide: 69 % 13

15 multaneous Detection of 7 Hotspots of p53 Gene Park et al. Nucleic Acids Research e90 (1995) Codon G A Relative 20 Intensity (%) 0 C T ngle Nucleotide Intensity less than 16% was observed for the all mutations. 14

16 Characteristics of the Surface in RNA Expression Profiling 1-1) Chip Design Chip design-4 10 Columns x 10 Rows x 4 Blocks Up-regulated genes Down-regulated genes No differentially expressed genes US patent application (2004) 15

17 1-2) Hybridization Condition: Probe concentration of 50 pmole/μl No blocking, 42 o C with 30% DMF No blocking, 65 o C Blocking, 42 o C with 30% DMF Blocking, 65 o C For gene expression analysis with NSB slides, blocking agent was not needed, while use of BSA was required for Corning slides. 16

18 1-3) Detection Limit: RNA from 293 Cell Line labeled with Cy3, HeLa Cell Line with Cy5 100 μg 50 μg NSB PSTECH 30 μg 10 μg 1.0 μg The signal intensity decreases as the target concentration decreases. It seems possible to detect the signal with very small amount of total RNA as low as 1.0 μg. 17

19 1-4) Correlation Analysis: Pearson s Correlation, p = 0.01 NSB PSTECH Slide Superb correlation was maintained even with 1 μg of total RNA. 18

20 Detection Limit of Total RNA by Microarray For adequate fluorescence, the total RNA required per array, is μg. Nat. Genetics Suppl. 21, 10 (1999) 19

21 DNA-DNA Interaction Measured with Picoforce AFM Yu Jin Jung and Joon Won Park In collaboration with School of Pharmacy, Nottingham University 20

22 Force Distance Curve from AFM F = -kd (Hook s Law) 21

23 Force Distance Curve for DNA-DNA 48 ± 2 pn a = 5'-G-G-C-T-C-C-C-T-T-C-T-A-C-C-A-C-T-G-A-C-A-T-C-G-C-A-A-C-G-G-3' b = 3'-C-C-G-A-G-G-G-A-A-G-A-T-G-G-T-G-A-C-T-G-T-A-G-C-G-T-T-G-C-C-5' Strunz et al. Proc. Natl. Acad. Sci. USA 96, (1999) 22

24 Proper Spacing mplifies Force-Distance Curve Tip Tip AFM Tip Retracting Force-Distance Curve ngle Dip : > 80 % US Patent Application (2005) 23

25 ptimized Spacing gives a ngle and Sharp Histogram 8 (A) pn (B) pn Counts pn Counts pn 55 pn Counts Force (pn) 30-base DNA immobilized on 3-acid/GPDES Force (pn) 30-base DNA immobilized on 9-acid/GPDES Force (pn) 30-base DNA immobilized on 9-acid/TPU 24

26 A Bigger Spacing Reduces Chance of the Binding 30-base DNA immobilized on 9-acid/TPU 30-base DNA immobilized on 27-acid/TPU ~ 60 % pn Counts ~ 20 % Counts pn Force (pn) Force (pn) The observed binding force is constant with a bigger spacing, while chance of the binding event is reduced significantly. 25

27 Unprecedented Binding Event (A) (B) pn 25 pn 120 Force (pn) 5 nm Approaching trace Counts Retracting trace 20 Distance (nm) Force (pn) 26

28 It is Now a Reversible Process Tip AFM Tip Approaching Tip AFM Tip Retracting Park et al. J. Am. Chem. Soc. in print 27

29 Resolved Binding Force Histogram 20 mer 30 mer 40 mer 50 mer 50 pn 59 pn 29 pn 39 pn GC content = 60 % 28

30 Discrimination of ngle Base Mismatched Pairs (-2) (-7) (-2) (-9) 29

31 Interaction Force between gnal Transducing Proteins AFM Tip AFM Tip : GST : Munc-18-1 : PLD1-PX 30

32 Interaction between PLD1 and Munc-18-1 EGF EGF : Epidermal Growth Factor EGFR : Epidermal Growth Factor Receptor EGFR PLC-γ1 SH3 PX PLD1 Munc-18-1 Munc-18-1 Physiological response 31

33 Spacing Matters! 9-acid (~ 3-4 nm) At a proper concentration 27-acid (~ 5-6 nm) % 53 ± 2 pn 100 pn 20 nm % 50 ± 2 pn 100 pn 20 nm Counts 100 Counts Force (pn) Force (pn) Ratio of force-distance curve ngle : Multiple = 2.4 : 1 Ratio of force-distance curve ngle : Multiple = 5.4 : 1 32

34 Loading Rate Dependence of the Unbinding Force PX-Munc-18-1 interaction F = k B T r x ln ( β ) x β k off k B T Slope : k B T/x β x β = 0.77 nm F (pn) Extrapolated to F =0 : k off = r x β / k B T r (pn/s) k off = s -1 K A = M -1 k on = M -1 s -1 K D = M 33

35 Modulation of the Force with Munc-18-1 AFM Tip Dendron GST PLC-γ1 PLD1-PX GST Munc-18-1 Dendron substrate k = 10.2 ~ 12.2 pn/nm r = 0.5 μm/s At 15 Cin PBS 34

36 Titration Curve with Munc nM 40 F T (pn) Concentration of Munc-18-1 (nm) Langmuir fitting shows that K A between PLD1 and Munc-18-1 is 1.3 x 10 9 M -1. Manuscript in preparation 35

37 NanoSurface Biosciences PSTECH August

38 NSB PSTECH At a Glance The first PSTECH-founded company. NSB PSTECH specializes on surface science for biological application. The first commercial products, dendron-coated glass slides for biochips, have been launched in the 4 th quarter of The second line of products, dendron-coated AFM tips, will be launched in the 3 rd quarter of Efficacy of our technology has been confirmed internationally and over 10 US patents have been filed. Revenue > 1M $ in US in the second year. 37

39 Short-Term Goals Sales of Biochip Plates and Coated AFM-Tips Cash Cows Expedite FDA Approval of Biochips Sales of Biochips (Non-FDA Approval Items) Research Uses Animal Diagnostics Biomarker Discovery Collaboration with Renowned US Medical Institutes 38

40 Homepage 39

41 Products verview 40

42 Technology verview Dendron-coated glass slides Dendron-coated AFM tips 41