Diamond in Nanoscale Biosensing
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1 Czech Nano-Team Workshop 2006 Diamond in Nanoscale Biosensing Bohuslav Rezek Institute of Physics AS CR
2 Acknowledgements Dr. Christoph Nebel Dr. Dongchan Shin Dr. Hideyuki Watanabe Diamond Research Center AIST, Tsukuba, Japan Bohuslav Rezek, Institute of Physics ASCR 2
3 Outline Why nanoscale biosensing? Why diamond? Hydrogen-terminated and oxidized diamond surfaces Attachment of DNA to diamond Photo- and electrochemical methods Fluorescence microscopy Structural and mechanical properties of DNA Atomic force microscopy in liquids Optimized detection of DNA thickness (phase shift) Detailed DNA morphology Geometric model DNA orientation and density Mechanical stability of DNA bonding Comparison with other substrate materials Conclusion Bohuslav Rezek, Institute of Physics ASCR 3
4 Biosensing Crucial for health care, medical treatment, drug development, Typical: recognition of DNA sequences Encoded genetic information and functions Unique matching of base pairs (A-T,C-G) Big machines can do it well, but Nanoscale biosensing higher sensitivity (<fm), lower cost portable and remote diagnostics (aging society!) Needs substrate to carry DNA Needs new ways of detection probe A G T T C A $Mio target Genetic Analyzer (Applied Biosystems) Bohuslav Rezek, Institute of Physics ASCR 4
5 Diamond and biosensing Diamond is very interesting for bio-sensors semiconductor (wide band gap) considered highly biocompatible transparent (optical sensing) hard, durable, and stable well accepted by public diamond [W.Yang et al., Nature Mat. 1 (2002) 254] Bohuslav Rezek, Institute of Physics ASCR 5
6 Diamond surface functionalization How to make diamond? from methane using plasma assisted chemical vapor deposition (CVD) polycrystalline: on silicon or glass monocrystalline: on diamonds (homoepitaxy), advantageous for research How to attach molecules to inert diamond? Surface can be functionalized by atoms plasma techniques, wet chemical techniques we use H-terminated and oxidized surfaces Atoms can be replaced by organic molecules Photochemical reactions Electrochemical reactions H δ+ 2.1 δ O 3.5 C 2.5 C 2.5 C Bohuslav Rezek, Institute of Physics ASCR 6
7 Attachment of linker molecules to diamond Photochemical Electrochemical amino-decene aryl-diazonium 5-7 hours 1 minute reaction with hydrogen atoms conductive substrate required Bohuslav Rezek, Institute of Physics ASCR 7
8 Linking of DNA molecules linker cross-linker thiolated DNA sequence 5'HS-C6H12-T15-GC TTA TCG AGC TTT CG3' probe ds-dna length ~ 130 Å target probe fluorescent label length ~ 125 Å target Bohuslav Rezek, Institute of Physics ASCR 8
9 Probing DNA by Fluorescence Microscopy common technique Photochemical Electrochemical fluorescence (FAM) fluorescence (Cy5) Au oxidized H-terminated H-terminated oxidized no linker DNA present on H-terminated! (oxidized areas not fully dark) 100 μm DNA present! 100 μ m Crucial for bio-sensor functionality: morphology, arrangement, and stability of DNA beyond abilities of fluorescence Bohuslav Rezek, Institute of Physics ASCR 9
10 AFM in buffer solutions AFM scanner tip DNA diamond SSPE buffer BUFFERS AFM TIPS REGIMES SSPE/SDS buffer 2x SSPE/ 0.2% SDS buffer, ph=7.4 by NaOH advantages: bio-environment, no meniscus at tip silicon cantilevers (~75kHz in air, ~29kHz in liquid) force calibration: 56 nn/v contact (CM-AFM), oscillatory (OM-AFM), phase detection Bohuslav Rezek, Institute of Physics ASCR 10
![Thickness of DNA layers OM-AFM image in liquid height profile relative height [nm] 20 15 10 5 0 0 1 2 3 lateral distance [μm] - step in height ~ 70-80 Å](/docs-images/94/120064440/images/11-1.jpg "resolved - but DNA is soft matter true DNA layer thickness? AFM measurement optimized by monitoring phase contrast 27.4.")
11 Thickness of DNA layers OM-AFM image in liquid height profile relative height [nm] lateral distance [μm] - step in height ~ Å resolved - but DNA is soft matter true DNA layer thickness? AFM measurement optimized by monitoring phase contrast Bohuslav Rezek, Institute of Physics ASCR 11
12 Phase contrast in OM-AFM diamond (hard) DNA (soft) diamond-dna: phase contrast ~ difference in elastic properties phase contrast influenced by strength of tip-surface interaction adjusted by AFM setpoint ratio (A SP /A 0 ) Bohuslav Rezek, Institute of Physics ASCR 12
13 AFM phase images A SP /A 0 = 0.40 A SP /A 0 = 0.95 = 1 for free cantilever diamond DNA diamond DNA material contrast diamond DNA no contrast, DNA not affected by tip thickness? Bohuslav Rezek, Institute of Physics ASCR 13
14 Extrapolated DNA thickness Photochemical Electrochemical phase contrast [deg] A 0 ~ 6 nm phase contrast DNA thickness setpoint ratio A SP /A 0 [a.u.] DNA heigh [nm] phase contrast [deg] A 0 ~ 6 nm phase contrast DNA thickness setpoint ratio A SP /A 0 [a.u.] DNA height [nm] thickness (76 ± 8) Å thickness (82 ± 5) Å As tip-surface distance increases (setpoint ratio 1) DNA/diamond phase contrast decreases (less tip-dna interaction) thickness increases extrapolated DNA thickness (error bar ~ RMS surface roughness) Bohuslav Rezek, Institute of Physics ASCR 14
![DNA layer morphology OM-AFM image in liquid relative height [nm] 3 2 1 height profile 0 0](/docs-images/94/120064440/images/15-1.jpg "100 200 300 lateral distance [nm] substrate Features - surface modulations ~ 30 nm width")
15 DNA layer morphology OM-AFM image in liquid relative height [nm] height profile lateral distance [nm] substrate Features - surface modulations ~ 30 nm width (typical for closely packed DNA) - roughness RMS ~ 6-8 Å << thickness closely packed layer, no pinholes - fine structure ~ DNA? Bohuslav Rezek, Institute of Physics ASCR 15
16 Mechanical stability of DNA on diamond Bohuslav Rezek, Institute of Physics ASCR 16
17 Mechanical stability of DNA on diamond Photochemical H-terminated oxidized fluorescence: threshold (45 ± 12) nn covalent bonding error: interval, spring constant, statistics DNA removed threshold < (6 ± 4) nn non-covalent bonding (electrostatic?) H δ+ 2.1 δ O 3.5 C 2.5 C 2.5 C Bohuslav Rezek, Institute of Physics ASCR 17
![1 (2002) 254] diamond superior with regard to DNA bonding](/docs-images/94/120064440/images/18-2.jpg "stability, important for bio-sensor reproducibility note:")
18 Comparison of DNA removal forces [W.Yang et al., Nature Mat. 1 (2002) 254] diamond superior with regard to DNA bonding stability, important for bio-sensor reproducibility note: only approximate comparison, because the parameters and threshold values not clearly specified in the literature Bohuslav Rezek, Institute of Physics ASCR 18
19 Conclusions DNA attached to mono-crystalline diamond by photochemical and electrochemical methods Functionality as DNA-sensor demonstrated by fluorescence images of complementary DNA Properties of DNA layers on diamond resolved by AFM in liquids: closely packed, no pinholes, RMS roughness < 1 nm DNA molecules inclined, under angle of mechanically stable for forces up to 76 nn (good!), indicates covalent bonding threshold force DNA thickness angle DNA bonding photochemical process H-terminated surface (45 ± 12) nn (76 ± 8) Å 31 covalent oxidized surface < (6 ± 4) nn (20 ± 4) Å n/a non-covalent electrochemical process H-terminated surface (76 ± 18) nn (82 ± 5) Å 36 covalent oxidized surface (34 ± 9) nn (69 ± 5) Å 29 covalent [B. Rezek et al., J.Am.Chem.Soc. 128 (2006) 3884] Bohuslav Rezek, Institute of Physics ASCR 19
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