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Bio-recognition and Bio-sensing

Bio-recognition and Bio-sensing. 本研究與奈米所許鉦宗教授研究室合作完成. The Potential and Niche of Biosensors. The global market for biochips increased from $1,637.5 millions in 2006 to $2,115.6 millions by the end of 2008.

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Bio-recognition and Bio-sensing

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  1. Bio-recognition and Bio-sensing 本研究與奈米所許鉦宗教授研究室合作完成

  2. The Potential and Niche of Biosensors • The global market for biochips increased from $1,637.5 millions in 2006 to $2,115.6 millions by the end of 2008. • It should reach $3,840.7 millions in 2013, a compound annual growth rate (CAGR) of 12.7%.

  3. Basic Structure of Biosensor Signal processing & data output Bio-recognition Energy transducer Interface • Techniques for transduce • Surface Plasma Resonance • Cantilever sensor • Field effect transistor • others

  4. Biomolecule Detection Methods Enzyme-linked Immunosorbent Assay Surface Plasmon Resonance < 1 pM (streptavidin) > 1 fM (antibody) Nature Reviews Drug Discovery 2002, 1, 515-528 Nature Protocols 2007 Nanowire Field-effect Transistor Cantilevers Sensor I < 1 fM (DNA, antibody) < 50 pM (DNA) Nature 2007, 446, 1066 - 1069 Nature2007, 445, 519-522

  5. Good for big molecule detection!! Yet, the detection of small molecule is a great challenge!!

  6. Comparison of Biosensing Techniques * Based on published data on SCI articles.

  7. + + + S D S D BOX BOX G (I/V) G (I/V) G0 G0 ΔG Time (s) Time (s) Operation Principle of SiNW-FET Biosensors

  8. Silicon nanowire field effect transistor (SiNW-FET) B Yi Cui, Qingqiao Wei, Hongkun Park, Charles M. Lieber, Science (2001) 293, 1289.

  9. Proc Natl Acad Sci U S A. 2005,102, 3208–3212.

  10. Single virus detection Nanomedicine 1(1), 51, 2006

  11. World Leading Groups Top-down: Nanoimprint Bottom-up: VLS James R. Heath (Caltech) Charles M. Lieber (Harvard) Nanolett., 2006 J. Am. Chem. Soc. 2000

  12. Our strategy • Silicon-based nanowires or nano-belt • Compatible with current industrial process

  13. Spacer Lithography Technology Removal of sacrificial Layer and etch sacrificial Spacer Etch Resist Resist SiNW SiNW E-beam lithography Spacer lithography SiNW SiNW

  14. SU-8 Microfluidics S D D S Array of Silicon Nanowire FETs • Sidewall spacer technique Dimensions: L > 1 μm, H = 20~70 nm, W > 40 nm

  15. SiNWs PDMS m-Fluidic Channel 1 cm Integration of SiNW-FETs with PDMS microfluidic Channel for Analytes Delivery PDMS microfluidics Sample size SiNWs with Pads Chips bonded onto a PCB board for measurement After UV/Ozone treatment, sample and microfluidic channel were bonded.

  16. The established biosensing platform---On the way for miniaturization

  17. How to overcome the intrinsic limitation of SiNW-FET? To detect a non-charged small molecule such as steroids.

  18. TESTOSTERONE 17α-ESTRADIOL The importance of steroids analysis • Estrone (E1) and estradiol (E2) determine the risk of breast cancer • Restricted drugs in sports • Hormone.

  19. Idea

  20. △5-3-KetoSteroid Isomerase (KSI) (1) Gene has been cloned and overexpressed. (2) A dimeric enzyme (m.w. 13.4 kDa/sub-unit) with a hydrophobic cavity containing Asp-38and Tyr-14 as the essential groups. (3) X-ray protein structure has been solved. (4)Strong affinity toward steroids

  21. Fluorescence of I14-labeled KSI and I14 in IPA I14

  22. Isolation of I14-labeled peptides

  23. MS/MS analysis of I14-labeled KSI (a) The result of MS/MS of M/Z = 822.7 (2+) was shown (b) The result of MS/MS of M/Z = 975.8 (2+) was shown ∆ m/z = 306 ( I14 – I )

  24. Asp-38 Tyr-14

  25. mA51

  26. 19-NT mA51

  27. steroids Design of a Nano-device for Steroids Detection

  28. Detection of Non-charge Analytes on SiNW-FET Protein design Reporting molecule (probe) Fabrication and modification of SiNWs Gene construction & Overexpression Protein modified by probe protein immobilization SiNWs Improve and Optimize protein function Detection system

  29. Experimental Design for Chemical modification Steps involving chemical reaction

  30. Cys : R-SH Link to reporter Link to SiNWs Lys : R-NH2 Y55F/K60R/F86C/Y88G/K92R/K108R/K119R/A125K

  31. Construction and Purification of KSI

  32. 4-ESTREN-17α-ETHYL- 17β-OL-3-ONE 19-NORANDROSTENDIONE TESTOSTERONE PROGESTERONE 17α-ESTRADIOL Dissociation constants of Steroids with KSI

  33. MW = 367 Da m/z of KSI_A51 [mod_A51] : [KSI] = 1:1 In pH 7.6 Tris – HCl buffer For 24 hrs. ∆ m/z = 367 Da m/z of KSI

  34. KSI Direct Immobilization of Enzyme onto the Si Nanowire Surface KSI-126C APTS BS3 KSI-126C NH2 Sulfo-NHS ester Citrate-passivation gold nanoparticles were deposited onto the KSI-modified surface

  35. Real-time Sensing on Steroid

  36. Linear correlation 1 fM~0.1 pM

  37. Conclusion We demonstrated the design, fabrication and testing of the world’s first SiNW-FET device for non-charged steroid detection with the sensitivity greater than 10-16 M.

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