1. Single Molecule Detection Technology Jun Qian BioE 494 Prof. Mansoori Dec. 6. 2007

2. SMD Introduction Advantages: –Ultra sensitive –High efficient –Very Direct Applications: –Biological system DNA, RNA, Protein Cancer cell detection –Chemical analysis Organics –Environment test pH pollutant

3. SMD Techniques Scanning Probe Microscopy –Scanning Tunneling Microscopy, STM –Atomic Force Microscopy, AFM Optical Spectroscopy –Single Molecule Fluorescence –Surface-enhanced Raman Scattering Manmade Nano Structure –Nano electronics, Nano-FET based on Nanowire –Nano mechanics –Nano fluid

4. SPM techniques STM –Invented in 1981 by Binning and Rohrer –Tunneling current between tip and sample –Conductive sample AFM –Invented in 1986 by Binning, Quate and Gerber –Atomic force between tip and sample –All sample surface Conductor, semi-conductor, insulator Solid, liquid NaCl crystal By AFM Au (100) Surface By STM

5. Optical spectrum techniques Single Molecule Fluorescence Single molecule on solid surface A pentacene-doped p-terphenyl crystal stem –Cooling and trapping single molecule in a solid Fluorescence excitation as a function of the excitation frequency O1,O2 fluorescence excitation spectrum in p-terphenyl crystals Two successive scans of the excitation spectrum of a single molecule M. Orrit and J. Bernard, “Single Pentacene Molecules Detected by Fluorescence Excitation in a p-Terphenyl Crystal”, Physical Review Letters, Vol.65, pp: 2716-2719, 1990

6. Optical spectrum techniques Single Molecule Fluorescence Study dynamic structural changes of macromolecules undergoing biochemical reactions Single pair fluorescence resonance energy transfer (sgFRET) is used to measure the distance changes between two sites on a macromolecule. Nature Structural Biology, Vol.7, pp: 724-729, 2000

7. Optical spectrum techniques Surface-enhanced Raman Scattering First paper describing single molecule detection using surface- enhanced Raman scattering Single R6G adsorbed on silver colloid “ Hot particles ” observe Stokes- shifted light –Not every colloid light Advs compared to fluorescence –Strong intensity –Long lifetime Shuming Nie, and Steven R. Emory, “ Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering ”, Science, Vol.275, pp: 1102-1106, 1997

8. Optical spectrum techniques Surface-enhanced Raman Scattering First observation of single molecule Raman scattering –Extremely large effective cross sections –Non-resonance excitation Single crystal violet molecule in aqueous colloidal silver solution Statistical Result: –Poisson distribution for actually measuring 0, 1, 2, or 3 molecules. Physical Review Letters, Vol.78, No.9, pp: 1667-1670, 1997

9. Optical spectrum techniques Surface-enhanced Raman Scattering Detect molecular vibrations in single oxygen transport protein hemoglobin (Hb) protein attached to isolated and immobilized silver nanoparticles Electromagnetic field effects dominate the surface enhancement Single molecule Hb SERS is probably only for molecules sited between Ag particles. Physical Review Letters, Vol.83, No.21, pp: 4357-4360, 1999

10. Some Developments about SERS-1 Surface-Enhanced Resonance Raman Scattering (SERRS) of R6G combined with Spectrum and CCD- camera SERRS-spectra as a function of time reveal inhomogeneous behaviors –On/off blinking –Spectral diffusion –Intensity fluctuations –Split of lines Journal of Luminescence, Vol.94-95, pp: 147-152, 2001

11. Some Developments about SERS-2 Combine SERS with coherent anti-stokes Raman scattering for high-sensitivity detection Provide3 orders of signal enhancement compared with SERS dGMP and dAMP Optics Letters, Vol.30, No.9, pp: 1024-1026, 2005

12. Some Developments about SERS-3 R6G adsorbed on colloid silver clusters Huge SERRS spectra observed –Burst of intensity ten times the average value Intrinsic complicated interaction between R6G and environment Applied Physics Letters, Vol.88, pp: 034104-034106, 2006

13. Some Developments about SERS-4 Silver reduction on and by nanostructured Si films Laser-activated –Ostwald ripening –formation of aggregtes Common Dye Molecule Fluorescein (C20H10Na2O5) Large Ag particles –Ostwald-ripened particles as dimer or trimer Applied Physics Letters, Vol.89, pp: 233103.1-233103.3, 2006

14. SMD by Nanostructure Nano electronics, Nano-FET based on Nanowire –Selective adsorption of specific virus or chemicals –Measure Electrical properties Nano fluid –Nanosize channel allow only single molecule through open pores –Directly see the image or Measure electrical properties Nano mechanics –Resonance Frequency Shift after single molecules attach to cantilever

15. Nano electronics Direct, real-time detection of single virus particle with high selectivity by using nanowire field effect transistors Nanowire modified with antibodies for influenza A shows conductance changes of binding and unbinding in present of influenza A but not paramyxovirus or adenovirus. PNAS, Vol.101, No. 39, pp: 14017-14022, 2004

16. Nano fluid Nano Letters, Vol.7, pp: 2901-2906, 2007 Applied Physics Letters, Vol.88, pp: 033106, 2006

17. Nano fluid Nano Letters, Vol.4, No.3, pp: 497-501, 2004 Nano Letters, Vol.5, No.2, pp: 403-407, 2005

18. Nano mechanics APL 84, 1796-1978, 2004

19. Summary of SERS No. YearAuthorsMethodType of MoleculeMediumFeatures Raman Shift (cm- 1) 11997K. KneippSERS, nonresonant near-IR excitation Crystal violetAqueous colloidal silver solution 700, 1700 21997Shuming NieSERS, Fluorescence optical spectroscopy R6Gsilver colloidal nano particles, 110-120 nm 1184, 1310, 1385, 1514, 1578, 1657 31998K. KneippNonresonant SERS,AdenineSilver clusters 100nm, 20um 735, 1335 41999Hongxing XuSERSHemoglobin (Hb)Silver nanoparticles, Hb between them 1375, 1586, 1640 52001A. MeixnerSERRS, CCD and Spectrometer R6GSilver cluster, < 500 nm 1180, 1300, 1375, 1500, 1560, 1640 62005TW. KooSERS, anti-Stokes Raman scattering dAMPColloidal silver suspension730 72006ZH. ZhouAnisotropy SERRS, Huge SERRS R6GColloidal silver clusters1250, 1309, 1358, 1650 82006A. Kann Kalkan SERSDye fluorescein (C20H10Na2O5) Silver reduction on and by nanostrutured Si films, aggregates 1180, 1325, 1550

20. Summary of Nanofluid Device No. YearAuthorDeviceFabrication Approach Target MoleculeSignal Detection 12004A. MaraAsymmetric Polymer Nanopore Latent ion track etching technique DNACurrent change when DNA block the pore 22005M. Karhanek NanopipettePulled glass capillary 24-base DNA modified by GNP Blockades of ion current when DNA translocate through the nanopipette tip 32006M. WangSilicoa nanochannel Scanned coaxial electrospinning 5- Iodoacetamidoflu orescein (IAF) Monitor fluorescence signal when single molecule translocating 42007G. Chansin Nanopore members Semiconductor film and etch fabrication DNAOptical detection of DNA translocation through the holes