1. 1 Lecture 1: Biodetection using Silicon Photonic Bandgap Devices Philippe M. Fauchet University of Rochester Supported in part by the National Science Foundation, the Infotonics Center of Excellence, and the Center for Future Health Biophotonics Winter School 2007

2. 2 Toronto New York Where is Rochester? California Italy > 3000 km >6000 km

3. 3 Rochester: the campus and the city

4. 4 Organization Long-Term Goal Materials Science of Porous Silicon Sensing Principle using Microcavities Examples of Biosensing In lecture 2: Ultimate Performance of these Biosensors Futuristic Application

5. 5 The state of the art…yesterday and today 1860 2002

6. 6 The state of the art…tomorrow

7. 7 Bio Meets Nano In nanometer 10 -1 1 1010 2 10 3 10 4 10 5 10 6 10 7 10 8 nm µm cm WaterGlucoseAntibodyVirus Bacteria Cancer Cell Fruit Fly Tennis Ball Chip

8. 8 Objectives Biosensor platforms capable of detecting the presence of harmful pathogens, including public health hazards and biowarfare agents, are under development. These biosensors rely on advances in molecular recognition, nanoscience, nanotechnology, and optics. They are can be used for lab-on-chip applications or form intelligent systems that can be used by untrained personnel.

9. 9 Porous Silicon Materials Science

10. 10 Porous Silicon: Etching Mechanism Porous Silicon Formation Electropolishing

11. 11 ~ 150 nm diameter ~200 nm pore-to-pore spacing 5  m 200 nm Intermediate Pore Size (150 nm) H. Ouyang et al., SPIE 5511, 71 (2004)

12. 12 Material: Porous Silicon Chemicals, short DNA strands, small molecules Macromolecules, proteins Viruses, bacteria Mesopores 20 nm Small Macropores 200 nm 2000 nm Large Macropores H. Ouyang, M. Lee, B. L. Miller, and P. M. Fauchet, in Tuning the Optical Response of Photonic Bandgap Structures II, SPIE Proc. (2005)

13. 13 10 µm Pore Size and Morphology Engineering From nanopores to mesopores to macropores and from “spongy” to directional and smooth 100 nm

14. 14 Bruggeman approximation to simulate refractive index of porous silicon n 2 =  Porosity (%) Effective index Index of Refraction Tunability Refractive index is a function of porosity, refractive index of silicon, refractive index inside the pores

15. 15 Biosensing Principles

16. 16 Biosensing with PSi Microcavities  The optical properties of a porous silicon microcavity are governed by the refractive index of the porous silicon layer(s)  The refractive index of a porous silicon layer depends on what is inside the pores  The functionalized internal surface of porous silicon can bind the desired biological objects (“targets”)  Binding is detected through a change in refractive index, hence a change in optical properties (luminescence, transmission or reflectivity)

17. 17 Exposed to species Sensing Principle Wavelength (  m) Reflectivity Internal surface modification Specific binding Red shift n n +  n

18. 18 Reflectivity Wavelength (  m) A single porous silicon layer n n +  n Single layer When the index of refraction of the porous layer changes, the position of the interference fringes changes The air/porous silicon/silicon structure forms a Fabry-Perot interferometer

19. 19 White light reflection from a porous Si film Sailor’s group, UCSD: C.L. Curtis et al., Electrochem. Soc. 140, 3492 (1993) L

20. 20 AB CD EF GHI JK L M Streptavidin b-Prot. AIgGRinseIgGRinse Protein A/Human IgG Binding to Porous Si Sailor’s group, UCSD

21. 21 Reflectivity Wavelength (  m) Reflectivity Wavelength (  m) Reflectivity Wavelength (  m) More sophisticated structures n n +  n Single layerRugate filter Microcavity

22. 22 C-Silicon Subtrate j t [mA/cm 2 ] [s] Electrolyte Multilayer Structures H. Ouyang et al, Adv. Funct. Mater.15, 1851 (2005)

23. 23 Bragg mirror Defect layer 75% porosity layer (n = 1.44): 50mA/cm 2 for 8 sec 2µm 70% porosity layers (n = 1.57) : 35mA/cm 2 for 11sec 50% porosity layers (n = 2.16) : 5mA/cm 2 for 32sec Porous Silicon Microcavity

24. 24 CONTROL OVER THREE LENGTH SCALES PSi Multilayer Mirror PSi Central Layer Porous Silicon Microcavity

25. 25 Reflectivity (%) Wavelength (nm) Max R ~ 100% Min R ~ 10% FWHM ~ 15nm Only 5 period Bragg mirrors Reflectivity of a PSi Microcavity

26. 26 0 20 40 60 80 100 600800100012001400160018002000 Reflectivity (%) Wavelength (nm) Quality Factor  >> 1000 Near Zero Reflectivity Dip Uniformity Over Large Areas High-Quality Microcavities Large index of refraction contrast (from >2.5 to <1.3)

27. 27 Reflectivity Spectra The number and sharpness of reflectivity dips increase as the thickness of the active layer increases:  At ~200 nm, one reflectivity dip is present  At ~3.5 mm, up to seven reflectivity dips are present Reflectivity (%) Wavelength (nm) 234 nm 1170 nm 2340 nm 3520 nm S. Chan et al., Mat. Sci. & Eng. C15, 277-282 (2001)

28. 28 Sensitivity on Refractive Index Wavelength (nm) Reflectivity (%) DIGITAL SENSOR on-state “1” off-state “0” n pore = 1.00 n pore = 1.03

29. 29 Porous silicon can emit light PSi Bragg Reflector PSi Active Layer Wavelength (nm) Photoluminescence Intensity (a.u.) FWHM ~ 4 nm

30. 30 Examples of Biosensing  DNA  Proteins  Bacteria

31. 31 DNA Biosensor - Details Si O-Si-CH 2 -CH 2 -CH 2 -O-CH 2 -CH-CH 2 O O O + + N- 3 DNA H H Silanized Porous Silicon DNA Strand.. Si O-Si-CH 2 -CH 2 -CH 2 -O-CH 2 -CH-NH- 3 DNA O O CH 2 OH 3 DNA = 5 TAG CTA TGG AAT TCC TCG TAG GCA 3

32. 32 Microcavity DNA Biosensor  50 µM of DNA is exposed to the porous silicon microcavity sensor  1 µM of cDNA binds to the DNA sensor for one hour  7 nm PL red-shift is observed after binding  No PL shifting is observed when two non- complementary strands of DNA are in contact Wavelength (nm) Normalized PL Intensity (a.u.) PSi / DNA PSi / DNA / cDNA Differential Signal S. Chan et al., Phys. Stat. Sol. (a) 182, 541 (2000). S. Chan et al., Mat. Sci. & Eng. C15, 277-282 (2001)

33. 33 PL Red-Shift (nm) 1 HOUR OF DNA HYBRIDIZATION 10  M 100 nM 1000 pM 10 pM 10 -5 10 -7 10 -9 10 -11 10 -13 Concentration of cDNA (moles/L) PL Red-Shift (nm) DNA-cDNA Recognition & Binding Time (min) Allot one hour of hybridization time for cDNA to seek out its DNA counterpart DNA: Sensitivity and Response Time S. Chan et al., Mat. Sci. Eng. C15, 277 (2001)

34. 34 Bacteriophage Lambda 100 nm  NUMBER OF BASE PAIRS  48,502 base pairs  TOTAL MOLECULAR WEIGHT  31.5 x 10 6 g/mol  SIZE DIMENSIONS  length:190 nm  width: 18 nm  GENOMIC MATERIAL  double-stranded linear DNA  KNOWN HOST  E. Coli

35. 35 Viral Microcavity Biosensor 12 nm PL red-shift is observed upon DNA recognition and binding No induced PL shift through subsequent heat treatments No detectable PL shift is observed when cDNA is not immobilized Wavelength (nm) Normalized Photoluminescence Intensity (a.u.) Immobilized cDNA Phage Lambda DNA S. Chan et al., Mat. Sci. & Eng. C15, 277 (2001).

36. 36 Gram Negative Bacteria Detection TETRATRYPTOPHAN (TWTCP) LIPID A silanized PSi + NH 2 TWTCP (R. D. Hubbard, S. R. Horner, B. L. Miller, JACS 123, 5810 (2001)) + lipid A TWTCP bound to PSi failure to capture lipid A TWTCP bound to PSi + lipid A success in capturing lipid A NH 2 TWTCP : glycine methyl ester mixture

37. 37 BACTERIUMCLASSPL RED-SHIFT E. coliGram-(-)4 nm Bacillus subtilisGram-(+)none detected L. AcidiophilusGram-(+)none detected SalmonellaGram-(-)3 nm Pseudomom. AeruginosaGram-(-)3 nm E. coli Bacillus subtilis Salmonella Pseudomonas Aeruginosa Gram-Negative Bacteria Detection Principle: detect Lipid A using TWTCP probe molecules S. Chan et al., J. Amer. Chem. Soc. 123, 11797 (2001)

38. 38 Biotin concentration (mg/ml) Red shift (nm) Biotin-Streptavidin 1. Thermal oxidation 2. Amino silane 3. Sulfo-NHS-LC-LC-Biotin 4. Streptavidin Wavelength (nm) Reflectance Sensitivity: 1~2  M concentration, which is equivalent to 300 pg/mm 2 in the porous internal surface (~ 20,000 mm 2 ). Simulation: ~ 10 - 30 pg/mm 2 H. Ouyang et al. Adv. Funct. Mater. 15, 1851-1859 (2005)

39. 39 Immunoglobulin G (IgG) Biotinylated Goat AntiRabbit IgG Rabbit IgG Goat IgG Rabbit IgG Goat IgG Red Shift (nm) Biotin + Streptavidin

40. 40 EHEC (pathogenic E-coli) Detection Tir Intimin Tir Intimin Y. Luo et al. Nature Vol 405, 1073 (2000) Intimin ~10x5x5 nm 3

41. 41 Purified Intimin Detection Tir No TirTir + Intimin Intimin Wavelength (nm) Reflectivity (%) 8 nm red shift Wavelength (nm) Reflectivity (%)

42. 42 E. coli Cells from Culture w/o Intimin JM 109 w/ Intimin EPEC Tir-Intimin No Tir-Intimin Tir-JM108 No Tir-JM109 Red shift (nm) No false positive H. Ouyang, L. DeLouise, B.L. Miller and P.M. Fauchet, Anal. Chem. 79, 1502-1506 (2007)

43. 43 Quantitative Analysis Dissociation constant K d = 10 -4 This indicates a much lower binding than for Tir-Intimin in solution H. Ouyang, L. DeLouise, B.L. Miller and P.M. Fauchet, Anal. Chem. 79, 1502-1506 (2007)