Surface-plasmon related ultra sensitive analytical methods and their

Surface-plasmon related ultra sensitive analytical methods and their bio- & nano- applications Fang Yu, Wolfgang Knoll Max-Planck Institute for Polymer Research, Mainz, Germany International Congress of Nanotechnology, Nov. 7 -10, 2004, San Francisco, USA.

Outline q Introduction q Surface plasmon fluorescence immunoassays q Bridge SPR technology and nano-world q Development of surface plasmon diffraction sensor q Summary

Biosensors additional time and costs for labeling unnatural binding nonlinear signal e. g. 1. Surface plasmon e. g. resonance (SPR) 1. Surface plasmon 2. Quartz crystal fluorescence microbalance spectroscopy Mass-labels? (QCM) Labeling Beacon tech? Label-free (SPFS) 3. Reflectometry 2. Total internal interference reflection spectroscopy fluorescence insufficient sensitivity (RIf. S) (TIRF) non-specific binding 4. Surface plasmon 3. . complex apparatus diffraction (SPD) 5. Microcantilever 6. .

Distinctive features of SPR 1. Short range phenomenon 2. Enhanced electromagnetic field 3. Propagating with high attenuation 4. p-polarized

Principle of Surface Plasmon Fluorescence Spectroscopy (SPFS) Prism Au Dipole-to-dipole coupling water prism 15 100 10 5 0 0 0 100 z /nm Fluorescence Yield % Field-enhancement Factor 20 metal dye dielectric Surface plasmon Back-coupling Free emission

The set-up photodiode Laser-shutter laser 632. 8 nm prism 2 goniometer chopper polarizers flow cell lens attenuator filter a) PMT b) FOS c) CCD camera PC shutter controller motorsteering photoncounter lock-in amplifier

Surface matrix for biosensing § § Lateral control of ligand density § Compatible with the physics of the biosensor § 2 D (SAM, lipid bilayer…) § Ideally… Good chemistry (for NSB, activation, regeneration…) 2. 5 D (Layer-by-layer, nano-particle, nanocapsule, nano-wire modified surface, porous or roughened surface…) § 3 D (brush type polymer, hydrogel network, plasma- or electro-polymerized matrix…) Dimension…

Interfacial design of sensing matrix Au : antibody : fluorophore water 2 D (e. g. layer-by-layer assembly) : streptavidin : SAM : dextran 3 D (e. g. dextran matrix)

Lb. L to clarify metal-induced quenching Alternating biotin-Ig. G and SA, decorate certain layer by Alexa fluor labeled SA IJ* : Ib* = 34

Surface preparation for dextran matrix (3) (2) (1) ~8 ng mm-2 (1) (2) (3)

Limit of detection (LOD) evaluation under mass-transport limited binding condition Baseline deviation

LOD at atto-molar level Correlation between SPR and fluorescence Translate the LOD level to molecular surface concentration ~10 molecules/(mm 2*min) Yu, F. , Persson, B. , Loefas, S. , Knoll, W. JACS, 126, 8902 -8903, 2004.

Prostate-specific antigen (PSA) sandwich assay slope km= 0. 98(D/h)2/3(v/bx)1/3 -2/3 D=k. T/6 a

LOD of PSA assay without plasma NSB Yu, F. , Persson, B. , Loefas, S. , Knoll, W. ANALYTICAL CHEMISTRY, in press.

Streptavidin-latex bead in SPR sensing SA doping ratio: 125 nm ~300 SA per bead Utilities: 1, Signal amplification 2, Introduce surface scattering 3, Being functional matrix itself (2. 5 D)

FOS (Fiber optic spectrometer) SA-Lx Biotin SAM (1: 9) Au Surface plasmon enhanced light-scattering 633 nm laser

Coverage dependent scattering (1) (2) (3) (4) (5)

One step SPR detection of 15 mer oligonucleotide by latex-amplification

DNA conjugated core/shell QDs Core/shell QDs supplied by QDot Corp. : high stability in PBS before and after conjugation 5’-biotinylated target DNA wavelength 565 nm (green), 585 nm (yellow), 605 nm (orange) and 655 nm (red) are all excitable with 543 nm (green laser)

Color Multiplexed hybridization detection test Excitation-Filter (543 nm) +QD 565 -T 2 (MM 0 for P 2) Microarray image from SPM P 1 P 2 P 1 P 3 P 1+ P 2 P 3 P 1 P 2 P 1 (MM 0 for P 1) P 2 P 1+ P 2 +QD 655 -T 1 P 2 Robelek, R. , Niu, L. , Schmid, E. L. , Knoll, W. ANALYTICAL CHEMISTRY, in press

Principle of SPDS -2 -1 0 1 2 Diffraction orders (m) Functional area Nonfunctional area Au Dielectric grating nd, the grating amplitude

TIR diffraction vs. SPR diffraction -2 -1 0 1 2 -2 glass -1 0 1 2 Au Polystyrene pattern TIR mode ATR/SPR mode

Diffraction patterns Surface plasmon microscopy images Diffraction photographs

Micro-contact printing for SAM patterning 4 Si Photoresist 1 PDMS 5 Si Photoresist pattern PDMS Au 2 6 PDMS Si Au 3 Functional SAM Nonfunctional SAM 7 PDMS Au

Quadratic property of the diffraction signal Biotin SAM Anti-biotin antibody Yu, F. , Tian, S. , Yao, D. , Knoll, W. ANALYTICAL CHEMISTRY, 76, 3530 -3535, 2004.

Self-referencing property of the diffraction sensor - a temperature variation test Yu, F. , Knoll, W. ANALYTICAL CHEMISTRY, 76, 1971 -1975, 2004.

h. CG SAM SA Fab SPDS for label-free detection of human chorionic gonadotropin (h. CG) (a) (b) (c) (d) 500 n. M Fab-biotin 50 n. M h. CG in 1 mg/m. L BSA

SPDS for oligonucleotide detection 1, surface preparation target DNA biotin SAM SA probe DNA functional nonfunctional

SPDS for oligonucleotide detection 2, kinetic analysis Name HE* koff (s-1) kon (M-1 s-1) KD (M) T 15 -MM 0 84% 1. 3 10 -4 6. 6 104 2 10 -9 T 15 -MM 1 62% 1. 1 10 -3 2. 4 104 4. 6 10 -8 T 15 -MM 2 ~0% N/A N/A HE: Hybridization efficiency Yu, F. , Yao, D. , Knoll, W. NUCLEIC ACIDS RESEARCH, 32, e 75, 2004.

SPDS for oligonucleotide detection 3, adsorption isotherm analysis KD 0 KD 1

Summary q Ultra-sensitive SPFS immunoassay is established with the aid of three-dimensionally extended matrix q Initial attempts of SPR based nano-sensing q SPDS is developed for label-free analysis of protein interactions and oligonucleotide hybridizations

Acknowledgements Neal Armstrong (University of Arizona) Akira Baba (University of Texas at Houston ) Shengjun Tian (MPIP) Lau King Hang Aaron (IMRE, Singapore) (for helps in the diffraction work) Björn Persson (Biacore) Stefan Löfås (Biacore) Renate Sekul (Graffinity) Holger Ottleben (Graffinity) (for collaborations) Danica Christensen (MPIP) (for the LBL work) Pierre Thiébaud (MPIP) Darick Ding (MPIP) (for the set-up engineering ) Danfeng Yao (MPIP) Thomas Neumann (Graffinity) Eva - Kathrin Sinner (MPI biochemistry) Peter E. Nielsen (Panum Institute, Denmark) Keiko Tawa (AIST Osaka) Rudi Robelek (IMRE, Singapore) Lifang Niu (IMRE, Singapore) (for the DNA/QDs part)