Lecture #5: Surface Plasmon Resonance (SPR) Biosensing Objectives

Transcription

1 Laboratory of Biosensors and Bioelectronics Lecture #5: Surface Plasmon Resonance (SPR) Biosensing Objectives Understand the basic concepts of surface plasmon polariton Discuss the principles of operation of surface plasmon resonance (SPR) sensors Understand the principles of kinetic analysis by SPR Discuss the operation of SPR imaging View a few applications

2 What is Surface Plasmon resonance (SPR)? Spectroscopic method for the measurement of changes in refractive index near the metal layer surface Principle: Light is reflected from a thin metal layer Causing a collective vibration of the electron gas within the metal -> plasmon 1968 Kretschmann and Otto: optical excitation of surface plasmons by the method of attenuated total reflection

3 Gold is the most practical metal: it produces a strong, easy to measure SPR signal in the near infrared region very resistant to oxidation and other atmospheric contaminants compatible with a lot of chemical modification systems the thickness of the gold should be 50 nm.

4

5

6

7 Theory of Surface Plasmon Resonance x d 50 nm s mr p Surface plasmon wave (K sp ) Evanescent wave (K ev ) z x Light (ω) 800 nm 2D-detector array Condition of c mr s mr s Resonance : K = sp K ev sin * p c Reflectivity angle sin sin * mr p s 2 mr p 2 * ns s

8 where is the electric permittivity of free space. ε r is relative permittivity, also called dielectric constant In electromagnetism, the electric displacement field D represents how an electric field E influences the organization of electrical charges in a given medium, including charge migration and electric dipole reorientation.

9 What is a surface plasmon polariton? Transverse EM wave coupled to a plasmon (wave of charges on a metal/dielectric interface) = SPP (surface plasmon polariton) Note: the wave has to have the component of E transverse to the surface (be TM-polarized, or p- polarized).

10

11

12 Surface plasmon dispersion relation: p ck x d k Radiative modes x m d c m d ' m > 0) 1/ 2 real k x real k z p 1 d Quasi-bound modes d < ' m < 0) imaginary k x real k z z x Dielectric: d Metal: m = m ' + m " Bound modes (' m < d ) real k x imaginary k z Re k x

13 Dispersion Relation k s k ev k s sin k sp k 0 K sp k ev Dispersion of evanescent wave vector (k ev ) and surface plasmon wave vector (k sp )

14 3 Total reflection on a prism prism reflectance 1 0 c angle prism

15 4 Evanescent Wave prism evanescent field reflectance 1 0 c angle evanescent wave: - nearfield standing wave, - extends about 1/2, - decays exponentially with the distance

16 5 Surface Plasmon Resonance detector reflectance 1 50 nm Au 0 c o angle (Kretschmann)

17 6 Surface Plasmon Resonance Spectroscopy detector reflectance 1 Au analyte 0 c o 1 angle To measure: - thickness changes, - density fluctuation, - molecular adsorption

18 8 Coupling of Light to Surface Plasmon Prism coupler (Kretschmann) Waveguide coupler Grating coupler Homola, Chem. Rev. 2008, 108, 462

19 Plasmon resonance frequency strongly depends on geometry

20 Note: we cannot excite SPP by simply illuminating the surface! i k s k ev k s sin k i k SPP k k sp 0 K sp k ev k Excitation condition i sin i k SPP Impossible to satisfy! k i is always less than k SPP Calculated dispersion of surface plasmon-polaritons propagating at a Ag/air, Ag/glass, and Ag/Si interface, respectively. Maier & Atwater, JAP 2005

21

22 Exciting SPP (or any mode of your choice) by scattering light off grating d k i i k SPP This is effectively a (quasi-)momentum conservation Grating changes longitudinal wave vector of a photon by K g m Coupling to SPP is achieved when 2 ; m 1,2,... d k i sin K i g k SPP

23 Biacore SPR instrument SPR (surface plasmon resonance) technology enables real-time detection and monitoring of biomolecular events and provides quantitative information on: 1. Specificity how specific is the binding between two moelcules? 2. Concentration how much of a given molecule is present and active? 3. Kinetics what is the rate of association/dissociation? 4. Affinity how strong is the binding? 5. Binding partners - provide identification of binding targets by linking SPR to MS

24 Biacore instrument

25 Biacore Sensor Chips Sensor Chip CM5 for applications from basic research to quality control Sensor Chip SA for capture of biotinylated peptides, proteins and DNA Sensor Chip NTA for capture of ligands via metal chelation Sensor Chip HPA for membrane biochemistry and the study of membrane associated receptors in a near native environment Pioneer Chips as tools for users novel applications in terms of chemistry or binding specificity.

26

27

28 Evaluation of SPR detection sensitivity Angular Sensitivity: SPR detection using the Kretchmann configuration measures the angle of incident light at which the surface plasmon resonance takes place. The shift of the resonance angle provides a sensitive measurement of a molecular binding event onto the sensor surface or a change in the index refraction of the fluid medium near the sensor surface. For this reason, the minimum detectable angular shift, in unit of degree, may be used to describe the sensitivity.

29 Evaluation of SPR detection sensitivity Surface coverage: If one is interested in using SPR to detect molecular binding taking place on a sensor surface, then the surface coverage in terms of mass, e.g., pg/mm², is an appropriate way to define sensitivity. The unit, RU (termed Resonance Unit or Response Unit) is defined as 1 RU = 1 pg/mm², and is also often used to determine surface coverage.

30 Simulation based on n-layer system p-polarised light To detector z z n-1 z j z j-1 n (ε n ) n-1 (ε n-1 ) j+1 (ε j+1 ) j (ε j ) j-1 (ε j-1 ) Incident d j = z j z j-1 Reflected surface plasmon at interface, wavevector k Simulation based on Fresnel reflectivity equations for an n- layered system System consisted of a BK7 hemispherical prism, a 50nm gold layer and an analyte layer Simulation produced R vs θ curves from system parameters (incident light wavelength λ and angle θ, layer thicknesses d and permittivities ε) 2 (ε 2 ) z 1 x 1 (ε 1 ) Transmitted R 2idn1kzn1 2 rn, n1 rn 1, le n, l 2idn1kzn1 1 rn, n1rn 1, le r 2

31 Evaluation of SPR detection sensitivity %R conventional gold calcinated, d=2 nm calcinated, d=10 nm n Theoretical change in percent reflectivity vs. change in refractive index obtained with WINSPALL. Bare gold has a sensitivity of 4352% RI/RIU. This is known as bulk sensitivity

32 Evaluation of SPR surface sensitivity

33 Biological System: CT/GM1 Quantitative CT binding Regenerated surface Large dynamic range

34 Objectives of SPR Experiments Kinetic Rate Analysis: How FAST? k a, k d K D = k d /k a, K A = k a /k d Concentration Analysis: How MUCH? Active Concentration Solution Equilibrium Inhibition Affinity Analysis: HOW STRONG? K D, K A Relative Ranking Yes/No Data Ligand Fishing

35

36

37

38

39

40

41

42

43

44

45

46

47

48 Imaging SPR Measurement of the reflectivity at a fixed angle Rothenhausler, B.; Knoll, W. Nature, 332:615 17, 1988; Frutos, A. G., and Corn, R. M. Anal. Chem. 70, A449-A455, 1998.

49 Instrument Setup laser He-Ne Laser Incoherent Light Source LED Beam expander Band-pass Filter Prism CCD camera Turn table Flow Cell Spatial Filter Polarizer Outlet Inlet Schematic of the home-built imaging SPR spectrometer Wilkop, T.; Wang, Z.; Cheng, Q., Langmuir, 2004, 20,

50 Incoherent light source: LED Intensity (AU) Measured Simulated Fullwidth Reflectivity (100%) Wavelength (nm) 0.0 Monochromic 24 nm fullwidth Angle of incidence ( o ) Comparison of a theoretical SPR reflectivity spectrum of a bare Au substrate in air for a monochromatic source and for the LED used in the experiment. Wilkop, T.; Wang, Z.; Cheng, Q., Langmuir, 2004, 20,

51 Differential reflectivity for a 10-nm film Monochromatic source 648 nm Led with fullwith of 24 nm Reflectivity Change (100%) Angle of incidence ( o ) Simulated differential reflectivity after deposition of a 10 nm organic thin film for a monochromatic and LED light source.

52 Filter or not to filter Unfiltered LED 1.0 Transmitted light intensity (AU) nm bandpass filter 10 nm bandpass filter 5 nm bandpass filter 1 nm bandpass filter Total spectral energy Fullwidth of lightsource (nm) Wavelength (nm) Influence of a band-pass filter on the light intensity spectrum of a LED source Wilkop, T.; Wang, Z.; Cheng, Q., Langmuir, 2004, 20,

53 Photopatterned C 18 SH monolayer on gold Reflectivity (AU) Pixel 200 mesh grid SPR image of photopatterned C 18 SH monolayer on gold Bar Width: 35 µm Hole Width: 90 µm The lateral resolution is estimated to be 15 μm

54 Thiol derivatized PEG pattern on gold SPR image of a PEG-thiol (MW 5000) pattern after immersion in 0.1 mg/ml BSA Patterned PEG Adsorbed BSA on patterned PEG (~7 nm)

55 Printed SAMs for supported membranes Preparation of SLMs: inking the PDMS stamp with 10 mm 1- octadecanethiol (ODT) dry in air for 30 seconds gently applied to the gold substrate for 1 min to create a hydrophilic surface in the wells, the chip was then treated with 10 mm 2- mercaptoethanol solution for 60 minutes SPR image of a 100x100 μm ODT array: 2D (above) and 3-D (below) The vesicles were prepared with DMPC and cholesterol (molar ratio 2:1) in 10 mm Tris buffer (ph 7.5 containing 0.15 M NaCl) DMPC Formation of SLM

56 SPR Spectra from images of SAMs Normal and inverted images: position of imaging angle Bare Au C 18 SH monolayer Angular scan: Angular scan allows for determination of SPR angle Highly useful for quantification An angular scan for the printed pattern

57 Real time monitoring of vesicle fusion in wells t = 0 s t = 6 s t = 12 s t = 18 s Fusion occurs on both hydrophilic and hydrophobic surfaces Fusion completed in about 18 seconds injection Resulted membrane are stable against rinsing What do we have in the wells? Quantification is necessary with reflectivity intensity and angular shift Vesicle: DMPC with 20% CHO

58 Angular scan in imaging SPR for quantification Reflectivity Intensity (AU) C18SH matrix layer before vesicle incubation Open well (Au+ME) before vesicle incubation Open well after vesicle incubation C18SH matrix layer after vesicle incubation 1.7 nm Angle of incidence / o SAM monolayer thickness used as internal standard for thickness quantification The thickness of the membrane in the pocket is nm Single planar bilayer in "hydrophilic pockets" unlikely Wang, Z. et al Langmuir, 2005, 21,

59 Fluorescence microscopy for SLMs on Au A) B) a) R e la tive in ten sity Figure A shows the fluorescence image of the micropatterned membrane after vesicle fusion. The micropattern membrane was fabricated the same way as before. The DMPC vesicles contained cholesterol and 1% NBD-PC Pixel Fluorescence measurements suggest relatively flat structure in the hydrophilic pocket b) Figure B shows the fluorescence image for the intact vesicles. The DMPC vesicles contained cholesterol, 1% NBD-PC, and 1% ODT Suggested model

60 Thank You!