n i f F i i , , 1 , ) ( n i g G i i , , 1 , ) ( i i i G F D Surface plasmon resonance Surface plasmon resonance sensing sensing Dielectric Metal Evanesce nt Field Z θ i ≈ 50nm Prism Surface Plasmon θ i = θ r θ i > θ c θ r θ i = θ sp 2 1 2 1 1 1sin c c Conventional sensor system Conventional sensor system Surface plasmon Surface plasmon resonance sensors resonance sensors are important for are important for ultrasensitive ultrasensitive immunoassays immunoassays with applications with applications in health in health diagnostics diagnostics Principle of immunoassays: Principle of immunoassays: • Reactions between two protein Reactions between two protein molecules can be extremely specific. molecules can be extremely specific. • One type of molecule (antibody) can be One type of molecule (antibody) can be immobilised on gold sensor surface immobilised on gold sensor surface • The second (antigen) will bind The second (antigen) will bind changing the refractive index changing the refractive index • This change is detected by changed This change is detected by changed angle of surface plasmon resonance angle of surface plasmon resonance Surface plasmon waves extend few hundred nanometres above the metal Surface plasmon waves extend few hundred nanometres above the metal film. They are affected by the refractive index in this region. film. They are affected by the refractive index in this region.
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Surface plasmon resonance sensing Dielectric Metal Evanescen t Field Z θiθi ≈ 50nm Prism Surface Plasmon θ i = θ r θ i > θ c θrθr θ i = θ sp Conventional.
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Conventional sensor systemConventional sensor system
Surface plasmon Surface plasmon resonance sensors resonance sensors are important for are important for ultrasensitive ultrasensitive immunoassaysimmunoassayswith applications in with applications in health diagnosticshealth diagnostics
Principle of immunoassays:Principle of immunoassays:
• Reactions between two protein Reactions between two protein molecules can be extremely molecules can be extremely specific.specific.• One type of molecule (antibody) One type of molecule (antibody) can be immobilised on gold sensor can be immobilised on gold sensor surfacesurface• The second (antigen) will bind The second (antigen) will bind changing the refractive index changing the refractive index • This change is detected by This change is detected by changed angle of surface plasmon changed angle of surface plasmon resonanceresonance
Surface plasmon waves extend few hundred nanometres above Surface plasmon waves extend few hundred nanometres above the metal film. They are affected by the refractive index in this the metal film. They are affected by the refractive index in this region.region.
Simulation based on n-layer systemSimulation based on n-layer system
• Simulation based on Fresnel Simulation based on Fresnel reflectivity equations for an reflectivity equations for an n-layered systemn-layered system
• System consisted of a BK7 System consisted of a BK7 hemispherical prism, a 50nm hemispherical prism, a 50nm gold layer and an analyte gold layer and an analyte layerlayer
• Simulation produced R vs Simulation produced R vs θθ curves from system curves from system parameters (incident light parameters (incident light wavelength wavelength λλ and angle and angle θθ, , layer thicknesses d and layer thicknesses d and permittivities permittivities εε))
z
x
zn-1
zj
zj-1
z1
n (εn)
n-1 (εn-1)
j+1 (εj+1)
j (εj)
j-1 (εj-1)
2 (ε2)
1 (ε1)
Incident Reflected
To detector
Transmitted
dj = zj – zj-1
surface plasmon at interface, wavevector k||
p-polarised light
2
2,11,
2,11,2
,11
11
1
znn
znn
kidlnnn
kidlnnn
lnerr
errrR
Source: S. Orfanidis, “Electromagnetic waves and antennas” pp.81-108
R.U.S. Kurosawa et al, PRB 33,789 (1986)
Challenges:Challenges:
• LED must be stable, preferably at LED must be stable, preferably at a level 10a level 10-7-7
• Detector response must be linear, Detector response must be linear, with similar accuracywith similar accuracy• Readout must be very fastReadout must be very fast
Opportunities:Opportunities:
• Surface plasmons probe ultrathin Surface plasmons probe ultrathin regions (monolayer is enough)regions (monolayer is enough)• System can use inexpensive System can use inexpensive components, can run on Palmcomponents, can run on Palm• Sample can be extremely small Sample can be extremely small (microfluidics)(microfluidics)
Surface Plasmon Resonance Surface Plasmon Resonance Sensing SystemSensing System
produce difference curve produce difference curve DDii
From the average value of D From the average value of D and the variance S define a and the variance S define a standardised Z value standardised Z value
Central Limit Theorem states Central Limit Theorem states that Z has approximately that Z has approximately standard normal distributionstandard normal distribution
Hypothesis: that Hypothesis: that two curves two curves FFi i and and GGi i are the sameare the same
Choose to reject hypothesis Choose to reject hypothesis for significance level =0.05: for significance level =0.05: (Z>1.96)(Z>1.96)
Probability that this was Probability that this was wrong decision is less than wrong decision is less than 5%5%
Test parameters usedTest parameters used• Input wavelength 632.8nmInput wavelength 632.8nm• 3 layered system (BK7 glass 3 layered system (BK7 glass
prism, 50nm gold layer, prism, 50nm gold layer, analyte layer of water and analyte layer of water and isopropanol solutions)isopropanol solutions)
• Collection device - array Collection device - array detector from Ames detector from Ames Photonics Inc. Photonics Inc. – 384 simulation points 384 simulation points
(3mm laser diameter over (3mm laser diameter over 1024 pixels contained in 1024 pixels contained in 7.99mm)7.99mm)
– Noise standard deviation 3 Noise standard deviation 3 x 10x 10-6-6 (based on noise (based on noise specifics for detector, total specifics for detector, total integration time of 100s integration time of 100s with 1ms integration time)with 1ms integration time) Test applied to curve regions within
front edge of reflectance curve
Nanotechnology approach for the Nanotechnology approach for the optical sensing of trace pathogensoptical sensing of trace pathogens
• Aim:Aim:Develop a new Develop a new optical based sensor optical based sensor technology for rapid technology for rapid detection of trace detection of trace pathogens and pathogens and chemicals in the chemicals in the environment. environment.
• Novel approach:Novel approach: Apply surface nano-Apply surface nano-patterning patterning techniques to a techniques to a Surface Plasmon Surface Plasmon Resonance (SPR) Resonance (SPR) sensing system to sensing system to achieve achieve unprecedented unprecedented sensitivity levelssensitivity levels
Surface plasmons Surface plasmons are are electromagnetic electromagnetic waves excited by waves excited by light in metal light in metal films.films.
Surface Surface plasmons plasmons sense the sense the analyteanalyte
Nanostructures increase device sensitivity
Sensor readoutSensor readout
5 µm
2.5 µm
0 µm
5 µm
2.5 µm
0 µm
105.16 nm
52.58 nm
0 nm
Examples of nanostructures Examples of nanostructures
EXAMPLES OF NANOMODIFIED SENSOR SETUPS
Nanomodification of SPR sensors is achieved through the binding of noble metal colloids near the sensor surface or through direct nanostructuring of the sensing surface via lithographic or direct writing processes. Examples of two nanomodified SPR setups are presented below.
Figure 2: Two nano-modified SPR sensor configurations reported in the literature:
(a) 1. Prism for coupling to SP, 2. Thin metal layer(s), 3. Self Assembled Monolayer (typically 1,6-hexanedithiol or 2-mercaptoethylamine, 4. Attached metal colloids (typically Au or Ag, between 10 nm and 50 nm diameter) [11]
(a) 1. Prism for coupling to SP, 2. Thin metal layer(s), 3. Metal nanowires formed usually by nanolithography, 4. Self Assembled Monolayer either on top of structure or between nanowires and thin metal layers [12]
1.2.
3.4.
(a)
1.2.
3.4.
(b)
Rigorous coupled wave analysis for Rigorous coupled wave analysis for modelling nano-modified surface plasmon modelling nano-modified surface plasmon based sensing systemsbased sensing systems
OUTLINE OF MODEL:RCW approach was developed by Moharam and Gaylord [20] and is a full vectorial solution of Maxwell's equations.The approach is based on the following steps:
1. Representation of periodically varying permittivity (for example in a grating structure) using Fourier series expansion:
2. Simplification of Maxwell’s wave equations for incident light polarisation, through consideration of the orientation to the electromagnetic field with respect to the periodicity of the grating, using vector identities:
3. Equation * is simplified for p-polarised (transverse magnetic) light incidence as (from figure 1) H is perpendicular to so and Using the vector identity and producing:
4. The electric or magnetic field within the grating is written using a space-harmonic representation:
5. As the Fourier harmonics within the grating are a function of the grating perpendicular direction only, this allows the Maxwell equations within the grating to be written as a set of ordinary coupled differential equations with constant coefficients (in the case of a rectangular grating), allowing an eigenvalue approach to their solution.
6. From continuity considerations of the electromagnetic field at the boundary of the grating, the Fourier harmonics may be matched to the Rayleigh expansion of the fields beyond the grating region to determine the efficiency of each propagating order.Boundary conditions for p-polarisation are:
Reflectance results showing the effect of variation of nanowire period Λ. Insert: Diagram of setup under consideration.
Simulation results Simulation results
These simulations were These simulations were carried out using carried out using DiffractMODDiffractMOD, an RCWA , an RCWA based software package based software package from RSoftfrom RSoft
• Fluorescence emission is strongly directionalFluorescence emission is strongly directional• Applications for fluorescence bioassaysApplications for fluorescence bioassays
Excitation scheme Excitation scheme adapted for microscopyadapted for microscopy
Two photon Two photon SPCE SPCE demonstratdemonstrateded
0
100
200
300
400
640 680 720
WAVELENGTH, nm
SP
CE
INT
EN
SIT
Y a
t 66
5 n
m, a
.u.
BUFFER
SERUM
WHOLE BLOOD
Feasibility of bioassays in dense media
Schematic diagram of a model Schematic diagram of a model bioassaybioassay
Relevant publicationsRelevant publications
• ““Plastic Versus Glass Support for an Immunoassay on Metal-Coated Surfaces Plastic Versus Glass Support for an Immunoassay on Metal-Coated Surfaces in Optically Dense Samples Utilizing Directional Surface Plasmon-Coupled in Optically Dense Samples Utilizing Directional Surface Plasmon-Coupled Emission Evgenia G. Matveeva, Ignacy Gryczynski, Joanna Malicka, Zygmunt Emission Evgenia G. Matveeva, Ignacy Gryczynski, Joanna Malicka, Zygmunt Gryczynski, Ewa Goldys, Joseph Howe, Klaus W. Berndt, and Joseph R. Gryczynski, Ewa Goldys, Joseph Howe, Klaus W. Berndt, and Joseph R. Lakowicz, Lakowicz, Journal of Fluorescence vol.15, no.6 : 865-71, Nov. 2005Journal of Fluorescence vol.15, no.6 : 865-71, Nov. 2005
• “ “Ultrasensitive detection in optically dense physiological media: applications Ultrasensitive detection in optically dense physiological media: applications to fast reliable biological assays” . Matveeva, Evgenia G.; Gryczynski, Ignacy; to fast reliable biological assays” . Matveeva, Evgenia G.; Gryczynski, Ignacy; Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt. Berndt, Klaus W.; Lakowicz, Joseph R.; Goldys, Ewa; Gryczynski, Zygmunt. Proceedings of SPIE-The International Society for Optical Engineering (2006), Proceedings of SPIE-The International Society for Optical Engineering (2006), 6092 125-133.6092 125-133.
• “ “Detection limit improvement of surface plasmon resonance based Detection limit improvement of surface plasmon resonance based biosensors using statistical hypothesis testing”, Barnett, Anne; Goldys, Ewa biosensors using statistical hypothesis testing”, Barnett, Anne; Goldys, Ewa M.; Dybek, Konrad, Proceedings of SPIE-The International Society for Optical M.; Dybek, Konrad, Proceedings of SPIE-The International Society for Optical Engineering (2005), 5703(Plasmonics in Biology and Medicine II), 71-78. Engineering (2005), 5703(Plasmonics in Biology and Medicine II), 71-78. “Strategies for noise reduction and sensitivity increase for a Surface Plasmon “Strategies for noise reduction and sensitivity increase for a Surface Plasmon Resonance (SPR) based biosensing system”, A. Barnett, E.M. Goldys, K. Resonance (SPR) based biosensing system”, A. Barnett, E.M. Goldys, K. Dybek. OWLS Conference, Optics Within Life Sciences” Melbourne 28 Nov Dybek. OWLS Conference, Optics Within Life Sciences” Melbourne 28 Nov 2004 – 1 Dec 20042004 – 1 Dec 2004