Page 1
1
Lecture 1: Biodetection using Silicon Photonic Bandgap Devices
Philippe M. Fauchet
University of RochesterSupported in part by the National Science
Foundation, the Infotonics Center of Excellence, and the Center for Future Health
Biophotonics Winter School 2007
Page 2
2
Toronto
New York
Where is Rochester?
California
Italy
> 3000 km >6000 km
Page 3
3
Rochester: the campus and the city
Page 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
Page 5
5
The state of the art…yesterday and today
1860 2002
Page 6
6
The state of the art…tomorrow
Page 7
7
Bio Meets Nano
In nanometer
10-1 1 10 102 103 104 105 106 107 108
nm µm cm
Water Glucose Antibody Virus BacteriaCancer
Cell Fruit Fly Tennis Ball
Chip
Page 8
8
ObjectivesBiosensor 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.
Page 9
9
Porous Silicon Materials Science
Page 10
10
+ ++
+
F-
+ ++ +
++
++
F-
F-F-
F-
F-
F-
F-F-
F-
F-F-
F-
F-
F-
F-
F-F- Hydrofluoric Acid
Crystalline Silicon
++ ++++
++
F-F-
++ ++++ ++
++++
++++
F-F-
F-F-F-F-
F-F-
F-F-
F-F-
F-F-F-F-
F-F-
F-F-F-F-
F-F-
F-F-
F-F-
F-F-
F-F-F-F- Hydrofluoric Acid
Crystalline Silicon
+ ++
+
F-
+
+
+++
+ + ++
++
++
F-
F-
F-F-
F-
F-F-F-
F- Hydrofluoric Acid
Crystalline Silicon
F-
+
++
+++ ++
+
F-
+
+
+++
+ + ++
++
++
F-
F-
F-F-
F-
F-F-F-
F- Hydrofluoric Acid
Crystalline Silicon
F-
+
++
++
a.) b.)
Porous Silicon: Etching Mechanism
Porous Silicon Formation ElectropolishingPorous Silicon Formation Electropolishing
Page 11
11
~150 nm diameter
~200 nm pore-to-pore spacing
5 m
200 nm
200 nm
Intermediate Pore Size (150 nm)
H. Ouyang et al., SPIE 5511, 71 (2004)
Page 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)
Page 13
13
10 µm
Pore Size and Morphology Engineering
From nanopores to mesopores to macropores and from “spongy” to directional and smooth
100 nm
Page 14
14
n-void=1n-void=1.3
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1
neff
0 20 40 60 80 100
Porosity (%)
n-void=1n-void=1.3n-void=1n-void=1.3
1
1.5
2
2.5
3
3.5
4
0 0.2 0.4 0.6 0.8 1
neff
0 20 40 60 80 1000 20 40 60 80 100
Porosity (%)
Bruggeman approximation to simulate refractive index of porous silicon
n2 =
Porosity (%)
Eff
ectiv
e in
dex
Index of Refraction Tunability
Refractive index is a function of porosity, refractive index of silicon, refractive index inside the pores
Page 15
15
Biosensing Principles
Page 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)
Page 17
17
Exposed to species
Sensing Principle
Wavelength (m)
Ref
lect
ivity
0
0.2
0.4
0.6
0.8
1
0.6 0.7 0.8 0.9 1
Internal surface modification
Specific bindingRed shift
n n +n
Page 18
18
0
0.2
0.4
0.6
0.8
0.6 0.7 0.8 0.9 1
Ref
lect
ivity
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
Page 19
19
White light reflection from a porous Si film
Sailor’s group, UCSD: C.L. Curtis et al., Electrochem. Soc. 140, 3492 (1993)
800
600
400
200
Rel
ativ
e In
tens
ity
1000900800700600500400
Wavelength (nm)
a
porous Si
silicon wafer
light source
to spectrometer
L
Page 20
20
0
10
20
30
40
50
60
70
80
0 200 400 600 800
Time (min)
A B C D E F G H I J K LM
Streptavidin b-Prot. A IgG RinseIgG Rinse
Experimental Conditions [EOT shift]: A: PBS buffer pH = 7.4: B: 1mg/mL streptavidin [17 nm]: C: PBS rinse: D: 2.5 mg/mL biotinylatedProtein A [14 nm]: E: PBS rinse: F and J: 2.5 mg/mL Human IgG [34nm]: G and K: PBS rinse: H and L, 0.1 M acetic acid: I and M: PBSrinse. All data acquired under peristaltic flow of 0.5 mL/min in a flowcell.
Protein A/Human IgG Binding to Porous Si
Sailor’s group, UCSD
Page 21
21
0
0.2
0.4
0.6
0.8
1
0.6 0.7 0.8 0.9 1
Ref
lect
ivity
Wavelength (m)
0
0.2
0.4
0.6
0.8
1
0.6 0.7 0.8 0.9 1
Ref
lect
ivity
Wavelength (m)
0
0.2
0.4
0.6
0.8
0.6 0.7 0.8 0.9 1
Ref
lect
ivity
Wavelength (m)
More sophisticated structures
n n +n
Single layer Rugate filter Microcavity
Page 22
22
C-Silicon Subtrate
j
t
[mA
/cm
2 ]
[s]
Electrolyte
Multilayer Structures
H. Ouyang et al, Adv. Funct. Mater.15, 1851 (2005)
Page 23
23
Bragg mirror
Bragg mirror
Defect layer75% porosity layer (n = 1.44):
50mA/cm2 for 8 sec
2µm
70% porosity layers (n = 1.57):
35mA/cm2 for 11sec
50% porosity layers (n = 2.16):
5mA/cm2 for 32sec
Porous Silicon Microcavity
Page 24
24
CONTROL OVER THREE LENGTH SCALES
PSi Multilayer Mirror
PSi Multilayer Mirror
PSi Central Layer
Porous Silicon MicrocavityPorous Silicon Microcavity
Page 25
25
Ref
lect
ivit
y (%
)
Wavelength (nm)
0
20
40
60
80
100
1000 1200 1400 1600
Max R ~ 100%
Min R ~ 10%
FWHM ~ 15nm
Only 5 period Bragg mirrors
Reflectivity of a PSi Microcavity
Page 26
260
20
40
60
80
100
600 800 1000 1200 1400 1600 1800 2000
Reflect
ivit
y (
%)
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)
Page 27
27
Reflectivity SpectraReflectivity 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
600 650 700 750 800 850 900 950
Ref
lect
ivit
y (%
)
Wavelength (nm)
234 nm
1170 nm
2340 nm
3520 nm
S. Chan et al., Mat. Sci. & Eng. C15, 277-282 (2001)
Page 28
28650 675 700 725 750 775 800
Sensitivity on Refractive Index Sensitivity on Refractive Index
Wavelength (nm)
Ref
lect
ivit
y (%
)
DIGITAL SENSORDIGITAL SENSOR
on-state“1”
off-state“0”
npore = 1.00
npore = 1.03
Page 29
29
Porous silicon can emit lightPorous silicon can emit light
PSi Bragg Reflector
PSi Bragg Reflector
PSi Active Layer
0
2000
4000
6000
8000
10000
12000
14000
16000
650 700 750 800 850 900 950
Wavelength (nm)
Pho
tolu
min
esce
nce
Inte
nsit
y (a
.u.)
FWHM ~ 4 nm
Page 30
30
Examples of Biosensing
DNA Proteins Bacteria
Page 31
31
DNA Biosensor - Details
Si
Si
Si
O-Si-CH2-CH2-CH2-O-CH2-CH-CH2
O
O O
++ N-3DNA
H
H
Silanized Porous Silicon DNA Strand
..
Si
Si
Si
O-Si-CH2-CH2-CH2-O-CH2-CH-NH-3DNA
O
O
CH2OH
3DNA = 5TAG CTA TGG AAT TCC TCG TAG GCA3
Page 32
32
Microcavity DNA BiosensorMicrocavity 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
600 650 700 750 800 850 900Wavelength (nm)
Nor
mal
ized
PL
Int
ensi
ty
(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)
Page 33
33
0
1
2
3
4
5
6
0 30 60 90 120 150 180
PL
Red
-Shi
ft (
nm)
0
1
2
3
4
5
6
7
8
1.0E-13 1.0E-11 1.0E-09 1.0E-07 1.0E-05
1 HOUR OF DNAHYBRIDIZATION
10 M
100 nM
1000 pM
10 pM
10-510-710-910-1110-13
Concentration of cDNA (moles/L)
PL
Red
-Shi
ft (
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)
Page 34
34
Bacteriophage LambdaBacteriophage 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
Page 35
35
Viral Microcavity BiosensorViral 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
650 700 750 800 850 900Wavelength (nm)
Nor
mal
ized
Pho
tolu
min
esce
nce
Inte
nsit
y (a
.u.) Immobilized cDNA
Phage Lambda DNA
S. Chan et al., Mat. Sci. & Eng. C15, 277 (2001).
Page 36
36
Gram Negative Bacteria DetectionGram Negative Bacteria DetectionGram Negative Bacteria DetectionGram Negative Bacteria Detection
TETRATRYPTOPHAN (TWTCP) LIPID A
silanized PSi
+ NH2 NH2
NH2NH2
NH2 NH2
NH2NH2
NH2 NH2
NH2NH2
TWTCP
(R. D. Hubbard, S. R. Horner, B. L. Miller, JACS 123, 5810 (2001))
+
lipid A
TWTCP bound to PSi failure to capture lipid ATWTCP bound to PSi
+
lipid A
success in capturing lipid A
NH2
NH2NH2
NH2
NH2
NH2
NH2
NH2
TWTCP : glycine methyl ester mixture
Page 37
37
BACTERIUM CLASS PL RED-SHIFT
E. coli Gram-(-) 4 nmBacillus subtilis Gram-(+) none detectedL. Acidiophilus Gram-(+) none detectedSalmonella Gram-(-) 3 nmPseudomom. Aeruginosa Gram-(-) 3 nm
E. coli Bacillus subtilisSalmonellaPseudomonas Aeruginosa
Gram-Negative Bacteria Detection
Principle: detect Lipid A using TWTCP probe molecules
S. Chan et al., J. Amer. Chem. Soc. 123, 11797 (2001)
Page 38
38
0
2
4
6
8
10
12
14
0 0.4 0.8 1.2 1.6 2
Biotin concentration (mg/ml)R
ed s
hift
(nm
)
Biotin-Streptavidin
1. Thermal oxidation 2. Amino silane 3. Sulfo-NHS-LC-LC-Biotin
4. Streptavidin
0
20
40
60
80
100
600 650 700 750 800 850 900
Wavelength (nm)
R
efle
ctan
ce
Sensitivity: 1~2 M concentration, which is equivalent to 300 pg/mm2 in the porous internal surface (~ 20,000 mm2).
Simulation: ~ 10 - 30 pg/mm2
H. Ouyang et al. Adv. Funct. Mater. 15, 1851-1859 (2005)
Page 39
39
Immunoglobulin G (IgG)
Biotinylated Goat AntiRabbit IgG
Rabbit IgG
Goat IgG
0
1
2
3
4
5
6
7
8
1 2Rabbit IgG
Goat IgG
Red
Shi
ft (
nm)
Biotin + Streptavidin
Page 40
40
EHEC (pathogenic E-coli) DetectionEHEC (pathogenic E-coli) Detection
Tir
Intimin
Tir
Intimin
Y. Luo et al. Nature Vol 405, 1073 (2000)
Intimin ~10x5x5 nm3
Page 41
41
0
10
20
30
40
50
60
70
80
90
100
550 600 650 700 750 800 850 900 950
0
10
20
30
40
50
60
70
80
90
100
550 600 650 700 750 800 850 900 950
0
10
20
30
40
50
60
70
80
90
100
550 600 650 700 750 800 850 900 950
0
10
20
30
40
50
60
70
80
90
100
550 600 650 700 750 800 850 900 950
Purified Intimin Detection
Tir No TirTir + Intimin Intimin
Wavelength (nm) Wavelength (nm)
Ref
lect
ivity
(%
)
Ref
lect
ivity
(%
)
0
10
20
30
40
50
60
70
700 720 740 760 780 800
8 nm red shift
Wavelength (nm)
Ref
lect
ivity
(%
)
Page 42
42
E. coli Cells from CultureE. coli Cells from Culture
w/o Intimin
JM 109
w/ Intimin
EPEC
0
1
2
3
4
5
6
1 2 3 4Tir-Intimin No Tir-Intimin Tir-JM108 No Tir-JM109
Red
sh
ift (
nm)
No false positive
H. Ouyang, L. DeLouise, B.L. Miller and P.M. Fauchet, Anal. Chem. 79, 1502-1506 (2007)
Page 43
43
Quantitative Analysis
0
2
4
6
8
10
12
14
0 2 4 6 8 10 12Tir red shift (nm)
Inti
min
re
d s
hif
t (n
m)
0
0.2
0.4
0.6
0.80.00 0.10 0.20 0.30 0.40 0.50
Tir Concentration (mM)
Bo
un
d In
tim
in (
nm
ol)
Inimin (60 uM)
Intimin (30 uM)
Intimin (15 uM)
Intimin (5uM)
Dissociation constant Kd = 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)