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

2

Toronto

New York

Where is Rochester?

California

Italy

> 3000 km >6000 km

3

Rochester: the campus and the city

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

The state of the art…yesterday and today

1860 2002

6

The state of the art…tomorrow

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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

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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.

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Porous Silicon Materials Science

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

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~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)

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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)

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10 µm

Pore Size and Morphology Engineering

From nanopores to mesopores to macropores and from “spongy” to directional and smooth

100 nm

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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

15

Biosensing Principles

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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)

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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

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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

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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

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

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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

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C-Silicon Subtrate

j

t

[mA

/cm

2 ]

[s]

Electrolyte

Multilayer Structures

H. Ouyang et al, Adv. Funct. Mater.15, 1851 (2005)

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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

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CONTROL OVER THREE LENGTH SCALES

PSi Multilayer Mirror

PSi Multilayer Mirror

PSi Central Layer

Porous Silicon MicrocavityPorous Silicon Microcavity

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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

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)

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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)

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

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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

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Examples of Biosensing

DNA Proteins Bacteria

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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

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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)

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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)

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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

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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).

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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

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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)

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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)

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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

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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

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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

(%

)

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)

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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)