Nanoscale Engineered Plasmonic Nanostructures for

Biosensing and Bioimaging

Dr Fang Xie Department of MaterialsImperial College London

7th Asia Pacific Biotech Congress13th July 2015, Beijing, China

Imperial College London

Department of Materials

Overview

Plasmonic Materials

Engineered Material Fabrication

Material Design

Theoretical Simulation

Healthcare Technologies

Solar Energy Harvesting

Manipulating Light

Large scale Long range homogeneity Tunable optical properties Reproducibility

Background: What is fluorescence?

Jablonski diagram showing fluorescent absorption and emission processes.

Image courtesy of Photomatrics

Jabłoński, Aleksander "Efficiency of Anti-Stokes Fluorescence in Dyes" Nature 1933, volume 131, pp. 839-840.

The father of fluorescence spectroscopy

Background: What is plasmonic materials?

The optical properties of Nanoparticles—localized surface plasmon resonances (LSPRs):

Oscillations of the conduction electrons coupled to the E-field

The frequency and intensity of the oscillations are sensitive to the geometry and surrounding media

The artwork was crafted from glass stained with colloidal noble metal particles in the 4th Century; their strong interaction with visible light due to the excitation of LSPRs, gives rise to the vibrant green and red colours.

Adv. Mater. 2007, 19, 3771–3782

Lycurgus Cup

Reflection Transmission

Background: How we use them for sensing/Imaging?

Enhanced Fluorescence on metal nanostructured surface

Fluorescence on glass surface

Early Diagnosis by Metal Induced Fluorescence Enhancement

Light interacting with a metal nanoparticle: the possible spectroscopic responses for sensitive biomolecular detection

Metal Induced Fluorescence Enhancement: Fluorophores near metal nanostructures experience:

Greater quantum yields Reduced lifetimes

(a) Excitation Enhancement

Fluorophore in Free Space Condition

Fluorophore near Metallic particles

E: excitation; Em: metal enhanced excitation rate; m:radiative rate in the presence of metal; Knr: non-radiative rate.

(b) Emission Enhancement

Background: Mechanism of MIFE

Lakowicz et al. Analytical Biochemistry 301, 261–277 (2002)

Effect of metallic particle on fluorescence signal as a function of distance from particle

The effect of distance between metal surfaceand the fluorophore on MIFE:

(a) 0 – 5 nm, quenching;

(b) 5-20 nm, enhancement;

(c) > 20 nm, free space fluorescence.

Background: Mechanism of MIFE

Nanostructures/Nanoparticles Fabrication/Synthesis

Nanostructures by Nanosphere Lithography

SubstratePolystyrene Monolayer Metal

Deposition

Polystyrene Removed

Physicochem. Eng. Aspects 219 (2003) 1 /6

Nanostructures by Nanosphere Lithography

PS Deposition and Shrink

Metal Deposition

PS Removal

Metal Deposition

PS Deposition and Shrink

Argon Ion Milling PS

Removal

NanopillarsNanopillars

NanoholesNanoholes

Nanostructures by Nanosphere Lithography

Nanostructures by Synthesis

Plasmonic Materials for Fluorescence Enhancement Investigation

1) Au-core Ag-shell Nanoparticles:

Method: Produce gold colloid and use silver enhancing step:2AgNO3 + C6H4(OH)2 CO(CHCH)2CO + 2HNO3 + 2Ag

47 nm Au Core Ag-shell NP: Fluorescence enhancement of 10

MIFE Substrates – bottom up methods

A CB

Au Colloid Surface 10 nm

Glass SurfaceGlass Surface

Glass Surface

Au Core Ag-shell NP Surface

19 nm

F. Xie, M. Baker, E. Goldys, J. Phys. Chem. B 2006, 110, 23085-23091

Au-Core Ag-shell NP Surface 47 nm

MIFE Substrates – bottom up methods

2) Au Nanoparticles: Au colloids of 40, 59, and 81 nm in Radius with 24 h incubation to form self-assembled layers on glass substrates.

SEM images (24 hours) of (A) 80 nm Au colloid; (B) 118 nm Au colloid; (C) 162 nm Au colloid.

540 560 580 600 620 640 660 680 7000

500000

1000000

1500000

2000000

2500000

Fluo

resc

ence

Inte

nsity

(a.u

.)

Wavelength (nm)

Glass surface 80 nm - 24 hours 118 nm - 24 hours 162 nm -24 hours

F. Xie, M. Baker, E. Goldys, Chem. Mater 2008, 20, 1788-1797

Gla

ss

su

rfa

ce

Metallic surface

Average Lifetime (ps)

Frequen

cy (a.u.)

A colour coded lifetime image (FLIM) for the sample 161 nm Au – 24h

Distribution of the average lifetimes at the metallic surface and glass surface. Lifetime reduced on metal.

Two orders of fluorescence enhancement were observed for NIR dye by nano-engineering of Ag triangular arrays.

F, Xie et al., Nano Res., DOI 10.1007/s12274‐013‐0327‐5

MIFE Substrates – top down method

Fluorescence spectra of Alexa Fluor 790 monolayer on sample PS300-15s (blue), PS500-15s (red), and PS620-15s (green) as well as on glass as control (black)

Sample

Fluorescence

Enhancement Ef

Sample

Fluorescence

Enhancement Ef

PS300-0s /

AF488-SA7.8

PS300-15s /

AF790-SA5.5

PS500-0s /

AF680-SA5.7

PS500-15s/

AF790-SA83.0

PS620-0s /

AF750-SA10.0

PS620-15s/

AF790-SA33.8

Plots of e-field enhancement around the NPs at 780 nm, for (a) PS300 (b) PS500 (c) PS620 with 15s etching.

MIFE Substrates – top down method

1 2/ /1 2( ) t t t tI t a e a e

00

0 nr

Qk

00

1

nrk

m 0m

m 0 m,abs nr

Qk

m

m 0 m,abs nr

1

k

Samplem

(ps)/m (m +)/ Qm Eem =Qm/Q0

PS300-15s 238 2.14 2.4 21.0% 2.1

PS500 -15s 113 4.51 26.1 72.3% 7.2

PS620-15s 177 2.88 9.8 52% 5.2

Lifetime measurements for each sample and the calculated values of lifetime, radiative rate, and quantum yield ratios on metallic surfaces versus clean glass surface. (Q0 =10%)

Sample Ef Eem Eex

PS300-15s 5.5 2.1 2.6

PS500 -15s 83.0 7.2 11.5

PS620-15s 33.8 5.2 6.5

Values of the Excitation Enhancement and Emission Enhancement for Each Sample

MIFE Substrates – top down method

Plasmonic Materials for Protein Microarray

Plasmonic gold-on-gold nano-island films enhance the fluorescence of near-infrared fluorophores. Enhancement factor for IR800: 16

Dai et al, DoI: 10.1038/ncomms1477

MIFE biosensing Application – Protein Microarray

MIFE biosensing Application – Protein Microarray

Near-infrared fluorescence enhanced protein microarrays on gold substrates probed by IR800.

Model colon cancer biomarker: carcinoembryonic antigen (CEA), CEA sandwich bioassay probed by IR800. (excitation at 785 nm), CEA spiked into whole, undiluted serum

Sensitive enhancement: ~5000 fold;

Dynamic range: 6 orders of magnitude

MIFE biosensing Application – Protein Microarray

Multiplexed Protein Microarray

Multiplex capacity of using Plasmonic Chips: 32 human antigens and controls’ assay was printed (triplicated); Probed with IR800-conjugated goat anti-human IgG

Advantage of Plasmonic Au chip:

Increased feature intensities due to fluorescence enhancement 10 fold lower background than NitrocelluloseAdditional auto-antigen features can be distinguished on plasmonic chip from the intensity heatmap.

Broad dynamic range High sensitivity

Plasmonic Materials for in vitro Bioimaging

in vitro live cell imaging – fluorescence enhancement by Au nanostructres

MIFE Application – Bioimaging

Nano Res. 2010, 3(10): 738–747

Microplates hosting KB cells (oral cancer cell line) with Hoechst 33528 staining with the fluorescence enhancement being shown quantitatively (10 fold)

Confocal images of a Au microplate hosting mouse 3T3 cells, stained with Alexa 488 for the acetylated histone H3 visualization

Fluorescence Enhancement for biosensing/bioimaging

Applications:ELISA, Protein Microarray, in vitro bioimaging in IR region II (1100 -1400 nm)

Expected Detection limit: ~ Femtomolar range

Current Detection limit: ~Picomolar range ~103 more sensitive

Glass slide

Nanoengineering of Plasmonic Material

Bioimaging

Fluorescent Dyes in IR region II: Ag2S QDs and SWCNs

Ag2S QDs

Benefit of working in NIR region II: Increasing the sensitivityDeeper penetration

MIFE biosensing Application – Bioimaging

In Vivo Fluorescence Imaging with Ag2S Quantum Dots in the Second Near-Infrared Region

Dai et al, DOI: 10.1002/ange.201206059

A steady increase of NIR-II fluorescence of 6PEGAg2S QDs in the tumor region and a decrease of NIR-II fluorescence in other organs and skin was observed from30 min p.i. to 24 h p.i.

Plasmonic enhanced bioimaging using super bright NIR II probes?Plasmonic enhanced bioimaging and therapy ??Clinical questions where MEF can help??

MIFE technology – potential applications

Nanostructures by Nanosphere Lithography

Sensing and in vitro bioimaging

Nanostructures by Synthesis

Superbright in vivo bioimaging probes

Collaborators

The research areas include electromagnetic modeling, light harvesting, bioengineering, synthetic biology, biomaterials, bioimaging, and biosensing.

Acknowledgement

Acknowledgement

Team members

Ioannis Theodore (Postdoc); Jing Pang (PhD student); Daniel Price (PhD

student); Heng Qin (PhD student); Zaynab Jaward (PhD student);

Danyang Wang (MSc); Justin Lim (MEng); Amed Shamso (MSc)

Thank you