The ability to monitor cellular chemical environments in vivo has become a source of interest for diagnostic and analytical science. Biosensors, consisting of biosensitive molecules that can interact with target analytes, can be used to sense chemical environments and perturbations that can then be converted to measurable values. 1 Abnormal chemical environments at the cellular and sub-cellular levels, including imbalances in pH and ion concentrations, are an indicator of many disease states.2,3 The development of a surface-enhanced Raman scattering (SERS) active optical fiber probe that could measure in vivo chemical environments in a minimally intrusive way would assist in diagnosing these disease states.4

The sensitivity of SERS-devices for sensing is highly dependent on the morphology and uniformity of the metal coatings. Block copolymer thin films can be used to fabricate SERS active arrays of nanoparticles (NPs) in a reproducible manner. Gold NPs particle morphology, size and uniformity are easily defined by the selection and composition of the block copolymer. Currently there is a need for establishing routes to SERS active optical fibers with a uniform coating of gold nanoparticles for optimal enhancement of Raman scattering.

1. T. Vo-Dinh, P. Kasili, Anal. Bioanal. Chem. 2005, 382, 918-925. 2. S. M. Simon and M. Schindler, Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3497. 3. J. Poschet, E. Perkett, and V. Deretic, Trends Mol. Med. 2002, 8, 512. 4. F. L. Yap, P. Thoniyot, S. Krishnan, and S. Krishnamoorthy. Nano 2012, 6,3, 2056-

2070. 5. C.L. Haynes, A.D. McFarland, R.P. Van Duyne, Anal. Chem. 2005,339-346. 6. R. A. Jensen, J. Sherin, and S. R. Emory, Applied Spectroscopy. 2007, 61, 832-838.

Introduction

Figure 1. SERS-Active Optical Fiber. Uniform Au nanoparticle arrays are arranged on the surface of an optical fiber, which can then provide a surface to which Raman receptors (crystal violet or mercaptopyridine) can adhere.

Raman Spectroscopy

Step 1 Step 2

Figure 7. Block copolymer micelles of polystyrene-block-poly(4-vinylpyridine)

Step 4: Load with HAuCl4 in H2SO4 bath and reduce in NaBH4 solution Step 5: Ar plasma-etch away polymer micelles Step 6: Dip coat Au NPs in crystal violet solution

Methodology - Fabrication of Size and Shape Controlled Au NP Arrays

PS(57500)-b-P4VP(18500)

Clean As Cast MeOH Opened

Au -loaded

NaBH4

Reduced

Plasma Ion-Etch

PS(145000)-b-P4VP(50000)

References Acknowledgments

Characteristic CV Peak Enhancement Factor for PS(145K)-b-P4VP(50K)

Enhancement Factor for PS(57.5K)-b-P4VP(18.5K)

806 6 x 1010 6 x 1011

913 1 x 1010 8 x 1010

1175 1 x 1011 6 x 1011

1380 5 x 1011 5 x 1012

Scheme 1. Method of Au NP Fabrication.

Characterization of Fiber Morphology

Figure 9. AFM Characterization. AFM images of optical fibers after each step in the fabrication process illustrated in Scheme 1 (scan size = 1 x 1 µm2; height scale = 50 nm).

Transformation of Block Copolymer Films into Au NP Arrays - Atomic Force Microscopy (AFM)

Consistent and Uniform Arrays - Scanning Electron Microscopy

Figure 11. Scanning Electron Microscopy. SEM images of PS(145K)-b-P4VP(50K) etched Au nanoparticles along the fiber’s length (a) 0 mm from tip (b) 2 mm from tip (c) 4 mm from tip (d) 6 mm from tip (e) 8 mm from tip (scale = 5 μm).

Unenhanced Crystal Violet PS(57.5K)-b-P4VP(18.5K) Au Nanoparticles

Figure 4. Raman Spectrum. Raman spectra (i.e. unenhanced) of 1 mM crystal violet solution. λex = 785 nm and tint = 4 s.

Figure 5. SERRS Spectrum. Surface-enhanced resonance Raman scattering (SERRS) spectra of a monolayer of crystal violet on gold nanoparticle array of PS(145K)-b-P4VP(50K). λex = 633 nm and tint = 3 s.

Figure 6. SERRS Spectrum. SERRS spectra of a monolayer of crystal violet on gold nanoparticle array of PS(57.5K)-b-P4VP(18.5K). λex = 633 nm and tint = 3 s.

I = intensity from baseline n= number of molecules ν= frequency t= integration time

Table 1. SERS Enhancement Factors of Au NP arrays. Enhancement factor calculations according to the above equations for both polymer nanoparticle arrays.

Determination of SERS Enhancement Factors

PS(145K)-b-P4VP(50K) Au Nanoparticles PS(57.5K)-b-P4VP(18.5K) Au Nanoparticles

Assessment of Au NP Density Gradient - Inductively Coupled Plasma Mass Spectroscopy

Figure 12. ICP-MS Levels of Au on PS(145K)-b-P4VP(50K) Au NP Array Coated Fiber.

Figure 13. ICP-MS Levels of Au on PS(57.5K)-b-P4VP(18.5K) Au NP Array Coated Fiber.

-20-10

0102030

-50 50 150 250

He

igh

t (n

m)

Distance (nm)

Height Profile of Reversed Polymer Micelles

PS(145K)-b-P4VP(50K) Au Nanoparticles

Figure 10. Cross-sectional AFM Analysis of Opened Polymer Micelles.

Gold Nanoparticle Arrays for Surface-Enhanced Raman Spectroscopy on Optical Fibers

Rachel DeMayo,1 Steven R. Emory1 and David A. Rider1,2 1Department of Chemistry, Western Washington University, Bellingham, WA 98225

2Department of Engineering Technology, Western Washington University, Bellingham, WA 98225

E hnex

h(nex – nvib)

Vibrational Energy States

Virtual States

hnex

h(nex + nvib)

Stokes

hnex

hnex

Anti-Stokes Rayleigh

Figure 2. Raman Scattering. The incident photon (green) excites a ground state molecule to a virtual excited state. Upon relaxation, the molecule can scatter a photon of longer (red) or shorter (blue) wavelength. In elastic Rayleigh scattering, the molecule scatters a photon of the same wavelength, providing no molecular information.

Nanoparticle

Charge Cloud of Conduction Electrons

Electric Field

Figure 3. Electromagnetic SERS Enhancement by Metal Nanoparticles. When the incident light strikes the surface, localized surface plasmons are excited. The electric field enhancement is greatest when the plasmon frequency is in resonance with the radiation. In order for scattering to occur the plasmon oscillations must be perpendicular to the surface; if they are in-plane with the surface, no scattering occurs.

500 750 1000 1250 1500 1750 2000

0

2000

4000

6000

8000

10000

Inte

ns

ity

Raman Shift (cm-1)

807

923

1175

1381

1457

1629

(a)

(b)

Figure 8. SERS Reporter Molecules. (a) 4-mercapto-pyridine and (b) crystal violet.

Step 3 Step 4 Step 5 Step 6

Step 1: Declad and clean fiber Step 2: Dip coat fiber in polymer solution Step 3: Reverse polymer micelles in MeOH

Future Work • Optimize fabrication Au NP array processes

and Au NP ring formation. • Analyze Au NP SERS enhancement with 4-

mercaptopyridine (4-MPy). • Study pH-dependence of 4-MPy with SERS. • Analyze SERS enhancement of crystal violet

and 4-MPy with 633-nm fiber optic Raman spectrometer.

• Fabricate and characterize Au NP arrays with PS(75K)-b-P4VP(25K) block copolymer.

National Science Foundation Dr. Polly Berseth, AMSEC Manager Erin Macri, SciTech Coordinator Caileen Brison , SciTech Colin C. Hanson, Instrument Specialist Leah E. Bergquist, Rider group Blake M. Cassidy, Rider Group Alicia Mangubat, Emory Group

=

PS(145K)-b-P4VP(50K) PS(57.5K)-b-P4VP(18.5K)

PS(57.5K)-b-P4VP(18.5K)

As Cast MeOH

Opened Au-loaded NaBH4

Reduced Au NP

Period (nm) 83 ± 8 67 ± 8 60 ± 11 59 ± 9 83 ± 21

Height (nm) 31 ± 3 11 ± 2 17 ± 3 16 ± 2 4 ± 2

Particle Width (nm) 56 ± 4 N/A N/A N/A N/A

Ring Width (nm) N/A 31 ± 3 26 ± 4 26 ± 5 26 ± 2

PS(145K)-b-P4VP(50K)

As Cast MeOH

Opened Au-

loaded NaBH4

Reduced Au NP

Period (nm) 129 ± 15 114 ± 16 82 ± 14 101 ± 14 102 ± 27

Height (nm) 38 ± 9 28 ± 4 35 ± 6 25 ± 4 19 ± 7

Particle Width (nm) 96 ± 16 N/A 71 ± 7 N/A 57 ± 10

Ring Width (nm) N/A 34 ± 6 N/A 47 ± 7 N/A

Table 2. Nanoparticle Analysis. Measured period, nanoparticle height and width by AFM nanoscope analysis.

Figure 15. Collection of SERS Spectra Through Optical Fiber.

Enhancement Factor

(a) (b) (c) (d) (e)

Figure 14. pH-Dependent SERS Spectra of 4-MPy.6

500 750 1000 1250 1500 1750 2000

0

1000

2000

3000

4000

Inte

ns

ity

Raman Shift (cm-1)

801

916

1177

1357

1445