Raman measurements

Jussi HiltunenPhotonics Days 2017, Summer School

Outline

• Introduction to Raman scattering-Principles-Instruments-Surface enhanced Raman spectrosopcy (SERS)

• Microsystem integration• Analyte size effects

Raman spectroscopy§ Is a form of vibrational spectroscopy, measures inelastic light scattering§ Complementary to IR spectroscopy, different selection rules§ Most of molecular vibrations can be found in IR and Raman spectra

sample

sample

IR

Raman

Each material hasown finger print

Origin of Raman scattering

• Dipole moment is a measure of the separation of positive and negative electrical charges within a system• Electric polarizability is the relative tendency of a charge distribution displacement by an external electric• Coupling of light with dipole moment change induces absoprtion -> IR spectroscopy• Coupling of light with the change in polarizability induces Raman scattering

0( ) ( ) ( )

0 0

Mathematical formulation

Input field Induced polarization

Displacement dependent polarizabilityTime-dependent displacement

Coupling of input and vibrational frequences

Original frequency Coupled frequencies

Energy state diagram

• Rayleigh scattering is elastic process without energy transfer• In Stokes shift, the final state is higher in energy than the initial state.• In anti-Stokes shift, the final state is lower in energy than the final state.

Raman instrument

Laser

Laser filterSample

Emission filter

Spectrometer

• Raman instrument is essentially a spectrometer with a narrow band light source• System can be high-end laboratory instrument or hand-held device – and anything between

RingC-H

C-S

Benzenethiol spectrum

Wavenumber (1/cm)

Inte

nsity

Wikipedia

BWTekBWTekBaySpecTimeGate

Surface Enhanced Raman Scattering (SERS)

by Sanna Uusitalo

Surface plasmon polariton

by Sanna Uusitalo

• Strong field localization can occur due to the couplingof charge motion in the metal (surface plasmon) andelectromagnetic waves in the air or dielectric(polariton).

• Field effects can induce high enhancement of Ramanscattering 10^4 and 10^8

• Enhancement is localized near the surface

Nanoparticles

Nanostructured surfaces

UV-imprinted surface enhancedRaman spectroscopy substrates

Cut-out 96-well plate size sheets withSERS patterns

• A plastic SERS sensor was replicated by roll method• The plastic web was die-cut into sheets with 1cm*1cm

SERS patterned squares• Cut-out SERS sheets were coated with gold layer

www.photosens.eu

SERS sensor with integrated microfluidics

SERS layer

Fluidics layer (double sided tape)

Lid (polyolefin)

SERS enhancing structures were integrated to study the formation of signal in embedded structures1) Dynamics2) Background level

On-chip detection of Rhodamine 6G model analyte

• 1mM R6G model analyte sample

• The Raman spectrums recorded ontop of the SERS area and on thesmooth gold area for reference

→ The detected signal from theSERS area is surface enhanced

1100 1200 1300 1400 1500 1600 1700 1800

0

10000

20000

1187

1313

1361

1511

1600

1181

1309

1367

1513

Ram

anin

tens

ityRaman shift [1/cm]

1mM R6G on SERS Reference R6G on gold

Parameters used with Raman microscope:- 785nm laser- 20X objective- Integration time 15s- 2*3 point Image mapping- No signal accumulation

Continuous flow of 0.5mM R6G on-chip• Fluids were transferred from a sample

vial into the chip by underpressure• Water was first loaded into the chip

for base reference• R6G sample followed and the signal

rise was detected for flow velocities of25µl/min to 1000µl/min

• Trial was conducted under afluorescence and a Raman microscope

Continuous flow of 0.5mM R6G on-chip• Signal response for the convective flow of R6G molecules detected by fluorescence

microscopy• Diffusion affected flow at the sensor surface detected by SERS

0 2 4 6 8 10

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ised

sign

alin

tens

ity

Time (min)

25µl/min 30µl/min 50µl/min 250µl/min 500µl/min 1000µl/min

0 5 10 15 20

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ised

sign

alin

tens

ity

Time [min]

25µl/min 30µl/min 50µl/min 250µl/min 500µl/min 1000µl/min

SERS resultsFluorescence results

Effect of the polymer lid to the Raman spectrums• Polymer materials are Raman active

• Polyolefin lid has Raman peaks, no fluorescencebackground with 785nm

• The distortion of the R6G peaks can be removed bysubtracting the reference spectrum from the finalresults

Parameters used withRaman microscope:- 785nm laser- 20X objective- Integration time 30s- 2*3 point Image mapping- No signal accumulation

Background substracted 10µM and 100µM R6G spectraRaw spectra

Analyte size effects in SERS

Molecules

• Typically microbe size is in the order of µm while plasmonic features are below 1 µm-> Plasmon field can probe only the part of the ”analyte”

Sample preparation for size effect studies

• Listeria bacteria cells were cultured and immuno-magentic bead-separated (IMS) prior dispensing on the SERS substrate• Au nanoparticles were subsequently dispensed• As a result hybrid nanoparticle-nanosurface configuration was formed

Detectedlines

Raman shift(cm-1) Assignments

631 627/620 Phenylalanine (skeletal)

737 732

glycosidic ring mode of D-glucoseamine (NAG), adenine or

CH2 rocking968 955 C-N stretching

1142 1134/1130C-N and C-C stretch

(carbohydrates)1271 1230-1295 Amide III

13391334/1339/133

8

Deformation CH/Amide III/signature of adenosine;

monophosphate and guanosinemonophosphate, aromaticamino acids tyrosine and

tryptophan1374 1371 DNA

1397 1392/1398Symmetric deformation of CH3

groups1450 1453 CH2 deformation (lipids)

Raman signal intensity with different configurations

• Three configurations were tested1) Listeria on corrugated SERS substrate2) Listeria surrounded with Au nanoparticles, but without substrate enhancement3) Listeria surrounded with Au nanoparticles and located on SERS substrate

• Highest signal was obtained from the hybrid structure

AcknowledgementsSanna Uusitalo, PhD candidate

Thank you for your attention!