© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 1 Sample deposition on Klarite How Klarite works Sampling on Klarite Drop coat technique –Solvent role –Molecule distribution –Concentration vs molecules probed

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 2 How Klarite ® works Details of Klarite ® morphology –Regular array of inverted pyramids with carefully designed dimensions and engineered gold surface. Electric field distribution at surface –Depends on metal, its geometrical factors (hole size, shape, spacing) and excitation wavelength –Surface field distribution can be engineered to fit applications SERS intensity dependence –Molecular adsorption in areas of high electric field gives increased SERS signals. –Molecules must have an affinity for metal surface, and be intrinsically Raman active. 

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 3 Sampling on Klarite Delivering molecules in close proximity to metal surface is a prerequisite for measuring good SERS signal –Easy for vapours –Less straightforward for solutions Molecule attachment can be strong or weak depending on –Molecule affinity to metal –Presence/absence of surface chemistry Klarite ® Solution Vapor

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 4 Molecule distribution at surface For qualitative studies all three situations can give a good result. Quantitative studies require: - More uniform distribution of molecules at surface - Known volume of analyte in measurable area - Sampling of analyte deposition will be important evaporation rinse evaporation Aqueous solutions no surface chemistry Volatile solutions no surface chemistry Aqueous solutions with surface chemistry

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 5 Drop deposition on Klarite Volatile solvents (e.g. methanol, ethanol, acetone, etc) will spread across the whole surface even if small volumes are used 2 ul 1 ul Volatile solvents Water solutions 4mm Water solutions allow multiple points to be arranged on the same chip, depending on volumes. –1ul usually allows 5-6 drops onto Klarite (see picture)

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 6 How many molecules contribute to SERS signal? # molecule in solution (concentration) # molecules deposited on substrate? # molecules probed by laser? Intensity Raman shift

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 7 How many molecules contribute to SERS signal? (1) 2 ul Laser spot A TNT concentration of 4.4 x10 -5 M in acetonitrile equates to 2.3 ppm. BUT – contribution to SERS signal comes from sample underneath laser spot, so consider the droplet volume and the laser spot size. Case study: SERS of 4.4 x10 -5 M TNT in acetonitrile Active Excel Calculator double click to change values

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 8 How many molecules contribute to SERS signal? (2) 2 ul Laser spot 2 μl of 4.4 x M solution of TNT equates to 20 nanograms of analyte, but the laser spot interrogates only a mass of ~22 picograms. This means good SERS signals are observed from just ~1/1000 th of total amount of analyte deposited (see next slide). (assuming uniform coverage) Active Excel Calculator double click to change values

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 9 SERS of analyte in volatile solvents Experimental conditions: 785 nm, 150μm laser spot diameter (Mesophotonics SE1000) 10s acquisition time Sample deposition conditions are not optimised TNT signal shown is from a solution of 10 μg/ml in acetonitrile. This equates to 2.3 ppm, which is a concentration of 4.4 x M. TNT SERS of 4.4 x10-5 M TNT in acetonitrile

© 2008 D3 Technologies Ltd. All rights reserved. Private and Confidential. Slide 10 Conclusions SERS is capable of interrogating remarkably low concentrations of analyte molecules, (equating to low ppm ranges and analyte masses in the picograms range) even when simple instrumentation and basic deposition techniques are used Quantitative analysis of low concentrations is possible if sample deposition is controlled and substrate sampled accordingly. Surface chemistry is fundamental to improve wetting and quantitation