1. Real-Time Raman-Based Analytics for Micro-Systems: 1)Small-Scale Reactors 2)Lab-on-a-chip Separations Stuart Farquharson, Chetan Shende, Frank Inscore, and Wayne Smith www.rta.biz Contracts: NIH: 1R43CA94457-01 US Army: DAAD13-02-C-0015, NSF: DMI-0215819, DMI-0349687, DMI-0512701, NASA: NNA05CP04C, NNC05CA09C USDA: 2006-33610-1683

2. Mission: Provide analyzers that perform continuous, real-time, trace chemical detection with the ability to operate in harsh environments. Our fiber optic probes allow easy installation and use in all industries employing chemicals. Such as: Agriculture Petrochemical Biopharmaceutical Pharmaceutical Chemical Polymer Environmental Medical Homeland Security R&D We Do Raman – All analyses performed by Raman & SER Spectroscopy

3. Rapid Optimization of New Reactions/Processes Micro-Reactors => thermal & mass transport reduced Numbering-Up => 2 years earlier to market, Improve yield Process Analytical Technology => In spec, safety Project Goal:Project Drivers: New drugs improve health… A blockbuster drug makes >$1 million/day! Profits limited by patent expiration Eliminate delays to market (5-7 years lab-to-pharmacy) 16% of drugs out-of-specification (= $1.1 million/year loss) 50% of production time = analyzing intermediates/product

4. Esterification Reaction (Demonstration) Protection of a carboxylic acid group by esterification, represents one of the most often used reaction steps during the synthesis of pharmaceuticals.

5. Small-Scale Reactor: (5 mL coil)

6. Small-Scale Reactor: Photograph Volume = 5 ml Capillary Dr. Brian Marquardt University of Washington

7. RTA’s Raman Analyzer Advantages: No sample preparation Simple integration via fiber optics Remote analysis, multi-component No fluorescence interference Complete spectral coverage Wavelength stability Confident spectral subtraction and library search/match Real-time, On-demand analysis Long term stability Temperature and vibration immune Shock resistant It’s an Interferometer…no x-axis drift! $75,000

8. RTA Advantage: No Fluorescence 785 nm Laser Excitation 1064 nm Laser Excitation Diesel Fuel 8 Virtually all natural materials fluoresce using 785 nm

9. RTA Advantage: No X-Axis Shift Aspirin Aspirin Shifted Ibuprofen 9 X axis shifts limit: quantitation, multi-component analysis, and unknown identification

10. Sample Cell: 1 mm glass capillary 1-min, 375 mW of 785 nm Glass background = noise

11. Chemicals: Benzoic Acid Methanol Sulfuric Acid

12. Chemicals: Reactant & Product Benzoic Acid (BA) Methyl Benzoate (MBZ) 780 and 817 cm -1 => COO deformation mode

13. Benzoic Acid (0.5-4M) Methyl Benzoate (0.5-4M) Calibration:

14. Peak Height Peak Area Calibration:

15. Real-Time Monitoring by Raman Spectra generated using 375 mW of 785 nm and stored every 30 sec (every 3 min shown). 60 oC

16. Small discrepancy between rate constants Reaction Rates: 40 oC 50 oC 60 oC

17. Activation Energy: SPIE, 6371 (2006)

18. NeSSI-Based Small-Scale Batch Reactor Mike Cost Parker-Hannifin

19. Raman Probes: Swagelok

20. NeSSI-Based Small-Scale Batch Reactor BA Syringe Pumps H 2 SO 4 Methanol LOAD

21. NeSSI-Based Small-Scale Batch Reactor Monitor & Control Raman Probe REACT Peristaltic Pump

22. NeSSI-Based Small-Scale Batch Reactor MBZ Raman Probe Syringe Pumps EMPTY

23. Batch mode small-scale reactor built & used Esterification reaction successfully monitored Limits of detection: BA = 0.9%, MBZ = 0.75% Various acid catalysts used, sulfuric acid best Rate constants easily determined from 25-65 o C Activation Energy determined at 50.1 kJ/mole Best yield, 91% obtained at 60 o C Continuous Reactor requires 72 m tube Summary:

24. Lab-On-a-Chip Separations & Surface-Enhanced Raman Spectral Detection Basic SERS Concepts Anthrax Spores on Surfaces Pesticides in Fruits & Vegetables Chemotherapy Drugs in Saliva

25. Surface-Enhanced Raman Spectroscopy

26. Simple SERS Sample Vials 2001 U.S. Patent Number 6,623,977

27. SERS: silver particles in sol-gel 1 square micron = 12.6 silver particles Laser spot (325 micron diameter) = 83,000 square microns i.e. contains 1.04 million silver particles Or the equivalent of 1 perfect hot spot generating 10 12 enhancement 10 microns Which do you want: Sensitivity or Reproducibility?

28. Separation and Analysis 1 2 3 4 5 6 800 1000 1200 1400 1600 1800 2000 Wavenumbers (cm-1) 123456123456 PABA PA Farquharson & Maksymiuk Appl Spec, 57, 479 (2003), US Patents 6,943,031 & 2

29. Performance: reversibility 30 second “plug” of benzoic acid 8 second spectra, 100 mW 785 nm

30. Analysis: SERS of Pesticide Mixture Carbaryl on gold Chlorpyrifos on silver

31. Trace Detection of Bacilli Spores on Surfaces using a Portable SERS-Based Analyzer Professor Jay F. Sperry University of Rhode Island Anthrax LD 50 ~10,000 spores Goals: 100 spores/cm 2 1-min No False Positives

32. Approach: measure anthrax signature - CaDPA CaDPA

33. Raman Spectroscopy CaDPAB. cereus J Raman Spec, 35, 82-86 (2004) Pure samples 500 mW of 1064 nm 5-min acquisition time

34. SERS and RS of dipicolinic acid (DPA) SERS 1 mg/L (1 ppm) 150 mW, 10 sec Raman 5.5 g/L (55,000 ppm) DPA in KOH 300 mW, 25 min Appl Spec, 58, 351 (2004) Enhancement Factor = 10 5

35. Extraction & Identification of DPA in 2-min! 1. Added 10 micoliters of SporeDestroyer (1-min digestion) 2. Suck 1 microliter into SER-active capillary (10-sec) 3. Measure SERS of DPA (10% = 220 pg/microliter) (10-sec placement, 30-sec scan) 0. Dried 2200 spores from 1 microliter (~0.2 cm 2 )

36. RTA’s Raman Analyzer Advantages: No sample preparation Simple integration via fiber optics Remote analysis, multi-component No fluorescence interference Complete spectral coverage Wavelength stability Confident spectral subtraction and library search/match Real-time, On-demand analysis Long term stability Temperature and vibration immune Shock resistant RTA’s Portable Raman Analyzer SERS-Active Capillary

37. SERS: 220 Spores 100 pg/microL (ppb) reference spectrum 220 pg/microL DPA 1 spore = 10 pg, DPA =10% spore weight Internal Reference IJHSES, 20, 12-18 (2007)

38. Need pesticides to meet food demand US imports 40-45% fruit & vegetables 6.1% of imports exceed guidelines Only 1% is tested 3 Million tons of contaminated fruits & vegetables reaches the market each year Rapid Analysis of pesticides on or in fruit & vegetables by SERS Goals: 10 ppb-1 ppm (0.01-1 mg/L) 1-minute No false positives Minimum Reagents Field-usable

39. Sensitivity: 10 ppb 75 mW of 785 nm 1 min

40. Pesticide: chlorpyrifos (methyl) SERS: 10 & 5 ppm (mg/L) RS: 50,000 ppm (50g/L), 350 mW-25 min

41. Standard Method vs SERS Method 1 hour 10 min

42. Gas Chromatography After step 5a After step 7b Professor James Stewart, U. Connecticut

43. Chlorpyrifos: 50 ppb 1) In dichloromethane 2) In OJ after evaporation & SPE 3) In OJ after SPE

44. Method: 10-minute analysis sma

45. ABCABC SERS-Active Lab-on-a-Chip Sample Injection Syringe Prototyping SERS-chip ABCABC 500 750 1000 1250 1500 1750 Wavenumbers (cm -1 ) A) Endosulfan B) Thiabendazole C) Phosmet

46. Controlling Chemotherapy Drug Dosage 1,334,100 new cancer cases each year >0.5 million Americans died of cancer in 2003 Lung and bronchus – 28%Colon and rectum – 10% Breast – 7%Prostate – 6% Treatment involves Surgery, Radiation and Chemotherapy Chemotherapy drugs also kill non-cancer cells Dosage is critical No clinical trials to establish statistical based dosage Current analysis of drugs and metabolites use 10-20 ml blood Consequently, measurements made on an “as needed” basis

47. Detection of 5-fluorouracil in saliva using SERS 5-FU, one of most widely used chemotherapy drugs Wide genetic-based variation in metabolism,15-80% inactive 10% of patients die the first time administered Need: Monitor metabolism on individual basis Solution: SERS of saliva Concentrations in saliva 10-50% of blood plasma (1-10 microg/mL) Saliva is 99.5% water, few interfering physiological chemicals But Currently analyses require 10-20 cc - unsafe Raman – chemical specificity SERS – increased sensitivity Simple SERS Syringe - 100 microL, 5-min total analysis time

48. Analysis: 5-Fluorouracil Normal Raman Surface-Enhanced Raman J Raman Spec, 36, 208-212 (2005) Vibrational Spec, 38, 79-84 (2005)

49. Simple Separation Devices 5FU & 5FUH 2 5FdUrd Leucovorin ABCABC 600 800 1000 1200 1400 1600 Wavenumbers (cm-1) ABCABC

50. Simple Separation Devices 100 micron