Real-Time Raman-Based Analytics for Micro-Systems: Small-Scale Reactors Lab-on-a-chip Separations

Real-Time Raman-Based Analytics for Micro-Systems:

1) Small-Scale Reactors2) Lab-on-a-chip Separations

P rov id in g C h em ica l In fo rm a tion W h en & W h ere You N eed I t

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, NNC05CA09CUSDA: 2006-33610-1683


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 PharmaceuticalChemical PolymerEnvironmental MedicalHomeland Security R&D

We Do Raman – All analyses performed by Raman & SER Spectroscopy

• 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

Esterification Reaction (Demonstration)

OH

O

OCH3

O

+ +

Benzoic Acid + Methyl

Benzoate +

HOCH3H2O

Methanol Water

H+

acid

Protection of a carboxylic acid group by esterification, represents one of the most often used reaction steps during the synthesis of pharmaceuticals.


Small-Scale Reactor: (5 mL coil)

F ib e r O p tic C ap illa ry(O p tica l A cce s s)

B A /M eO H

S y rin g e P u m p

R a m a n A n a ly ze r O p t ic s P ro b e

P e r is ta lt ic P u m p

H 2 S O 4

L ase r

M ix in gC h a m b e r


Small-Scale Reactor: Photograph


Volume = 5 ml

Capillary

Dr. Brian Marquardt University of Washington

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


RTA Advantage: No Fluorescence

785 nmLaser

Excitation

1064 nm Laser

Excitation

Diesel Fuel

8P rov id in g C h em ica l In fo rm a tion W h en & W h ere You N eed I t

Virtually all natural materials fluoresce using 785 nm

RTA Advantage: No X-Axis Shift

Aspirin

Aspirin Shifted

Ibuprofen

9P rov id in g C h em ica l In fo rm a tion W h en & W h ere You N eed I t

X axis shifts limit: quantitation, multi-component analysis, and unknown identification

Sample Cell:

1 mm glass capillary

1-min, 375 mW of 785 nmGlass background = noise


Chemicals:

Benzoic AcidMethanolSulfuric Acid


Chemicals: Reactant & Product

Benzoic Acid (BA)Methyl Benzoate (MBZ)

780 and 817 cm-1 => COO deformation mode


Benzoic Acid(0.5-4M)

Methyl Benzoate(0.5-4M)

Calibration:


Peak Height Peak Area

Calibration:

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 1 2 3 4 5

Concentration (M)

Ra

man

Pe

ak

Inte

ns

ity

MBZ BA

0

2

4

6

8

10

12

14

0 1 2 3 4 5

Concentration (M)R

am

an

Pe

ak

Inte

ns

ity

MBZ (817)

BA (780)


Real-Time Monitoring by Raman

Spectra generated using 375 mW of 785 nm and stored every 30 sec (every 3 min shown).

60 oC


Small discrepancy between rate constants

Reaction Rates:

40 oC

50 oC

60 oC


Activation Energy:

y = 6.1561x - 12.008

R2 = 0.9964

6.00

6.20

6.40

6.60

6.80

7.00

7.20

7.40

7.60

7.80

8.00

2.95 3 3.05 3.1 3.15 3.2 3.25

1000/T (K)

Ln

k


SPIE, 6371 (2006)

NeSSI-Based Small-Scale Batch Reactor


Mike CostParker-Hannifin

Raman Probes:

5 ”

3 ”

1 .5 ”

1 .5 ”th re a d e d fo r

d e v ic e a tta c h m e n t

S E R S -C a p illa ryIn te rf a ce B lo c k

h o le to p a s sm o u n tin g

sc re w

1 /8 ” Ty g o ntu b in g

S E R S -a c tiv ec a p il la ry

o p e n in g to p a s s l ig h t

F ib e r O p tic c a b le

to in s tru m e n t

F O P ro b e& M o u n t


Swagelok


BA

Syringe Pumps

H2SO4

Methanol

LOAD



Monitor & ControlRamanProbe

REACT


PeristalticPump


MBZ

RamanProbe

Syringe Pumps

EMPTY


• 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 oC

• Activation Energy determined at 50.1 kJ/mole

• Best yield, 91% obtained at 60 oC

• Continuous Reactor requires 72 m tube

Summary:


Lab-On-a-Chip Separations&

Surface-Enhanced Raman SpectralDetection

Basic SERS ConceptsAnthrax Spores on Surfaces

Pesticides in Fruits & VegetablesChemotherapy Drugs in Saliva


Surface-Enhanced Raman Spectroscopy

h

P la sm o n F ie ld

H

NH

H

H

H

N

NN

N

A g

S u rfa c e -E n h a n ce dR a m a n P h o to n


M eta l P artic le

S o l-G e l M atrix

A d so rb edM o lec u les

M o lec u lesin S o lu tio n

L ase r

R am a nS ca tte rin g

Simple SERS Sample Vials

2001 U.S. Patent Number 6,623,977


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 1012 enhancement

10 microns

Which do you want: Sensitivity or Reproducibility?


Separation and Analysis

1 2 3 4 5 6

800 1000 1200 1400 1600 1800 2000

Wavenumbers (cm-1)

1

2

3

4

5

6

PABA

PA

Farquharson & Maksymiuk Appl Spec, 57, 479 (2003), US Patents 6,943,031 & 2


Performance: reversibility30 second “plug” of benzoic acid

8 second spectra, 100 mW 785 nm


Analysis: SERS of Pesticide Mixture

Carbaryl on gold

Chlorpyrifoson silver


Trace Detection of Bacilli Spores

on Surfaces using a Portable SERS-Based

Analyzer


Professor Jay F. Sperry University of Rhode Island

Anthrax LD50 ~10,000 spores

Goals: 100 spores/cm2 1-min

No False Positives

Approach: measure anthrax signature - CaDPA

C o re W all(p ro te in s-cy ste in e ,

)C a d ip ico lin a teC o rte x

(p ep tid og ly ca n )

E x o sp o riu m

S p o re C o a t

D N AR ib o so m es

C C

O O

O ON

2 -

C a 2 +

CaDPA


Raman Spectroscopy

CaDPA

B. cereus

J Raman Spec, 35, 82-86 (2004)

Pure samples500 mW of 1064 nm5-min acquisition time


SERS and RS of dipicolinic acid (DPA)

SERS1 mg/L (1 ppm)150 mW, 10 sec

Raman5.5 g/L (55,000 ppm)

DPA in KOH 300 mW, 25 min

Appl Spec, 58, 351 (2004)

Enhancement Factor = 105


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 cm2)


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


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)


• 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


Sensitivity: 10 ppb

75 mW of 785 nm1 min


Pesticide: chlorpyrifos (methyl)

SERS: 10 & 5 ppm (mg/L)

RS: 50,000 ppm (50g/L), 350 mW-25 min


Standard Method vs SERS MethodBlank Sample: 1 ppm pesticide

1100g sample + 200 mL CH3CN 50% add 50 microg pesticide test

50% 100%2 10g NaCl 10g NaCl test organic layer

3 Centrifuge Centrifuge test eluant

4a evap to 5 ml evap to 5 ml4b SPE: C-18 SPE: C-184c add CH2Cl2/CH3OH add CH2Cl2/CH3OH test eluant

5a evap to 5 ml evap to 5 ml5b SPE: amino-propyl SPE: amino-propyl5c add CH2Cl2/CH3OH add CH2Cl2/CH3OH test eluant

6a evap to 5 ml evap to 5 ml6b SPE: carbon-black SPE: carbon-black6c add CH3CN/Toluene add CH3CN/Toulene test eluant

7a evap to 5 ml evap to 5 ml 7b 1 microL in GC-FID 1 microL in GC-FID compare

1 hour

10 min


Gas Chromatography

After step 5a After step 7b


Professor James Stewart, U. Connecticut

Chlorpyrifos: 50 ppb

1) In dichloromethane2) In OJ after evaporation & SPE3) In OJ after SPE


Method: 10-minute analysis

sma


ABC

SERS-ActiveLab-on-a-Chip

Sample Injection Syringe

Prototyping SERS-chip

A

B

C

500 750 1000 1250 1500 1750

Wavenumbers (cm-1)


A) Endosulfan

B) Thiabendazole

C) Phosmet

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


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

O

O

F

H

H

H

N

N1

2

34

5

6

Analysis: 5-Fluorouracil

Normal Raman

Surface-Enhanced Raman

J Raman Spec, 36, 208-212 (2005)Vibrational Spec, 38, 79-84 (2005)


Simple Separation Devices

5FU & 5FUH2

5FdUrd

Leucovorin

A

B

C

600 800 1000 1200 1400 1600 Wavenumbers (cm-1)


ABC

3C h em ica lly-S e lec tiv e

S E R S -A ctiv e C a p illa rie s

L a se r F o c u s L e n s R a m a n In s tru m e n t S u rfa c e

1 m L S y rin g e

To p V iew

S id e /E n d V iew

S c a n S ta g e

P le x ig la s s S u b s tra te

Simple Separation Devices

100 micron


Documents

Real-Time Raman-Based Analytics for Micro-Systems: Small-Scale Reactors Lab-on-a-chip Separations