eas 2011 fei invited sers talk drugs in saliva
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EAS 2011RTA Booth #106
Frank E. Inscore, Chetan Shende, Atanu Sengupta, Hermes Huang and Stuart Farquharson
Rapid Detection and Identification of Drugs in Saliva by Surface-Enhanced Raman Spectroscopy
Relevant R&D Funding to RTA:
NIH CN: 1R43CA94457-01NSF CN: DMI-0215819
NASA CN: NNC05CA09C
Jet Propulsion Laboratories(Dr. Eric Wong)
UK Road Policing TechnologiesHome Office Scientific Development Branch
(Dr. Helen Turner, Dr. Audrey Carmichael)
Focus: SERS Applications in BiofluidsScreening Abused Drugs in Salivaat Road-Side & Emergency-Roomfor Driver Impairment & Overdose
The Need & Challenge: Drugs in saliva
Road-side screening for determining drug induced driver impairmentNational Highway Traffic Safety Administration Stats:
• 2007: 11% of drivers stopped tested positive for drugs• 2009: 18% of driver fatalities tested positive for drugs
Emergency room assessment of overdose and drug abuseU.S. Drug-Related Emergency Room (ER) Stats:
• 2004: 2.4 million visits• 2009: 4.6 million visits
2.1 million attributed to illicit drugsCocaine, Heroin, Methamphetamine, PCP, MDMA, LSD, Methadone2.3 million attributed to prescription & some to over-the-counter (OTC) drugsOxycodone, Diazepam & Acetaminophen
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Analysis of chemo-drugs and metabolites for dosage control• Dosage critical (to little noneffective, to much kills patient)• Current analysis requires large sample volume (10-20 ml blood and/or urine)• Traditional lab methods are labor intensive and time consuming• Viable alternative is saliva, parent drugs/metabolites adequately represented (but at lower levels)• Advantages of saliva, non-invasive (no needles) & 100X less potential interferents (99.5% water)
Challenge: Drugs in salivaCritical need to rapidly identify offending drug(s), so that impaired drivers can be arrested or appropriate medical care can be administered.
The analyzer must typically provide the following criteria:• Specificity – Identify and Discriminate Drugs (with No False Positives!)
• Sensitivity – Detect ~10-8 M or less (e.g. 30 ppb cocaine is threshold)
• Reproducibility – Accurate and Repeatable (with No False Negatives!)
• Speed – Rapid Response and Analysis within 8-10 minutes
• Field Usable – Battery Operated and Rugged
In an effort to meet this need we have been investigating the ability of surface-enhanced Raman spectroscopy (SERS) to detect and identify numerous drugs of abuse in saliva at ng/mL concentrations within 10 minutes.
We present successful measurement of representative illicit, prescribed, and over-the-counter drugs in saliva by SERS, with a focus on cocaine.
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The Solution: SERSSpecificity: all chemicals (drugs/metabolites) produce a unique Raman spectrum allowing unequivocal identification (no false-positives).
Sensitivity: Ag and Au nanoparticle substrates used to generate SERS amplify Raman signals (increase scattering efficiency) by 1 million times or more allowing required detection of 10-8 M (ppb) (no false-negatives).
Raman: Pure Cocaine
Gold SERS: 1 ppm Cocaine
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CH3N
CO2CH3
O2C
H Cl -
Cocaine Hydrochloride
+
Au
Ag
hνTransmitted
Absorbed
Scattered
Rayleigh
Raman
HH
H H
H H
Raman
How it works: Raman
(IR)
Laser light directed at a chemical generates Raman light.
o hνvib
hνvib
hνo hνscat
vib0
vib1
virt
hνscat
Light Chemical
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ALSO, chemical contribution can provide additional 103 enhancement
Sub part-per-billion detection becomes possible with SERS
SERS provides
Single Molecule Detection some argue this requires enhancement factor (EF)
of 1012 -1014
Surface-enhanced Raman Spectroscopy
Raman, although weak effect, provides molecular specificity
BUT, when a molecule is within a laser induced plasmon field,
the efficiency of Raman scattering can increase by 106 i.e. 1 million times!
Sub part-per million detection possible
h
Plasmon Field
ν
H
NH
H
H
H
N
NN
N
Ag
Surface-EnhancedRaman Photon
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• Electrochemically Roughened Electrodes• Metal Colloidal Hydrosols• Metal Islands or Nanoparticles on Solid Supports• Metal Coated Surface Structures• Self-Assembled Monolayers (SAMs)
Traditional:
Recent:• Metal-Doped Porous Media• Periodic Apertures in Metals• Metal Shells• Fiber Optic Tips
SERS-Active Media
Commercial SERS Substrates: Benzenethiol
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10-3M
10-5M
10-8M (~10ppb)
benzenethiol
RTA: LMC ~10-11M (0.01 ng/mL or 10 ppt)
(A)LOW SENSITIVITYSLOW RESPONSE
BUTREPRODUCIBLE
(B)LESS REPRODUCIBLE
SLOW RESPONSEBUT
MODERATE SENSITIVITY
(C)HIGH SENSITIVITY
ANDFAST RESPONSE
ANDOK REPRODUCIBLE
Conc. EF
102
104
107
Approach: RTA SERS Patented Sampling Systems provide instant response in seconds as opposed to 30-min or more!
Metal Particle
Sol-Gel Matrix
AdsorbedMolecules
Moleculesin Solution
Laser
RamanScattering
2001: Simple SERS Sample Vials
1 10
2004: SERS-Active Capillary
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silver gold
2003: SERS Microplate
High Throughput Screening Extraction and Pre-ConcentrationU.S. Patents for RTA
6,623,977; 6,943,031; 6,943,032; 7,312,088; 7,393,691; 7,393,692; 7,462,492; and 7,462,493
2007: Functionalized Sol-Gel SERS Capillary(affords greater selectivity and sensitivity)
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PC
OTC
Std chromatographic media
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R&D: SERS Lab-On-ChipDifferent wafer, glass and plastic Lab-on-Chip designs used
RTA’s SERSID - Trace Chemical Analyzer’sfor Field and Lab Use
Patents: 6623977, 6943031, 6943032, 7312088, 7393691, 7393692, 7462492, 7462493, 7713914
2010
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2011
The Proposal: The DeviceThe proposed SERS-RSSD-DD (Road Side Screening Device for Drug Detection) and SERS-ERSD-DD
(Emergency Room Screening Device for Drug Detection) will extract, identify, and quantify the presence of drugs (and metabolites) in driver or patient saliva at ~10-8M within 8-10 minutes.
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6
UK Road-Side Screening: Required Drugs
35 drugs & metabolites were measured31 active on gold, 4 (barbiturates) active on silver
Spectra search worked for all drugs Providing Chemical Information When & Where You Need It
150 Drugs in Expanded Gold SERS Library:Applicable to the ER
See recent RTA paperPharmaceutics 2011, 3, 425-439 doi:10.3390/pharmaceutics3030425
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SERS of Representative Illicit, Prescription & OTC Drugs in Expanded Gold Spectral Library (150) PCP
Methamphetamine
MDMA
LSD
Heroin
Diazepam
Ritalin
Demerol
Hydrocodone
Oxycodone
Acetaminophen
Acetylsalicylic acid
Ibuprofen
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Library Search: Measure Priority DrugsSpectra search and identification worked for all drugs
Hit Quality Name1 0.001 Nordiazepam2 0.010 Methadone3 0.010 Oxazepam4 0.010 Temazepam5 0.012 Norcodeine
Hit Quality Name1 0.036 Nordiazepam2 0.357 Temazepam3 0.363 Diazepam4 0.373 Methadone5 0.392 Oxazepam
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Spectral Match Results for Mixtures
500ppm cocaine/diazepam
Diazepam Ref
Cocaine Ref
500ppmnordiazepam /diazepam
Diazepam Ref
Nordiazepam Ref
Cocaine: Static Concentration Data
50 ppb Cocaine
Background = Luminescence from glass capillaryIt can be subtracted.
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25 ppb = 7x10-8M
Sensitivity: Static ROC Curves of Cocaine50 ppb
25 ppb
New Modified Gold Sol-GelROC Curves 95% Confidence = 48 ppb
(10 capillaries measured at 9 spots per concentration)
100 ppb
75 ppb
50 ppb
25 ppb
0 ppb
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Conc. # of substrates
Mean peak hgt
Std Deviation
Mean Std Deviation (α)
Blank 10 0.002 0.0014 0.00425 ppb 9 0.0113 0.00650 ppb 10 0.0157 0.003
Conc. C K value25 ppb 2.26350 ppb 3.343
Log C at K=3.29
LMC at 95 %
confidence
-7.31583 48 ppb
Improve sub-ppb Detection via Pre-concentration
A
B
25 ppb (7.3x10-8M) cocaineHCl
12.5 ppb (9.2x10-8M) amphetamine
25 ppb (8.7x10-8M) diazepam
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static 25 ppbcocaine in water
flowed 5 mL of 25 ppb cocaine in water
Sensitivity and Reproducibilityachieved with flowing less than 5 mL of 25 ppb (10-8M)
cocaineHCl in HPLC water on new gold SG4(3H); detected at 95% confidence (K > 3.29) in under 5-min!
extracted 5 mL of 25 ppb cocaine in water
static 25 ppbcocaine in water
Alsodetected 25 ppb cocaineHCl on gold SG4 after extracted
from 1 mL HPLC water with SPE capillary column 8-min;intensity 40x the static signal and is equivalent to a 1ppm
signal as observed on the static concentration curve.
Develop Saliva Extraction Method
1. Collect 0.5 ml in 1 min 2. Expel sample into buffer 3. Filter sample
4. Draw sample into microSPE 5. Elute cocaine from SPE 6. Inject sample in SERS Capillary
7. Measure spectrum
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Cocaine Extracted from Saliva 25ppb cocaine in 0.5mL saliva extracted & detected in 8-min (from start to finish)
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500 750 1000 1250 1500 1750Wavenumbers (cm-1)
25 ppb Cocaine in 0.5 ml Saliva
25 ppb Cocaine in 0.5 ml Water
Amphetamine
Diazepam
Methadone
PCP
Above drugs at 1ppm extracted from 0.5 mL saliva in 8-min
Spectral Search and Match Results for Drugs in Saliva
Unknown Oxycodone(0.1 mg/mL water)
Cocaine (50 ng/mL)
PCP(1 mcg/mL)
Diazepam(1 mcg/mL)
Acetaminophen(10 mcg/mL)
Rank HQI Chemical HQI Chemical HQI Chemical HQI Chemical HQI Chemical
1 0.073 Oxycodone 0.287 Cocaine 0.019 PCP 0.022 Diazepam 0.276 Acetaminophen
2 0.734 Hydrocodone 0.348 Ethyl- benzoyl-ecgonine 0.315 Fentanyl 0.317 Temazepam 0.639 Sulfadoxine
3 0.800 Trazadone 0.349 Benzoyl-ecgonine 0.325 EMDP 0.328 Nor-diazepam 0.704 Serotonin
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50 ppb cocaine
1ppm PCP
1ppm diazepam
10ppm acetaminophen
100ppm Oxycodone
Lib. Oxycodone
Lib. Hydrocodone
Further Test: 50ppb Drug in Saliva Samples
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Hit Quality Name1 0.236 Cocaine
1 0.171 Diazepam
1 0.171 PCP
1 0.066 Methadone
Cocaine 50ppb
Diazepam 50ppb
PCP 50ppb
Methadone 50ppb
Drug in Saliva Extraction: Potential Interferents
Correctly Identified Cocaine by Spectral Match!
50ppb in saliva via SPE method
Caffeine
QuininevitC
Mannitol
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50ppb equal mixturecocaine and caffeineextracted from 0.5mL saliva in 8-min
blank saliva extracted as control
30ppb cocaine and 50ppb mannitol mixture extracted from 0.5mL saliva in 8-min
Preliminary LOC Designs
SPE
Ag/AuPMMA with
Channels
Glass Plate
Plastic Cover
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Preliminary LOC Results: in Saliva at 50 ppb
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amphetamine 50ppb
cocaine 50ppb
diazepam 50ppb
PCP 50ppb
methadone 50ppb
All Drugs Correctly Identified by Spectral Match!
Next Generation LOC Design
Elution solvent reservoir
3mL syringe
0.2µm filter
Valve
Valve
Valve
Lab-on-chip SPE capillary SERS capillary
Vacuum
Solvent waste reservoir
Sample waste reservoir
Buffered saliva sample
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Summary
Critical need to rapidly identify offending drug(s) in impaired drivers and overdose in ER.
Demonstrated ability of surface-enhanced Raman spectroscopy (SERS) to detect and identify numerous drugs in saliva at ng/mL concentrations within 8 minutes.
Identification is provided by matching measured spectra to a SERS library comprised of over 150 different drugs, each of which possess a unique spectrum.
Trace detection is provided by gold nanoparticles trapped within a porous glass matrix that generate SERS.
Speed is provided by a syringe driven sample system that extracts the drugs from saliva AND provides SERS-activity.
Spectral collection is provided by a portable Raman analyzer.
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