eas 2011 fei invited sers talk drugs in saliva

Providing Chemical Information When & Where You Need It

EAS 2011RTA Booth #106

Frank E. Inscore, Chetan Shende, Atanu Sengupta, Hermes Huang and Stuart Farquharson

[email protected]

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


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.


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


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


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



• 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


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


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)


PC

OTC

Std chromatographic media


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


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.


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


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


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


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.


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


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


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


Cocaine Extracted from Saliva 25ppb cocaine in 0.5mL saliva extracted & detected in 8-min (from start to finish)


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


50 ppb cocaine

1ppm PCP

1ppm diazepam

10ppm acetaminophen

100ppm Oxycodone

Lib. Oxycodone

Lib. Hydrocodone

Further Test: 50ppb Drug in Saliva Samples


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


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


Preliminary LOC Results: in Saliva at 50 ppb


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


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.


eas 2011 fei invited sers talk drugs in saliva

Documents

sers applications

sers microplate

need challenge

identification of drugs

discriminate drugs

offending drugs

counter drugs

sersactive capillary