Sers Biofluids Fei Invited Talk Pittcon2010

Rapid Analysis of Drugs, Biomarkers and Pathogens in Urine, Saliva and Nasal Mucus

Using a Disposable Lab-on-a-chipFocus: SERS Applications in Bio-Fluids

Providing Chemical Information When & Where You Need It

Frank Inscore, Chetan ShendeAtanu Sengupta and Stuart Farquharson RTA

NIH CN: 1R43CA94457-01NSF CN: DMI-0215819

NASA CN: NNC05CA09CDoD

Dr. John Murren Yale New Haven HospitalDr. Eric Wong Jet Propulsion LaboratoriesProf. Jay Sperry University of Rhode Island

Dr. Steven Christesen ECBC

UK Road Policing Technologies Home Office Scientific Development Branch(Dr. Helen Turner )

www.RTA.bizBooth #2825 Pittcon 2010

http://www.rta.biz/�

Challenge: Offset negative effects of weightlessness in spaceSolution: Monitor indicators and associated counter-drugs in urine


Extended Weightlessness in Space Causes:• Muscle Loss, Bone Loss• Renal Stone Formation•Difficulty in Sleeping, Motion SicknessMinimize Effects with Drugs and Exercise

NASA desires a light-weight On-Board Analyzer-that is non-invasive-with fast response-and has necessary sensitivity specificity selectivity

Physiological Changes Reflected in Urine Composition:• Biomarkers (~1 mg/L)Drugs & Metabolites (~10 microg/L)

Challenge: Analysis of chemo-drugs and metabolites for dosage controlSolution: Monitor drugs and metabolites in saliva


• Chemotherapy drugs also kills non-cancer cells• Dosage is critical (to little noneffective, to much kills patient)• No clinical trials to establish statistical based dosage• Current analysis requires large sample volume (10-20 ml blood and/or urine)• These methods are labor intensive and time consuming

• Centrifugation to remove red blood cells• Extraction using organic solvents• Separation using chromatography• Detection with UV or Mass Spec

• Standards are needed to ensure measurement accuracy•Consequently, measurements made on an “as needed” basis• Viable alternative, parent drug/ metabolites represented in saliva

• Concentration ~10-50% of blood plasma • 1 microg/mL detection needed for chemo-drugs

• Advantage, Non-invasive (no needles)• Issues of clotting and platelet count in blood of chemo-patients

• Advantage, Saliva is 99.5% water• Interfering physiological chemicals 100X less

Challenge: Analysis for presence of prescription & illicit drugs of abuseSolution: Identify drugs and metabolites in saliva


2 scenarios: ER overdoses, road-side policing of impaired drivers

Speed - Rapid diagnosis of Drugs in Saliva.•Saliva analysis will increase speed by eliminating time consuming extraction and separation steps.•Rapid diagnosis for drug overdose in ER can lead to appropriate medical care.

Specificity - Correct Identification of abused drug.•Variety of overdose systems can lead to confusing diagnosis. •Explicit drug detection will lead to accurate treatment regime.

Sensitivity - Low detection limit Required.•Drugs in Saliva need to be measured at ng/mL concentrations

The Biofluid Analysis Challenge Need non-invasive technique that meets following criteria:

• Specificity – identify Bio-agents Biomarkers Drugs Metabolites • Uniquely, in the presence of of the biofluid matrix • With no False Positives (or False Negatives)!

• Speed – Field Applications require rapid analysis• 10 minutes or less

• Sensitivity – requirements to Monitor and Detect • Drugs Metabolites ~ 100 to 10 ng/mL (100-10 ppb) • Biomarkers ~ 1000 to 10 microg/L (1 ppm - 10 ppb)• Bioagents ~ 10-4 to 10-2 cfu/mL and 1 ng toxin/mL

Selective detection of target analyte(s) at parts-per-billion and lower is challenging:even under ideal conditions, more so in a complex bio-matrix!


Approach: Surface-enhanced Raman Spectroscopy

h

Plasmon Field

ν

H

NH

H

H

H

N

NN

N

Ag

Surface-EnhancedRaman Photon

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 becomes possible.


Single Molecule Detection: requires 1012 -1014

Chemical contribution can provide additional 103 enhancement

Raman, although weak effect, provides molecular specificity

SERS-Active Substrates


10-3M

10-5M

10-8M (~10 ppb)

benzenethiol

LMC ~10-11M (~10 ppt)

Approach: RTA SERS Patented Sampling Systems (rapid response)

Metal Particle

Sol-Gel Matrix

AdsorbedMolecules

Moleculesin Solution

Laser

RamanScattering

2001: Simple SERS Sample Vials

1 10

2004: SERS-Active Capillary


silver gold

More suited to extract and pre-concentrate

2003: SERS Microplate

2007: Functionalized Sol-Gel SERS Capillary(affords greater selectivity and sensitivity)


2nd generation

Std SERS vials

PC

OTC unreduced reduced

RTA’s Portable Raman AnalyzersAdvantages: • No sample preparation• Simple integration via fiber optics• Remote analysis, multi-component• 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

25 poundswater tight


1064 nmVials

Microplates

Capillaries

785 nm

Lab-on-Chips

Other Wavelengths1532, 976, 633, 532 nm

Analysis: SERS of Urine Matrix and Components

O

H2N NH2

N

N

O

NH2

N

N NH

HN

O

OH

HO

OH

OH

O


urea

creatinine

uric acid

lactic acid

•100 chemicals/biochemicals as potential interferents in urine were measuredUric acid: dominates urine SERS!

Simulated

Lyophillized

Real

Simulated urine = 20 g/L urea, 3 g/L NaCl, 1.5 g/L KCl, 1.4 g/L creatinine, 0.2 g/L lactic acid, 0.15 g/L uric acid

Analysis: Biomarker and Counter-Drug


100 mw 785 nm, 1-min

SERS

NR

OH

O

NH2

N

N

OH

PHO

OH

O

P

HO

HO

ON

Risedronate

0.005

0.025

0.045

0.065

0.085

0.105

0.125

0 200 400 600 800 10000.005

0.01

0.015

0.02

0.025

0.03

0 200 400 600 800 1000

Microgram/L Microgram/L

3-MethylHistidine

Analysis: doped reconstituted urine sample - multi-targets complicatedCan we detect directly? Pre-cleanup necessary? Extract and separate?


Raloxifene (organic extract)

3-Methylhistidine (aqueous extract)

Risedronate (aqueous extract)

Scopolamine (directly on gold)

Urine

With exception of drugs like Ral or Scop, further pre-treatment necessaryLOC is ideally suited for urine analysis

Analysis: 3-Methylhistidine

OH

O

NH2

N

N

OH

O

NH2

N

N


Histidine

3-MeHis

unknown

best match

• Unique vibrational features provide ability to identify and discriminate chemicals•RTA spectral search soft-ware identifies unknown chemical from library data base

Analysis: parent drug and metabolite


N

N NH

N

OH

OH

N

N

HO NH

N

Allopurinol

Oxypurinol

Mixture

•Unique vibrational features provide ability to identify and discriminate•RTA quantification and chemometric soft-ware can define composition

Component selection: for urine sample pre-treatment

1) Filter particulates

2) Exclude small inorganic ions

UARIS 3-MeHIS

3) Refine SG selectivity, extract and separate

3-MeHISRIS

3-MeHIS


Analysis: Demonstrate sub microg/L detection levels


Risedronate

3-MeHis

CaPAllopurinol

Oxypurinol

1 microg/L (1 ng/mL)

10 microg/L

IbandronateAlendronate

Pamidronate

Clodronate

Etidronate

N

OH

PHO

OH

O

P

HO

HO

O

CH3CH3

NH2

OH

PHO

OH

O

P

HO

HO

O

NH2

OH

PHO

OH

O

P

HO

HO

O

Cl

Cl

PHO

OH

O

P

HO

HO

O

CH3

OH

PHO

OH

O

P

HO

HO

O

N

H

H

O OH

O

O

S

N

CH3

NCH3H3C

Scopolamine

Promethazine

Uric Acid

Calcium Oxalate

Calcium Phosphate

Cystine Penicillamine

Hydrochlorothiazide

Allopurinol

NH

SNH

OO

Cl

S

O

O

H2N

O

HO

HS

NH2

H3C

H3C

Ca+2Ca+2

Ca+2

P

O

-O

O-

O-

PO

O--O

-O

O

SHO

HO

O

NH2

S

NH2

O-

O

O

O-Ca+2

H-Deoxypyridinoline

N

N NH

HN

O

OH

HO


R&D: Optimize Lab-On-Chip Components


R&D: SERS Lab-On-ChipDifferent Lab-on-Chip designs used

R&D: SERS Lab-On-Chip


Analysis of real human urine sample doped with 3-MeHis and Ris at 1 microg/L (1 ng/mL) in 10-min!


R&D: SERS Lab-On-Chip

Multiplexed Sample Analysis

on CD Style LOC

Base-line Analysis: Chemotherapy Drugs


5-Fluorouracil

Dacarbazine

Doxorubicin

• Incorrect dosage can be toxic or ineffective•Complex analysis: drug mixtures and metabolites

• LMC is 100-10 ng/mL

Dacarbazine

100 µg/mL mixture

5-FU

NR

SERS

Base-line Analysis: OTC and Prescription Drugs


NR

SERS

Aspirin

Acetaminophen

• Overdose with Acetaminophen is common in elderly

• Lowest Measured Concentration (LMC) is 100 - 10 ng/mL

Amobarbital

Secobarbital

PhenobarbitalHN

O

NH

O

O

NH

NH

O

O

O

NH

HN O

O

O

• Barbituates are one of the most abused of prescribed drugs

Diazepam on Ag

Oxycodone on Au

SERS

SERS

SERS

NR

Base-line Analysis: Illicit Drugs of Abuse


SERS Ag-SG

NRCocaine

• Most ER reported overdose cases occur with cocaine•Other drugs measured - Amphetamine, Heroine, Morphine, LSD, THC

• LMC and Reproducibilitymodify SG chemistry

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

00.10.20.30.40.50.60.70.80.9

11.1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Prob

abili

ty o

f Det

ectio

n

Probability of False Positive

ROC Curve

Log C at K=3.29

LMC at 95 %

confidence

-7.31583 48 ppb

SERS Au-SG


1. Mix sample with saliva

2. Suck sample into SER-active capillary (10-sec)

3. Measure SERS of sample (10-sec placement, 1-min scan)

Analysis: Single Drug in Saliva

Typical SERS Experiment

Single Component Analysis: 5-FU in Saliva


5-FU doped saliva pH adjusted

1 microg/mL weak acid

50 microg/mL HCl

• Filter improved SERS-response

• Small ions can be excluded

• Weak acid improved sensitivity


Proof of Concept: Multi-component analysis in saliva

Manual Flow Switch

AB

C

AB

CManual Flow Switch

cis-platinum

oxypurinol

• drugs extracted from saliva of chemotherapy patient


Non-Invasive Pathogen Detection in Nasal Mucus

Manual Flow Switch

AB

C

AB

CManual Flow Switch

(calcium dipicolinate)

C CO O

O ON

- -

Ca2+

Core

Outer Core Wall

Cortex

Exosporium

Spore Coat

DNA

Ribosomes

Inner Core Wall

B C

D

E

Raman Shift, cm-1 1800 500 800 1000 1200 1400 1600

A

F

G

H

I

JInternal intensity reference

digesting reagent

Dipicolinic acid 25 ppm B.

cereus

cysS-Ag

No DPA signature!

250 ppm

B. subtilis

spectrum same as before adding BS

BC spores in mucus

Summary: Non-invasive Urine Saliva Analysis


• Small sample volume: < 100 microL• Rapid & simple analysis: < 10 minutes (total)• Specificity: detect various drugs biomarkers• Sensitivity: at required detection in urine, but

in saliva needs improvement• Require 1 microg/mL to 10 nanog/mL

• Future work: improve extraction efficiencyRoad-side Policing with UK HOSB

LOC for Infectious Pathogens in Hospitals

Documents

Sers Biofluids Fei Invited Talk Pittcon2010