Sers Biofluids Fei Invited Talk Pittcon2010
Transcript of 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
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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
Challenge: Offset negative effects of weightlessness in spaceSolution: Monitor indicators and associated counter-drugs in urine
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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
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• 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
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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!
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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.
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Single Molecule Detection: requires 1012 -1014
Chemical contribution can provide additional 103 enhancement
Raman, although weak effect, provides molecular specificity
SERS-Active Substrates
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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
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silver gold
More suited to extract and pre-concentrate
2003: SERS Microplate
2007: Functionalized Sol-Gel SERS Capillary(affords greater selectivity and sensitivity)
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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
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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
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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
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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?
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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
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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
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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
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Analysis: Demonstrate sub microg/L detection levels
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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
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R&D: Optimize Lab-On-Chip Components
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R&D: SERS Lab-On-ChipDifferent Lab-on-Chip designs used
R&D: SERS Lab-On-Chip
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Analysis of real human urine sample doped with 3-MeHis and Ris at 1 microg/L (1 ng/mL) in 10-min!
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R&D: SERS Lab-On-Chip
Multiplexed Sample Analysis
on CD Style LOC
Base-line Analysis: Chemotherapy Drugs
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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
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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
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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
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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
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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
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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
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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
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• 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