chem symposium-haddow-oct-2012b
DESCRIPTION
Seminar - 2012 - UAEU Chemistry Symposium on "Chemistry and Health". Dr. Haddow presentation on Amperometric determination of sialic acid from bio-samples.TRANSCRIPT
Development of amperometric dual-channel FIA systems for the determination of
clinically important free-, bound- and total sialic acid
Jody D. Haddow a
Sayed A.M. Marzouk a
Amr Amin b
a Department of Chemistry, United Arab Emirates University, b Department of Biology, United Arab Emirates University
Symposium on Chemistry and Health, United Arab Emirates University, Oct, 2012.
OutlineHealth
Why sialic acid?
Chemistry
SA Biosensor
Single-channel IER FIA of SA
Dual-Channel IER FIA of SA
Conclusions
Acknowledgements
1
2
3
4
86
5
7
9
What is Sialic acid?N-Acteylneuraminic acid (Neu5Ac or NANA)
Sialic acids are found widely distributed in animal tissues. Important biological roles
43 derivatives of SA but NANA is the most common Very often the terminal sugar in a glycan Glycoproteins and Gangliosides
Bound sialic acid (SAb)
Importance of quantifying SANormal Function – many…- Sialic acid-rich glycoproteins bind lectins – cell adhesion, etc- Cell signaling/recognition, Siglecs – Lectin-Igs
Health ImplicationsViral/Bacterial Infection- Influenza viruses bind to sialic acids of the upper respiratory tract.
Cancer- Metastatic cancer cells often express a high density of sialic acid-rich
glycoproteins.
Pharmacodynamics- Epoetin (erythroprotein) used to treat anemia, due to renal failure and
cancer chemotherapy. - Baby Formulas- Sialic acid content of the glycan is central of in vitro and in vivo
functionality.
PyruvateN-Acetyl-D-mannosamine
La
cta
te d
eh
ydo
rge
na
se,
NA
D+
Fluorometric measurement of the generated NADH
Pyr
uva
te o
xisd
ase
, O
2
H2O2
Pe
roxi
da
se,
p-
chlo
rop
he
no
l-4-
am
ino
an
tipyr
ine
Colorimetric measurements of the
produced dye
Bound sialic acid
(neuraminidase (Sialidase)
Free sialic acid
(N-acetyl-neuraminic acid aldolase)
NADH
Amperometric detection
(present work)
dim
eth
yl-a
min
o-
be
nza
lde
hyd
e
Colorimetric measurements of the
product
Acy
lglu
cosa
min
e 2
-e
pim
era
se
N-Acetyl-glucosamine
N-a
cety
lhe
xosa
min
e
oxi
da
se
acetylglucosaminic acid + H2O2
pH sensor
Amperometric Biosensor/ IER FIA
Analysis of SA A Novel Approach
Bound-Sialic acid
Free Sialic acid
Pyruvate
H2O2
Sialidase
Sialic acid aldolase
Pyruvate oxidase
Free Sialic acid
Pyruvate
H2O2
Sialic acid aldolase
Pyruvate oxidase
Anal. Chem 2007, 79 1668-1974
Prototype Amperometric Biosensor for Sialic Acid DeterminationSayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari
Sensors & Actuators B 157 (2011) 647- 653
Flow injection determination of sialic acid based on amperometric detectionSayed A.M. Marzouk, Jody D. Haddow, and Amr Amin.
Research Progression
Batch SA biosensor
Flow Injection SA biosensor
Flow Injection Enzyme Reactor
Dual Channel – Bound and Free IER SA detection
Current Research
emannosaminDacetyl-NPyruavteacidSialicFree(SAA)AldolaseSA
AcidSialicFreebSA sialidase
222234 OHCOphateAcetylphosOPOPyruvate PyO
Current
signal
PYOSAA
+PYO
SD+
SAA+
PYO
Pyruvate + free SA + b-SA
(Py) (Py + SA) (Py + SA +bSA)
Enzyme strategies
H2O2 H2O2 H2O2
Anodic oxidationH2O2 --> O2 + 2H+ + 2e-
0 1000 2000 3000 4000 5000
0
2
4
6
8
10
12
14
Cu
rren
t, n
A
Time, sec
0 20 40 60 80 100 120 140 160 180 200
0
2
4
6
8
10
12
14
Cu
rre
nt,
nA
Sialic acid Conc, M
Pt disc, 2 mm
Kel-F insulating body, 6 mm
Microporous PolyEster membrane
Enzyme layer(face down)
Teflon cap
SA Amperometric Biosensor – batch mode
Anal. Chem 2007, 79 1668-1974
Prototype Amperometric Biosensor for Sialic Acid DeterminationSayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari
Linear response to SA
Stable and Steady-state response
SA Amperometric Biosensor – Optimizations
Anal. Chem 2007, 79 1668-1974Prototype Amperometric Biosensor for Sialic Acid DeterminationSayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari
1. Buffer Type - PB vs MOPS2. Temperature3. Cofactor concentration4. NANA Aldolase / Py Oxidase
ratio5. Buffer pH6. % Glutaraldehyde : Total
Protein crosslinking ratio (G/T)
7. Enzyme to BSA matrix ratio
Helped to lay foundation for current work
Thermostated Water in
SS rod with inlet channel
Teflon flow-cell
Au/Pt/enzyme layer
Polyethylene electrode body
Copper lead
Ref. electrode
Thermostated Water out
Flow in
Flow out
Copper tube 3 mm OD
Pt counter electrode
20 mm dia
13 mm dia
15 mm dia
A. Biosensor detector
Single-Channel Amperometric FIA of SA
Sensors & Actuators B 157 (2011) 647- 653
Flow injection determination of sialic acid based on amperometric detectionSayed A.M. Marzouk, Jody d. Haddow, and Amr Amin.
Water flow in
Water flow out
Copper tube – 10 turns
IER
Flow-through cell
Injection valve
PumpCarrier solution
B. Immobilized Enzyme Reactor
PYO – SAACo-immobilized
Single-Channel Amperometric FIA of SA
Sensors & Actuators B 157 (2011) 647- 653
Flow injection determination of sialic acid based on amperometric detectionSayed A.M. Marzouk, Jody d. Haddow, and Amr Amin.
• Longer operational lifetime which could be due the larger amount of immobilized enzyme
• longer residence time which results in almost complete conversion of the substrate
• Contrary to biosensors, enzyme immobilization and signal transduction are optimized independently
• IEF can be prepared and used by less experienced personnel compared to biosensors
Advantages IER vs Biosensor
Based on these points the SA analysis was further optimized with IER
0.1 mM0.25 mM
0.50mM1.00 mM
2.00 mM
5.00 mM
PYO – SAACo-immobilized
Sensors & Actuators B 157 (2011) 647- 653
Amperometric FIA of SA based on an IER – in situ heating
Easily controlled and rapid thermostating
Signal ≈ 3x
Reduced stability and linearity!!
0 500 1000 1500 2000 2500 30000
3
6
9
12
15
0.1 mM0.25 mM
0.1 mM0.25 mM
0.5 mM 0.5 mM
1.0 mM 1.0 mM
2.0 mM 2.0 mM
5.0 mMC
urre
nt, A
Time, sec
13 mm dia Pt electrode23 oC
Sensors & Actuators B 157 (2011) 647- 653
Amperometric FIA of SA based on an IER - repeatability
In another experiment, two SA solutions of 100 and 250 uM were injected (twenty injections each) and showed RSD of peak heights of 1.5 and 1.1%, respectively. Data not shown
Working 1 Working 2Ref
Counter
IER-1 IER-2
Construction of the dual-channel Flow Cell
2-channel system to allow simple and rapid quantitation of real bio-samples
Allow subtraction of a “spy” channel
2-CH Potentiostat
2-CH Potentiostat
Split ratio at the Y-connector?
R1 R2
W1 W2
Very stable – primarily controlled by the relative back pressures introduced by the IERs.
Not necessarily 50-50 but each channel is calibrated independently
FIA Systems based on dual IER and Two amperometric detectors
0 1000 2000 3000 4000 5000
0
2
4
6
8
10
12 0 1000 2000 3000 4000 50000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
PY injection
0.05 mM0.1 mM
0.25 mM
0.5 mM0.75 mM
1 mM
2.0 mM
Cu
rre
nt
x 1
06 , A
Time, s
SA injection
NANA+PyO
1.5 mM
0.05 0.10.25
0.5 mM0.75 mM
1 mM
1.5 mM
2 mM
Cu
rre
nt
x 1
06 , A
Relative sensitivities to SA and Py injections – NANA/PyO IER
Carrier PB pH 7.3,
T = 37oC
Sample loop =10 μL
Single channel
Carrier PB pH 7.3,
T = 37oC
Sample loop =10 μL
Single channel
8 X
Signal after two enzymatic conversions
Signal after one enzymatic conversion
1000 2000 3000 4000
0
1
2
3
4
5
Time, s
Cu
rre
nt, A
0.25 mMPy
0. 5 mMPy
1.0 mMPy
0.25 mMSA
0. 5 mMSA
1.0 mMSA
PY/SA ~ 6
PYO
SAA - PYO
Relative sensitivities to SA and Py injections – 2-channel
Channel that must be normalized and subtracted is too intense
Further optimization of SA detection in the presence of Pyruvate
R1 R2
W1 W2
PYO
Catalase
Depletion of pyruvate
2-CH Potentiostat
PyO = pyruvate + phosphate + O2 acetyl phosphate + CO2 + H2O2
Catalase = 2 H2O2 → 2 H2O + O2
R2= PYO
R1= SAA - PYO
FIA peaks simultaneously obtained for SA and PY
Pre-depletion Py/SA = 6Post-depletion SA/Py = 2.5
2000 4000 6000
0.00
0.08
0.16
0.24
B
C
Time, s
Cu
rre
nt, A
1.50 1.75 1.25 1.00 0.750.5
FR: mL/min
Effect of Flow Rate
Balance between sample residence time (in reactor and at electrode) with the rate sample dispersion
PYO
PYO-SAA
R1 R2
W1 W2
SialidaseIER
Analysis of bound sialic acid – Fetuin Protein
Fetuin Glycoprotein
Molecular weight: 48.4 kDa
The composition of bovine fetuin (weight %) is polypeptide 74%, hexose 8.3%, hexosamines 5.5%, and sialic acid 8.7%.
2-CH Potentiostat
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FIA peaks simultaneously obtained for SA, Fetuin and PY
PYO-SAA
PYO
R1 R2
W1 W2
PYO
Catalase
Simultaneous analysis of total SA and PY in simulated serum sample
Sialidase
Simulated Serum6% BSA – 140 mM NaCl10 mg/mL Fetuin1 mM Py – 2 mM SA
2-CH Potentiostat
0 1000 2000 3000 40000.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 1000 2000 3000 40000.00.20.40.60.81.01.21.41.61.8
5.0 mM PY
1.0 mMPY1.0 mM
PY
5.0 mM SA
2.0 mMSA
6% BSA - 140 mM NaCl10 mg/mL Fetuin
1.0 mM PY-2.0 mM SA
5.0 mMPY
5.0 mM SA
2.0 mM SA
1.0 mM SA
Cur
rent
x 1
06 , A
FR = 2.8 mL/minFR = 1.5 mL/minFR = 2.8 mL/min
Cur
rent
x 1
06 , A
Time, s
Simultaneous analysis of total SA and PY in simulated serum sample
PY signal diminished at reduced flow rate: More time for removal
bSA was completely hydrolyzed at the high FR
The problem of intrinsic high sensitivity towards pyruvate was resolved using PYO-catalase sequence.
The split ratio was stable as indicated by the calibration stability.
The flow cell design proved excellent to provide fast, sensitive and reproducible response.
The first simultaneous FIA analysis of PY, SA and or b-SA was successfully demonstrated.
The reliability of the analytical systems was evaluated by analyzing PY, SA and bSA in simulated serum sample
Conclusions
UAEU for the financial support
Prof. Sayed Marzouk and Dr. Amr Amin for a fruitful collaboration
Khawla A. Al Tayyari early optimization of biosensor
Thank-you
Acknowledgements
0.2 0.3 0.4 0.5 0.6 0.7 0.8
0
50
100
150
200
0.2 0.3 0.4 0.5 0.6 0.7 0.8
0
50
100
150
200
Cu
rre
nt
x 1
06 ,
A
Potential (E), V vs SCE
Electrode 1
Potential (E), V vs SCE
ELectrode 2
Cyclic voltammograms obtained for electropolymerization of 1,3-diaminobenzne (m-phenylenediamine) at two simultaneous Pt disc electrodes
Formation of the protective polymeric layer
Tested against oxidizable species: Thiamine pyrophosphate (TPP), acetaminophen (4-acetamidophenol) (AAP), and uric acid (UA) – blocked by polymer- data not shown
500 1000 1500 2000 2500
0.00
0.09
0.18
B
C
Time, s
Cu
rre
nt, A
1 mM SA – 1.5 mL/min – 100 µL injection
PYO
SAA - PYO
Signal Stability/Repeatability (2-ch split flow)
- no fluctuation in split ratio - actual ratio not critical - channels calibrated independently
Analyzing biological samples. Serum, breast milk, formula, etc.
Expanding the current study to more comprehensive multi-channel analysis.
Flow-through porous electrodes - connected as a
single detectorImmobilized enzyme
reactors
Sample in
Time
Sin
gle
ch
ann
el
resp
on
se
Waste
Immobilized enzyme reactors
Ch-1Ch-2
Ch-3Ch-4
Ch-5
Ch-1 Ch-2 Ch-3 Ch-4 Ch-5
Sample in
Waste
Time
nch
an
nel
re
spo
nse
(A)
(B)
Flow-through porous electrodes - connected as a
single detectorImmobilized enzyme
reactors
Sample in
Time
Sin
gle
ch
ann
el
resp
on
se
Waste
Immobilized enzyme reactors
Ch-1Ch-2
Ch-3Ch-4
Ch-5
Ch-1 Ch-2 Ch-3 Ch-4 Ch-5
Sample in
Waste
Time
nch
an
nel
re
spo
nse
(A)
(B)
Future Work