Seminar - 2012 - UAEU Chemistry Symposium on "Chemistry and Health". Dr. Haddow presentation on Amperometric determination of sialic acid from bio-samples.

1. 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.

2. Outline
Health
 Why sialic acid?
Chemistry
 SA Biosensor
 Single-channel IER FIA of SA
 Dual-Channel IER FIA of SA
 Conclusions
 Acknowledgements

3. What is Sialic acid?
N-Acteylneuraminic acid (Neu5Ac or NANA)
1
8
9 6 2
7 4
5 3
Bound sialic
acid (SAb)
 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

4. Importance of quantifying SA
Normal Function – many…
- Sialic acid-rich glycoproteins bind lectins – cell adhesion, etc
- Cell signaling/recognition, Siglecs – Lectin-Igs
Health Implications
Viral/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.

5. Bound sialic acid
Amperometric
pH sensor (neuraminidase (Sialidase) Biosensor/ IER FIA
Free sialic acid Analysis of SA
(N-acetyl-neuraminic acid aldolase)
A Novel Approach
N-Acetyl-D-mannosamine Pyruvate
dimethyl-amino-
Lactate dehydorgenase, NAD+
Acylglucosamine 2-
benzaldehyde
Pyruvate oxisdase, O2
epimerase
Colorimetric
measurements of the
N-Acetyl-glucosamine product
N-acetylhexosamine
H2O2 NADH
oxidase
aminoantipyrine
chlorophenol-4-
Peroxidase, p-
Amperometric Fluorometric
detection measurement of the
(present work) generated NADH
acetylglucosaminic acid + H2O2 Colorimetric
measurements of the
produced dye

6. Anal. Chem 2007, 79 1668-1974
Current Research Prototype Amperometric Biosensor for Sialic Acid
Determination
Bound-Sialic acid Sayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari
Sensors & Actuators B 157 (2011) 647- 653
Sialidase Flow injection determination of sialic acid based
on amperometric detection
Sayed A.M. Marzouk, Jody D. Haddow, and Amr Amin.
Free Sialic acid Free Sialic acid
Research Progression
Sialic acid
Sialic acid
aldolase Batch SA biosensor
aldolase
Flow Injection SA biosensor
Pyruvate Pyruvate
Pyruvate Flow Injection Enzyme
Pyruvate
oxidase Reactor
oxidase
H2O2 H2O2 Dual Channel – Bound and
Free IER SA detection

7. Enzyme strategies
sialidase
bSA Free Sialic Acid
SA Aldolase (SAA)
Free Sialic acid Pyruavte N - acetyl D mannosamine
3 PyO
Pyruvate PO4 O2 Acetylphosphate CO2 H 2O 2
Pyruvate + free SA + b-SA
SD
SAA +
PYO + SAA
Anodic oxidation PYO +
H2O2 --> O2 + 2H+ + 2e-
PYO
Current
H2O2 H2O2 H2O2
signal
(Py) (Py + SA) (Py + SA +bSA)

8. SA Amperometric Biosensor – batch mode
14 Stable and Steady-state response
12
Teflon cap
10
Microporous
8
Current, nA
PolyEster
membrane 6
4
2
0
Enzyme layer
(face down) Pt disc, 2 mm 0 1000 2000 3000 4000 5000
Time, sec
14
Linear response to SA
Kel-F insulating 12
body, 6 mm
10
8
Current, nA
6
4
Anal. Chem 2007, 79 1668-1974
2
Prototype Amperometric Biosensor for Sialic Acid
0
Determination
Sayed A.M. Marzouk, S.S. Ashraf, and Khawla A. Al Tayyari 0 20 40 60 80 100 120 140 160 180 200
Sialic acid Conc, M

9. SA Amperometric Biosensor – Optimizations
Anal. Chem 2007, 79 1668-1974 1. Buffer Type - PB vs MOPS
Prototype Amperometric Biosensor
for Sialic Acid Determination
Sayed A.M. Marzouk, S.S. Ashraf, and
2. Temperature
Khawla A. Al Tayyari
3. Cofactor concentration
4. NANA Aldolase / Py Oxidase
ratio
5. Buffer pH
6. % Glutaraldehyde : Total
Protein crosslinking ratio
(G/T)
7. Enzyme to BSA matrix ratio
Helped to lay foundation for current work

10. Single-Channel Amperometric FIA of SA
Sensors & Actuators B 157 (2011) 647- 653
A. Biosensor detector Flow injection determination of sialic acid
Ref. based on amperometric detection
Sayed A.M. Marzouk, Jody d. Haddow, and Amr Amin.
electrode
Copper
lead
Polyethylene
Flow electrode body
out
Teflon flow-cell 15 mm dia
Au/Pt/enzyme layer
20 mm dia
SS rod with inlet
channel
Pt counter Thermostated Water 13 mm dia
electrode in
Copper tube 3 mm
Thermostated OD
Water out
Flow in

11. Single-Channel Amperometric FIA of SA
B. Immobilized Enzyme Reactor
Sensors & Actuators B 157 (2011) 647- 653
Flow injection determination of sialic
acid based on amperometric detection
Sayed A.M. Marzouk, Jody d. Haddow, and Amr Flow-
Amin. through cell
IER
Water flow out
PYO – SAA Copper tube – 10 turns
Co-immobilized
Water flow in
Carrier Pump Injection
solution valve

12. Advantages IER vs Biosensor
• 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
Based on these points the SA analysis was further optimized with IER

13. Amperometric FIA of SA based on an IER – in situ heating
Easily controlled and rapid thermostating
PYO – SAA
Co-immobilized
Signal ≈ 3x
Reduced stability and linearity!!
5.00 mM
2.00 mM
1.00 mM
0.50mM
0.25 mM
0.1 mM
Sensors & Actuators B 157 (2011) 647- 653

14. Amperometric FIA of SA based on an IER - repeatability
15
5.0 mM
13 mm dia
Pt electrode
12
23 oC
9
In another experiment, two SA
Current, A
solutions of 100 and 250 uM
were injected (twenty injections
2.0 mM 2.0 mM each) and showed RSD of peak
6
heights of 1.5 and 1.1%,
respectively. Data not shown
1.0 mM 1.0 mM
3
0.5 mM 0.5 mM
0.25 mM 0.25 mM
0.1 mM 0.1 mM
0
0 500 1000 1500 2000 2500 3000
Time, sec
Sensors & Actuators B 157 (2011) 647- 653

15. Construction of the dual-channel Flow Cell
Ref
Working 1 Working 2
2-CH Potentiostat
2-channel system to
allow simple and
rapid quantitation of
real bio-samples
Allow subtraction of
a “spy” channel
Counter
IER-1 IER-2

16. FIA Systems based on dual IER and Two amperometric
detectors
2-CH Potentiostat
W1 W2
R1 R2
Split ratio at the
Y-connector?
 Very stable – primarily controlled by
the relative back pressures
introduced by the IERs.
 Not necessarily 50-50 but each
channel is calibrated independently

17. Relative sensitivities to SA and Py injections – NANA/PyO IER
Signal after two 2 mM
1.4
enzymatic conversions
1.2
NANA+PyO
Current x 10 , A
1.0 Carrier PB pH 7.3,
1.5 mM
6
0.8 SA injection T = 37oC
0.6 Sample loop =10 μL
1 mM
0.4
0.75 mM Single channel
0.5 mM
0.25
0.05 0.1
0.2
0.0
12 0 1000 2000 3000 4000 5000
Signal after one Carrier PB pH 7.3,
enzymatic conversion 2.0 mM
10 PY injection
T = 37oC
Current x 10 , A
1.5 mM
8
6
8X 1 mM
Sample loop =10 μL
6
0.75 mM
0.5 mM
Single channel
4
0.25 mM
2 0.1 mM
0.05 mM
0
0 1000 2000 3000 4000 5000
Time, s

18. Relative sensitivities to SA and Py injections – 2-channel
5
1.0 mM
Py
4
PY/SA ~ 6
0. 5 mM
Current, A
Py 3
Channel that must
0.25 mM
be normalized and 2
Py subtracted is too
intense
1
0
PYO
0. 5 mM 1.0 mM
0.25 mM SA
SA
SA
SAA - PYO
1000 2000 3000 4000
Time, s

19. Further optimization of SA detection in the presence of Pyruvate
2-CH Potentiostat
W1 W2
R1= SAA - PYO
R2= PYO
R1 R2
Depletion of
pyruvate
Catalase
PYO
PyO = pyruvate + phosphate + O2 acetyl phosphate + CO2 + H2O2
Catalase = 2 H2O2 → 2 H2O + O2

20. FIA peaks simultaneously obtained for SA and PY
Pre-depletion Py/SA = 6
Post-depletion SA/Py = 2.5

21. Effect of Flow Rate
0.24
1.50 1.75 1.25 1.00
FR: mL/min 0.75
Current, A
0.5 0.16
0.08
0.00
BPYO-SAA
C PYO
2000 4000 6000
Time, s
Balance between sample residence time (in reactor and
at electrode) with the rate sample dispersion

22. Analysis of bound sialic acid – Fetuin Protein
2-CH Potentiostat
W1 W2
R1 R2
Fetuin Glycoprotein
Molecular weight: 48.4 kDa
The composition of bovine fetuin
(weight %) is polypeptide 74%, Sialidase
hexose 8.3%, hexosamines
5.5%, and sialic acid 8.7%. IER
his file is in the public domain because it was solely created by NASA. NASA
copyright policy states that "NASA material is not protected by copyright unless
noted"

23. FIA peaks simultaneously obtained for SA, Fetuin and PY
PYO-SAA
PYO

24. Simultaneous analysis of total SA and PY in simulated serum sample
2-CH Potentiostat
W1 W2
R1 R2
Sialidase
Catalase
PYO
Simulated Serum
6% BSA – 140 mM NaCl
10 mg/mL Fetuin
1 mM Py – 2 mM SA

25. Simultaneous analysis of total SA and PY in simulated serum sample
1.6 6% BSA - 140 mM NaCl 5.0 mM PY
10 mg/mL Fetuin
1.4 1.0 mM PY-2.0 mM SA
1.2
Current x 10 , A
5.0 mM SA 5.0 mM SA
1.0
6
5.0 mM
0.8 PY
0.6 2.0 mM SA 2.0 mM
1.0 mM
SA
0.4 1.0 mM SA 1.0 mM PY
PY
0.2
0.0
1.8 0 1000 2000 3000 4000
1.6 FR = 2.8 mL/min FR = 1.5 mL/min FR = 2.8 mL/min
1.4
Current x 10 , A
1.2
6
1.0  PY signal diminished at reduced flow rate: More time for removal
0.8  bSA was completely hydrolyzed at the high FR
0.6
0.4
0.2
0.0
0 1000 2000 3000 4000
Time, s

26. Conclusions
 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

27. Acknowledgements
 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

29. Formation of the protective polymeric layer
Cyclic voltammograms obtained for electropolymerization of 1,3-diaminobenzne
(m-phenylenediamine) at two simultaneous Pt disc electrodes
Electrode 1 ELectrode 2
200 200
150 150
Current x 10 , A
6
100 100
50 50
0 0
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.2 0.3 0.4 0.5 0.6 0.7 0.8
Potential (E), V vs SCE Potential (E), V vs SCE
Tested against oxidizable species: Thiamine pyrophosphate (TPP), acetaminophen (4-
acetamidophenol) (AAP), and uric acid (UA) – blocked by polymer- data not shown

30. Signal Stability/Repeatability (2-ch split flow)
1 mM SA – 1.5 mL/min – 100 µL injection
0.18
Current, A
0.09
0.00
SAA - PYO
B
PYO
C
500 1000 1500 2000 2500
Time, s
- no fluctuation in split ratio
- actual ratio not critical - channels calibrated independently

31. (A)
Sample
Future Work Waste
in
 Analyzing biological samples. Serum, breast milk, formula, etc.
Single channel
response
 Expanding the current study to more comprehensive
multi-channel analysis. Time
Immobilized enzyme
reactors
(B)
Sample Waste
in
Ch-1 Ch-2 Ch-3 Ch-4 Ch-5
n channel
response
Time
Ch-1
Ch-2
Ch-3
Ch-4
Ch-5