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

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"

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