DETECTION OF GLYCATED HEMOGLOBIN USING

3-AMINOPHENYLBORONIC ACID MODIFIED GRAPHENE OXIDE

Siva Rama Krishna V., Bharadwaj Amrutur, Navakanta Bhat

Department of Electrical Communication Engineering & Center for Excellence in Nanoelectronics

Indian Institute of Science, Bangalore, India

Chakra Pani K., Sampath Srinivasan

Department of Inorganic Physical Chemistry, Indian Institute of Science, Bangalore, India

Keywords:

Graphene oxide, 3-Aminophenylboronic acid, Glycated hemoglobin, IR spectroscopy, Electrochemical

impedance spectroscopy.

Abstract:

This paper presents the chemical synthesis of 3-Aminophenylboronic acid (APBA) modiﬁed graphene oxide

(GO) and its application to the electrochemical detection of glycated hemoglobin (GHb). The compound

(GO-APBA) was synthesized by forming an amide linkage between the amino group (-NH

) of APBA and

the carboxylic group (-COOH) of GO. The compound was characterized using IR spectroscopy. Detection of

GHb was carried out using Electrochemical Impedance Spectroscopic (EIS) measurements with GO-APBA

modiﬁed glassy carbon electrode as the working electrode.

1 INTRODUCTION

Afﬁnity of glycated proteins and saccharides towards

boronic acid compounds is exploited quite exten-

sively in designing sensors for them (Takahashi and

Anzai, 2005; Fang et al., 2004; Zhao et al., 2009;

Rohovec et al., 2003; Liu et al., 2006; Son and

Yoon, 2008). The cis-diol bonds of these compounds

interact with boronic acids to form boronate esters

(Springsteen and Wang, 2002). D-glucose, the pri-

mordial saccharide in blood, undergoes a slow non-

enzymatic irreversiblereaction with hemoglobin (Hb)

to form glycated hemoglobin. The reaction hap-

pens throughout the life cycle time of erythrocyte

(RBC), which is 120 days. Measuring the concen-

tration of glycated hemoglobin as a percentage of to-

tal hemoglobin gives the average value of glucose

present in the blood over 120 days. The clinical ref-

erence range is between 4 and 20% with 4-6% being

considered as normal. Glycated hemoglobin test is

considered as gold standard for the long term moni-

toring of diabetes (Peterson et al., 1998). A dispos-

able, low cost biosensor will be handy in achieving

glycemic control, thereby reducing the risk of cardio-

vascular, retinal, renal complications that occur due to

improper control of glycemia. The present clinical

methods of measuring GHb include immunoassay,

ion-exchange chromatography (Halwachs-Baumann

et al., 1997), electrophoresis (Menard et al., 1980),

boronate afﬁnity chromatography (Fl¨uckiger et al.,

1984), and high pressure liquid chromatography

along with electrospray ionization mass spectrome-

try (Jeppsson et al., 2002). The equipments used in

these techniques are bulky and the procedures are

time consuming and are not cost effective. Frank

Frantzen et al., have reported a table top GHb mea-

surement unit based on boronate afﬁnity and col-

orimetry (Frantzen et al., 1997). This is also not cost

effective due to the use of optics. Electroanalytical

techniques have a unique advantage of having elec-

trical signals as both input and output. A dispos-

able sensor with a handheld electronic device similar

to that of a glucometer can be easily achieved using

this sensing methodology. Liu et al., have used fer-

roceneboronic acid to detect GHb(Liu et al., 2006).

The whole process is based on adsorption of Hb/GHb

onto Zirconia nanoparticles and is time consuming.

Son et al., have fabricated a lab on chip for the de-

termination of GHb (Son and Yoon, 2008). They

used m-aminophenyl boronic acid agarose beads to

bind GHb. The chip involves a complex fabrication

process. Park et al., formed a self assembled mono-

109

V. S., Amrutur B., Bhat N., K. C. and Srinivasan S..

DETECTION OF GLYCATED HEMOGLOBIN USING 3-AMINOPHENYLBORONIC ACID MODIFIED GRAPHENE OXIDE.

DOI: 10.5220/0003125401090113

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2011), pages 109-113

ISBN: 978-989-8425-37-9

 2011 SCITEPRESS (Science and Technology Publications, Lda.)

layer of thiophene-3-boronic acid to detect GHb. The

substrate used was gold which is not suited for low

cost applications. Carbon-based materials are bio-

compatible and cost effective. They have been ex-

tensively used in various electroanalytical techniques

to detect bio-molecules (Privett et al., 2008). Among

these, graphene has received much attention due to

its extraordinary electrical, thermal and mechanical

properties (Geim and MacDonald, 2007; Geim and

Novoselov, 2007). Graphene and its oxidized form,

graphene oxide (GO) have been extensively explored

for sensor applications (Schedin et al., 2007; Zuo

et al., 2009; Sun et al., 2008). GO has hydroxyl and

epoxy functional groups on the hexagonal network of

carbon atoms with carboxyl groups at the edges. The

presence of carboxylic group provides the possibility

of amide bond formation with an amine group. In this

work 3-aminophenylboronic acid (APBA) is chemi-

cally attached to carboxyl groups of GO as shown in

the Figure 1 and used in the detection of GHb.

COOH

HOOC

OHHO

CONH

COOH

HOOC

EDC

APBA

Figure 1: Schematic representation of amide linkage forma-

tion enabled by coupling reagent EDC.

2 EXPERIMENTAL

2.1 Chemicals

Natural graphite was obtained from Stratmin

graphite co., USA., N-(3-Dimethylaminopropyl)-

N’-ethylcarbodiimide hydrochloride (EDC), 3-

AminoPhenylboronic acid (APBA) were procured

from Sigma, USA. All other chemicals were procured

from Merck, India and used without further puriﬁca-

tion. All the solutions and buffers were prepared in

Millipore water with 18MΩ-cm resistivity.

2.2 Apparatus and Measurements

FTIR measurements were carried using Perkin Elmer

FT-IR Spectrometer. UV-Vis spectroscopy was car-

ried out using Perkin Elmer 35 UV-Vis spectrom-

eter. EIS measurements were carried out using

electrochemical workstation CHI660C, CH instru-

ments, USA. The electrochemical system used con-

sists of modiﬁed glassy carbon electrode as the work-

ing electrode, platinum foil as the counter electrode,

standard calomel electrode (SCE) as the reference

electrode and 0.15 mM phosphate buffer (pH 8.0) as

the supporting electrolyte.

2.3 Synthesis

Graphene oxide was synthesized from exfoliated

graphite using modiﬁed Hummers method (Ramesh

et al., 2004). Chemical modiﬁcation of GO with

APBA was carried out as follows. Required amount

of GO and EDC were added to DI water and was

stirred continuously for 36 hrs. EDC is a coupling

reagent which aids in the formation of amide bond.

After the stirring, of APBA was added to the mixture

and stirred at room temperature for another 24 hrs.

The suspension was ﬁltered and washed several times

with water and ethanol, to remove any physically ad-

sorbed APBA. The material was then dried in vacuum

using silicagel. The product was then characterized

using IR spectroscopy. For the EIS measurements, the

compound was dispersed in DI water and drop coated

onto a cleanly polished glassy carbon electrode.

2.4 Electrochemical Impedance

Spectroscopy

A glassy carbon electrode (GCE) (3mm dia.) was pol-

ished with 0.05µm alumina and washed thoroughly

with DI water. Depending on the experiment GO or

GO-APBA dispersion was drop cast on the electrode

and was allowed to dry for 2 hours. Impedance mea-

surements were carried out in 0.15 mM phosphate

buffer (pH 8.0, 3mL volume) containing 2.5 mM each

of K

Fe(CN)

and K

Fe(CN)

.The modiﬁed GCE

was used as working electrode. A potential of 0.2V

was applied between the working electrode and the

SCE. The impedance offered by the electrode for the

electron transfer was measured between the working

and counter electrodes. Freshly prepared hemoglobin

of known concentration was added in 30 µl steps to

this solution. Nyquist plots and the extraction of elec-

trical parameters were done using the software pro-

vided by the CH instruments.

BIODEVICES 2011 - International Conference on Biomedical Electronics and Devices

110

2.5 Hemoglobin Preparation

Ethylenediaminetetraacetic acid (EDTA) coated

blood sample pool of diabetic patients were collected

from Health Centre of Indian Institute of Science.

Plasma and leukocytes were removed by centrifuging

the sample at 5000 rpm for 5 min. The supernatant

was discarded. The remaining erythrocyte cells were

washed three times with 0.9% NaCl. The cells were

then lysed by adding 8 volumes of ice cold DI water

to one volume of cell solution. After lysis, the mix-

ture was centrifuged at 12000 rpm for 10 min. The

cell debris settles at the bottom of the centrifuge tube.

The supernatant was collected, labeled and stored at

-20°Cfor further use. UV-Visible spectroscopy was

carried to ﬁnd out the concentration of hemoglobin.

Based on the absorbance at 541nm Figure 2 and

Beer-Lambart’s law, the concentration of hemoglobin

was found to be 240 µM (Faniran and Shurvell,

1968). Based on the measurements carried in the

health centre, the glycated hemoglobin was found out

be 8% of the total hemoglobin.

Figure 2: UV-Visible Spectrum of Hb.

3 RESULTS & DISCUSSION

3.1 IR Spectroscopy

IR spectroscopy was carried out to conﬁrm the forma-

tion of amide bond between the amine group of APBA

and carboxylic group of GO. Figure 3 shows the IR

spectra of all the compounds. The region of interest

is between 1640 cm

−1

and 1580 cm

−1

. In the case

of GO, the band at 1617 cm

−1

corresponds to aro-

matic C=C stretching (Faniran and Shurvell, 1968).

In APBA the bands at 1608 cm

−1

and 1586 cm

−1

cor-

respond to C=C stretching as well as N-H stretching

(Faniran and Shurvell, 1968; Silverstein and Bassler,

1962). In the case of the synthesized compound new

bands appearing at 1625 cm

−1

and 1580 cm

−1

cor-

respond to amide-I band and amide-II stretching re-

spectively (Silverstein and Bassler, 1962). This con-

ﬁrms the covalent modiﬁcation of GO with APBA.

The other bands also match with the existing litera-

ture values (Silverstein and Bassler, 1962; Park et al.,

2009; Bard and Faulkner, 2006).

Figure 3: IR spectrum of (a) GO , (b) APBA (c) GO-APBA;

( Inset: Expanded region 1650 cm

−1

to 1500 cm

−1

. The

amide bands are labeled with a ‘

’).

3.2 Electrochemical Impedance

Spectroscopy: Detection of GHb

The interaction of GHb with GO-APBA compound

is shown in Figure 4. When GO-APBA modiﬁed

glassy carbon is used as working electrode GHb

gets immobilized on to the electrode surface through

afﬁnity of cis-diol bonds of glucose to boronic

acid moiety. The chemical adsorption of GHb in-

hibits the electron transfer rate of the redox cou-

ple [Fe(CN)

]

3−

/[Fe(CN)

]

4−

thereby increasing the

charge transfer resistance (R

). The Nyquist plots

are shown in Figure 5. The diameter of the semicir-

cle which is R

, increases with increase in the con-

centration of Hb. In order to eliminate the possible

reason that physisorption may play a role in increase

, an experiment was perfomed using GO modiﬁed

GCE. The corresponding Nyquist plots are shown in

the Figure 6. The variation in R

is small and is

not systematic in this case. This proves that the in-

crease in the charge transfer resistance is only due to

the chemisorption of GHb onto the electrode surface.

Hence this method can be used for detecting GHb.

3.3 Linearity

is calculated from Nyquist plots by ﬁtting the

curve with parameters in the Randles circuit shown

in Figure 7 (Bard and Faulkner, 2006). R is the so-

lution resistance and C

is the double layer capaci-

DETECTION OF GLYCATED HEMOGLOBIN USING 3-AMINOPHENYLBORONIC ACID MODIFIED GRAPHENE

OXIDE

111

CONH

COOH

HOOC

CONH

COOH

HOOC

GO-APBA

GHb

Figure 4: Schematic representation of GO-APBA and GHb

interaction.

Figure 5: Impedance data obtained for GO-APBA modiﬁed

GCE before and after the addition of Hb.

Figure 6: Impedance data obtained for GO modiﬁed GCE

before and after the addition of Hb.

tance. The values of R

with Hb were normalized to

t without Hb and are denoted by R

norm

. The varia-

tion of R

norm

with respect to the concentration of Hb

is shown in Figure 8 and it is linear. The concentra-

tion of Hb used in the present studies are in µM range

which implies that the concentration of GHb is in the

range of nM (8% of Hb).

Figure 7: Randles’ Circuit.

Figure 8: Variation of normalized charge transfer resistance

with respect to concentration of Hb.

4 CONCLUSIONS

Chemical synthesis of APBA modiﬁed GO was car-

ried out and was characterized with IR spectroscopy.

GCE surface modiﬁed with GO-APBA complex was

used to detect GHb using EIS. EIS results indicate

good sensitivity and linearity in the electrochemical

activity of GHb with respect to its concentration. En-

couraged by these results, we are currently fabricating

and testing GO-APBA modiﬁed screen printing elec-

trodes. Our efforts in this direction will result in a low

cost disposable GHb sensor.

ACKNOWLEDGEMENTS

we acknowledge sponsorship of this project by Soci-

ety for Biomedical Technology (SBMT), Bangalore,

India.We also acknowledgethe help from HealthCen-

tre, Indian Institute of Science four supporting us by

giving necessary blood samples for our experiments.

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OXIDE

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