Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/8352/12/12_chapter 2.pdf · Review of Literature 5 ... Glycation is a non-enzymatic reaction between free

Review of Literature

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2.1. Introduction

Diabetes mellitus is a chronic metabolic disorder characterized by rise in blood glucose

level called “hyperglycaemia” (Vinik and Flemmer, 2001). Monitoring the blood glucose

level is required for diabetic patients, to maintain it within a normal range, and thereby

reducing the risk of severe complications such as kidney failure, blindness and nerve

damage (Genuth et al., 1998). The worldwide prevalence of diabetes in 2000 was

approximately 2.8% and is estimated to grow by 4.4% by the end of 2030. This translates

to a projected rise of diabetes from 171 million in 2000 to well over 350 million in 2030

(Sultanpur et al., 2010). Hence, there is an urgent need for improved treatment of

hyperglycaemia and other risk factors associated with this metabolic syndrome. If left

untreated or improperly managed, diabetes can result in a variety of complications,

including heart disease, kidney disease, eye disease, neuropathy, gangrene, gastroparesis,

blindness, erectile dysfunction, impotence and nerve damage. Poor blood glucose control

also increases the risk of short-term complications of surgery such as poor wound

healing. Historically, measurement of glucose levels has been the method, universally

used to diagnose diabetes. Laboratory methods such as fasting plasma glucose (FPG) or

2-h plasma glucose (2HPG) level have been used for this purpose. However, this

approach still suffers from the same problems and difficulties associated with glucose

biosensors such as the need for fasting, biological variability and the effects of acute

perturbations (e.g., stress- or illness related) on glucose levels (Sheikholeslam et al.,

2011). Also, blood glucose reflects a fluctuating glucose level, and it is affected by daily

diet and requires frequent measurements. Currently, portable-sized glucose measurement

devices are being designed for self use, low costs, and quick results. Some of them adapt

invasive measurement methods for blood extraction, i.e., use a piercing component of a

microneedle or a lancet with a disposable strip for sensing and a handheld fixture for

readout (Yang et al., 1998; Wang and Zhang, 2001; Mukerjee et al., 2004).

The glycated hemoglobin (HbA1C) level, defined as the ratio between HbA1C


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concentration and total Hb concentration, is considered to be a very useful diagnostic

marker for diabetic patient in addition to the measurement of the glucose level (John,

2003). Also, the use of this approach solves many of the problems associated with FPG

or 2HPG methods based on glucose measurements such as no need for fasting,

substantially less biological variability and relative insensitivity of HbA1C levels to acute

perturbations (Sheikholeslam et al., 2011). Compared with blood glucose, HbA1C level is

a more stable diagnostic index and is recommended as a baseline test for diabetes. HbA1C

is the stable glucose adduct to the N-terminal group of the β-chain of HbA0. Since it

reflects the average blood glucose level over the preceding 2-3 months and is not affected

by the daily fluctuation of the glucose level, HbA1C level provides a more accurate index

for diagnosis and long term control of the disease. HbA1C level correlates linearly with

mean blood glucose (MBG). 1% change of HbA1C level reflects a fluctuation of MBG

concentration by about 2 mM (MBG (mmol/L) = (1.98.HbA1C)-4.29). The clinical

reference range of HbA1C level is 5-20%, with 4.0-5.7% considered as normal. Since the

lifetime of Hb in blood is approximately 2-3 months, the HbA1C level provides a good

indication of the glucose level over this period of time. Consequently, the measurement

of HbA1C level is important for the long-term control of the glycemic state in the diabetic

patients (Jeppsson et al., 2002).

Normal adult Hb consists primarily of Hbs A (90-95%), A2 (2-3%), F (0.5%), Ala

(1.6%), Alb (0.8%) and A1c (3-6%). HbA1C are the minor Hb molecules separable by

chromatographic techniques into three major components: Ala, Alb, and A1c.

Hemoglobin Al refers to a combination of these three components (Bunn et al., 1979).

Diabetes Control and Complication Trial (DCCT), has demonstrated that 10% stable

reduction in HbA1C determines a 35% risk reduction for retinopathy, a 25-44% risk

reduction for nephropathy and a 30% risk reduction for neuropathy (Calisti and Tognetti,

2005).

Underlying principle

In the normal 120-day lifespan of the red blood cell (RBC), glucose molecules react with

Hb, forming HbA1C. In individuals with poorly controlled diabetes, the quantity of


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HbA1C is much higher than in healthy people.

Once a Hb molecule is glycated, it remains that way. A buildup of HbA1C within

the RBC, therefore, reflects the average level of glucose to which the cell has been

exposed during its life-cycle. Measuring HbA1C assesses the effectiveness of therapy by

monitoring long-term serum glucose regulation. The HbA1C level is proportional to

average blood glucose concentration over the previous four weeks to three months. Some

researchers state that the major proportion of its value is related to a rather shorter period

of two to four weeks (HbA1C fact sheet, http://www.med.umich.edu/mdrtc/cores/

ChemCore/hemoa1c.htm.).

The 2010 American Diabetes Association Standards of Medical Care in Diabetes

added the A1c ≥ 48 mmol/l (≥ 6.5%) as another criterion for the diagnosis of diabetes

(Executive Summary, 2010).

Glycation is a non-enzymatic reaction between free aldehyde group of glucose

and free amino groups of proteins (here β chain of Hb). A labile aldiminic adduct (Schiff

base) forms at first, then, through a molecular rearrangement, a stable ketoaminic product

slowly accumulates (Fig. 1). In Hb, the preferential glycation site is the amino-terminal

valine of the β chain of the globin (about 60% of glycosylated globin). Other sites are:

lysine 66 and 17 of the β chain, valine 1 of the α chain. The term HbA1C refers to the Hb

fraction of the glucose bound stably (ketoamine) to beta terminal valines.

2.2. Fructosyl amino acid oxidase

Fructosyl amino acid oxidases (FAOs) (fructosyl amine: oxygen oxidoreductases or

fructosylamine: oxygen oxidoreductases, EC 1.5.3.X) are a group of enzymes that

catalyses the oxidative deglycation of fructosyl amino-acids, which are formed by non-

enzymatic glycation reaction, in which biological amines (i.e., free amino acids, amino

acid residues of proteins, and amino groups of nucleic acids) react with reducing sugars

at an early stage of the Maillard reaction and are the model compounds for glycated

proteins, yielding corresponding amino acids, glucosone and H2O2 (Horiuchi et al., 1989;

Horiuchi and Kurokawa, 1990, Sakai et al., 1995; Yoshida et al., 1995; Takahashi et al.,

http://www.med.umich.edu/mdrtc/cores/%20ChemCore/hemoa1c.htm

http://www.med.umich.edu/mdrtc/cores/%20ChemCore/hemoa1c.htm


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Fig. 1 Chemical reactions involved in glycation of hemoglobin

1997a). The resultant Amadori compounds repeat dehydration and condensation to form

stable compound called advanced glycation end products (AGEs) (Ulrich and Cerami,

2001). Glycation affects the function of the proteins in vivo, and protein cross-links with

advanced glycation end product cause the development of diabetic complication and

aging (Schmidt et al., 1994; Singh et al., 2001; Goldin et al., 2006). Thus, it is important

to investigate the intervention against the Maillard reaction. Most FAOs show this type of

oxidation and require FAD as cofactor, but the enzyme found in Pseudomonas sp. has a

somewhat different reaction mechanism, utilizing Cu2+

(Saxena et al., 1996). This

enzyme produces fructosamine residue from fructosyl-amino acid, indicating that C-N

bonds suffered from the cleavage are different in these two types of enzymes. The first

FAOs was isolated by Horiuchi et al. (1989) from Corynebacterium sp. 2-4-1.

2.2.1. Sources of FAO

2.2.1.1. Fungi

The presence of fungal FAOs has been reported most extensively, in strains of various

genera Achaetomiella, Achaetomium, Acremonium, Aspergillus, Chaetomium,

Coniochaeta, Eupenicillium, Fusarium, Gelasinospora, Gibberella, Microascus,

Fructosyl valine

or ketomine

Aldimine

(labile)

Glucose

β-val-NH2 +

H O

C

H-C-OH

HO-C-H

H-C-OH

H-C-OH

H-C-OH

H

CH(CH3)2

CH2NHCH

C=O

HO-C-H

H-C-OH

H-C-OH

CH2OH

CO2H

H N-Val-β

C

H-C-OH

HO-C-H

H-C-OH

H-C-OH

H-C-OH

H


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Penicillium, Thielavia and Ulocladium (Staniford et al., 1993; Yoshida et al., 1995;

Takahashi et al., 1997a, b; Sakai et al., 1999, Fujiwara et al., 2006; Hirokawa et al.,

2003). Akazawa et al., 2004 studied the application of fungal FAOs in the clinical

diagnosis of diabetes mellitus (Yoshida et al., 1996), since the amounts of glycated

proteins such as Hb and albumin in blood reflect the level of blood glucose, and

fructosyl-amino acids are model compounds for the glycated proteins. They also found

that FAO is a peroxisomal enzyme and widely distributed in filamentous fungi. Thus, it

was concluded that FAO plays an important role in fungal cells. However, the definite

physiological role of the enzyme is still unknown.

2.2.1.2. Bacteria

FAO has also been found in the bacterial genera Arthrobacter, Corynebacterium,

Klebsiella, and Pseudomonas (Ferri et al., 2004; Ferri et al., 2005; Gerhardinger et al.,

1994; Saxena et al., 1996).

2.2.1.3. Yeast

FAO occurred in Yeast genera Debaryomyces and Pichia (Sode et al., 2001a; Staniford

et al., 1993). There is no report for the existence of FAO in mammalian organisms.

2.3. Applications of FAOs

FAOs have been explored for use in diabetes diagnosis, detergents and food processing.

The application for examination of diabetes was first realized around the time, enzyme

was discovered (Tatsuo and Yoshiko, 1987), and now FAO-based assays have become a

“gold standard” diagnostic tool (Hirsch and Brownlee, 2005) for determining the levels

of HbA1C, an important indicator for diabetes patients (Kobold et al., 1997; Hirokawa et

al., 2004; Nakamura et al., 2007). In this scheme, FAO reacts with fructosyl-valine (FV),

which is released from the N terminus of HbA1C upon protease treatment. The resulting

H2O2 is measured by peroxidase reaction. Several research groups have also developed

electrode-based biosensor systems which are capable of measuring FV, based on the

FAO from Pichia sp. N1-1 (Fang et al., 2009; Ogawa et al., 2002; Tsugawa et al., 2000;

Tsugawa et al., 2001) or Arthrobacter sp. FV1-1 (Sakaguchi et al., 2003).


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Detergent additive is a second promising area of application for FAOs, as

evidenced by a recently filed patent application (O'Connell et al., 2008), which relies on

the ability of FAOs to enzymatically degrade amadori products and also release in situ

hydrogen peroxide, which in itself is a bleaching agent. A preliminary performance test

of an engineered amadoriase II on American gravy stains has already been demonstrated

(Zheng et al., 2009).

While the Maillard reaction also exerts an important influence on the food

properties, the commercial use of FAOs in the food processing and quality control

remains to be seen (Kato et al., 1998; Ishida et al., 2002; Yagi et al., 1997). Lastly, as

amadori compounds and advanced glycation end products (AGEs) have been implicated

in aging and several diseases, the therapeutic use of this class of enzymes should be

worth exploring. For all these applications, FAOs that can react directly with lager

glycated proteins instead of smaller glycated amino acids or short peptides would be of

greater value, and therefore it is interesting to engineer and expand the limited

accessibility of the substrate binding sites of FAOs.

2.4. Various methods for HbA1C determination

A number of chromatographic methods are currently available for the determination of

HbA1C level such as electrophoresis/electroendosmosis (Hageman and Kuehn, 1977), ion

exchange chromatography (Goldstein et al., 1986), high performance liquid

chromatography (HPLC) (Brunnekreeft and Eidhof, 1993; Ellis et al., 1984; Turpeinen et

al., 1995), boronate affinity chromatography (John, 1997), immunoassay (Turner et al.,

1999) and liquid chromatography associated in tandem with mass spectroscopy (LC-

MS/MS) (Jeppsson et al., 2002) prior to fluorometric (Gallop et al., 1981) and

colorimetric (Fluckiger and Winterhalter, 1976) quantification of HbA1C.

2.4.1. Capillary electrophoresis

Principle: Basically, two possibilities exist for separation of HbA1C in capillary

electrophoresis (CE) according to charge-to-mass ratio. Firstly analysis as cations in

acidic buffers of pH below pI of Hb, which is approximately 7.0. Separation of


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hemoglobins A1C and A0 occurs due to a charge difference coming from elimination of

one positively charged amino group in the HbA1C molecule by attachment of glucose

moiety. Secondly, Hb analysis as anions in alkaline conditions with selectivity to HbA1C

induced by a cis-diol interaction of its glucose unit with a borate anion from background

electrolyte (BGE) (Fig. 2).

CE includes method for separating Hb derivatives and Hb variants making use of

a dynamic coating technique that allowed rapid separation of Hb variants and derivatives.

CE was performed on a Beckman P/ACE System 5000. Before sample injection, the

capillary is first rinsed with initiator solution (containing a polycation, albumin, pH 4.5),

followed by buffer solution containing a polyanion (chondroitin sulfate, pH 4.5), at the

same pressure. Sample is injected, followed by an injection with buffer solution to rinse

the outside of the capillary. The capillary is then transferred to another vial containing

buffer solution, in which the electrophoresis is performed. Negatively charged molecules

(chondroitin sulfate, pH 4.5) in the buffer solution bind to Hb. Electrophoresis is

performed with the negative electrode at the detector site. Detection is executed with a

UV/VIS absorbance detector at 415 nm. Peak integration for peak area measurement is

performed by a Beckman System Gold chromatography data system. Stability and

coefficient of variance of the method were found to be 20 days and 1.4 to 3.7%. No

interferences were observed from carbamylated or acetylated Hb or from the labile

HbA1C fraction. A good linear correlation coefficient with r=0.98 was found with Bio-

Rex 70 HPLC (Doelman et al., 1997).

A CE method was also developed for measurement of HbA1C by exploring the

potential of cis-diol interactions for separation of HbA1C (Koval et al., 2011). First, a CE

analysis was performed with a sodium tetraborate buffer (pH 9.3) as background

electrolyte in a neutrally coated capillary (Shen and Smith, 2000). HbA1C was separated

from HbA0 due to specific interactions of borate anions with the cis-diol pattern in the

saccharide moiety of glycohemoglobin.

Merit: Easy experimental setup.


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Demerit: Not much selective because of some interfering species among the multitude of

known Hb variants.

Fig. 2 Schematic representation of determination of HbA1C by capillary electrophoresis

2.4.2. HPLC method

Principle: HPLC typically utilizes different types of stationary phases, a pump that

moves the mobile phase(s) and analyte through the column, and a detector to provide a

characteristic retention time for the analyte. For HbA1C, automated HPLC employs a

weak cation exchange column. The support material is silica with carboxymethyl

functional groups. The analyzer forms a stepwise gradient composed of three phosphate

buffers of increasing ionic strength. The system is optimized for quantitation of HbA1C,

while HbA2 coelutes with HbA2. Quantitation is based on comparison of the absorbance

values at 415 and 690 nm. Before analysis, the samples are incubated at 37oC for 30 min.,

to remove the labile fraction (Fig. 3).


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International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)

Working Group developed HPLC, as a standard reference method for HbA1C. Different

values for HbA1C can be obtained when the same blood samples are measured,

depending on the chromatographic system, e.g. the kind of resin, lotto-lot variation of

resins, column size, buffer composition and elution times. The peak considered to be

HbA1C may contain variable proportion of substances which have the same elution

behaviour as HbA1C but are not HbA1C according to definition, since they lack

immunological activity or do not bind during affinity chromatography.

In an automated HPLC assay 45-min. in acetate buffer (pH 5.5) is required to

eliminate labile HbA1C. The chromatographic column contained “polyCAT” (a weak

cation-exchanger, polyaspartic acid linked to silica). Run time was 14 min per sample

(Ellis et al., 1984).

HbA1C and total Hb were simultaneous determined by isotope dilution and

HPLC-inductively coupled plasma mass spectrometer (ICP-MS). HbA1C was separated

by cation exchange chromatography, employing a Mono S column. Then, the detection

was carried out by measuring the Fe contained in the heme-group of the protein, using an

ICP-MS as the Fe selective detector. Thus, speciation of glycated and non-glycated

haemoglobin was investigated with detection limits around 1 mg mL-1

of protein (Busto

et al., 2008).

Merits: It provides good precision and long-term stability and results correlated very

well with by other ion-exchange method. Also, it gas high resolution, sensitivity,

reproducibility, accuracy and automation.

Demerits: Historically HbA1C is defined as a certain peak in an HPLC system, but this is

no longer scientifically acceptable, as it lacks specificity. Different values for HbA1C in

the same blood were obtained depending on the conditions used, because the HbA1C

peaks contain different kinds and amounts of substances that are not HbA1C. Also, HPLC

require special instrumentation and training and result in patterns that are relatively

complex.


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Fig. 3 Schematic representation of determination of HbA1C by HPLC

2.4.3. Capillary isoelectric focusing

Principle: Capillary isoelectric focusing (CIEF) separates the analytes according to their

pI differences. The pI of HbA1C is slightly shifted to the acidic direction with respect to

HbA0 due to one amino group blocked by the glucose moiety. This subtle pI difference

underscores the need for a very well-tuned CIEF method to separate both species.

CIEF methodology for the analysis of HbA1C, in dimethylpolysiloxane coated

fused-silica capillaries was carried out, using a narrow pH ampholyte mixture. In the

focusing procedure, a 0.100 mol L-1

phosphoric acid solution was used as analyte and a

0.040 mol L-1

NaOH solution was used as catholyte. Chemical mobilization was used

which allowed the complete baseline resolution of the HbA1C in less than 8 min.

Correlation coefficient was found to be 0.872 (n=31) with standard cation-exchanger

columns (Jager and Tavares, 2003).

Another CIEF method, exploited a difference in pI values of HbA0 and HbA1C,

performed with a range of carrier ampholytes commercially available like Servalyt pH 6-


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8, Biolyte pH 6-8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by

preparative fractionation of Servalyt pH 4-9 carrier ampholytes (Koval et al., 2011). They

adopted optimization of experimental parameters in CIEF by Beckman Coulter (Mack et

al., 2009). The relative standard deviation (RSD) values of migration time in the interday

study were initially 4.3-5.7% before dropping to 0.1-0.3% for pI after electropherogram

normalization with the aid of pI markers.

Merits: Outstanding separation power, due to the pH gradient being flattened by a

narrow pH cut of carrier ampholytes. Efficient, expressive, economic (no sophisticated

equipment required), easy (clear, one-dimensional separation principle), fast, sensitive.

Separation in CIEF does not require denaturation of proteins, thus any kind of subsequent

investigations, such as activity staining (e.g. to find separated enzymes) or antibody

detection, is not hindered.

Demerits: More experimentally demanding.

2.4.4. Boronate affinity chromatography

Principle: It is based on use of a “biological interaction” for the separation and analysis

of specific analytes within a sample. For HbA1C, boronate affinity chromatography is a

glycation specific method based on boronate binding to the unique cis-diol configuration

formed by stable glucose attachments to Hb. This method thus measures all four stable

species, altogether. The combined measure of only the four stable species has been

referred to as "Total HbA1C" or by some as "True HbA1C ". Since only two fractions are

present in these methods (glycated and non-glycated), the glycated portion is compared to

the total and results are expressed as % HbA1C (Fig. 4).

The technique was first used for the determination of glycohemoglobin (Mallia et

al., 1981). A low-performance agarose gel was used as the support and absorbance

detection was recorded at 414 nm used to quantify the retained and non-retained Hb

fractions in human hemolysate samples. Elution was performed by passing through the

column a soluble diol-containing agent (i.e., sorbitol) that displaced the retained

glycohemoglobin from the column. After the initial report by Mallia et al., similar low-


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performance methods were reported or evaluated by other groups (Gould et al., 1982;

Klenk et al., 1982; Fluckiger et al., 1984; Johnson and Baker, 1988). The same approach

has since been adapted for use in HPAC and HPLC-based systems (Gould et al., 1982;

Hjerten and Li, 1990; Kitagawa and Treat-Clemens, 1991; Singhal and DeSilva, 1992).

Merits: Analytical advantages of this method are low susceptibility to variations in

temperature and pH. It is the only interference-free method for quantifying HbA1C. This

method is specific for all HbA1C species regardless of charge. It provides very good

precision and accuracy.

Demerit: Boronate affinity does not measure A1a1, A1a2, A1b or the labile fraction.

Fig. 4 Chemical reactions involved in determination of HbA1C by affinity

chromatography

2.4.5. Electrospray ionisation mass spectrometry (ESI-MS)

Principle: It is based on formation of polyprotonated or polydeprotonated ions of intact

analyte molecules that are produced from a fine spray of an aqueous solution of the

analyte, assisted by a strong electrical field at atmospheric pressure. The ions have low

internal energy and are not prone to fragmentation. The measurement of mass-to-charge

values of these multiple charged ions gives the inherent molecular mass of the analyte

molecules. The ion currents produced by ESI are dependent on the analyte concentration

(Fig. 5).


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A method was developed for quantification of HbA1C, based on the specific N-

terminal residue of the Hb β-chains. Enzymatic cleavage of the intact Hb molecule with

endoproteinase Glu-C was optimized to obtain the β -N-terminal hexapeptides of HbA1C

and HbA0. These peptides were first separated by reversed-phase HPLC and quantitated

by ESI-MS. The stability, reproducibility and repeatability of the total analytical system

were found to be 1 month with coefficient of variance 2.0%, 1.2%, and 2.5% (n=3) for

250 uses. This demonstrates a very good reproducibility of the system. This method

distinguished the smaller N-terminal parts of the β-chains of HbA1C from those of the

HbA0 molecules, thus avoiding the heterogeneity created by modifications of other

glycation sites of the Hb molecule. With these peptides and hyphenated separation

techniques, it was possible to overcome the insufficient resolution of currently used

protein separation systems for HbA1C (Kobold et al., 1997).

Merit: Relatively new mass spectrometric technique suited for the analysis of polar

biomolecules of low and high molecular mass. ESI-MS is ideally suited for an on-line

coupling with HPLC, and the combination of these techniques might become a powerful

instrument for the development of new reference methods for a variety of analytes.

Demerit: High cost of ESI-MS equipment.

Fig. 5 Schematic representation of determination of HbA1C by Electrospray ionization

mass spectrometry


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2.4.6. Colorimetric method

Principle: Colorimetric approach of detection of HbA1C is based on conversion of

hexose moiety of HbA1C to 5-hydroxymethylfurfural (HMF) by heating at 100oC in the

presence of a weak acid (oxalic acid). HMF then reacts with 2-thiobarbituric acid (TBA),

and the resulting color is measured colorimetrically.

In the colorimetric method, glucose moiety of HbA1C is converted to 5-

hydroxymethylfurfural (5-HMF) by heating with oxalic acid for 60 min. in an autoclave

at 124 kPa (18 lb/in.2). The adduct thus formed by reaction of TBA with HMF was

measured photometrically and results were expressed either as nanomoles of HMF or as

fructose equivalents. Within-assay and between-assay coefficients of variation were <2%

and <3%, respectively. The method showed a very good correlation coefficient with

r=0.98 (n=50) of the present method and as analyzed by liquid chromatography (Parker et

al., 1981).

In another colorimetric assay for, HbA1C was formed from HbA by the chemical

condensation of a molecule of glucose specifically with the NH2-terminal of the chain of

HbA (Bunn et al., 1975). In this method, the carbohydrate moiety was cleaved by acid

hydrolysis to yield 5-HMF, which is subsequently complexed with TBA. The resulting

chromogen has a maximal absorbance at 443 nm. The normal mean (±SD) for HbA1C

was found to be 5.85% ± 0.79 for males (70) and 5.88% ± 0.79 for females (n=30). A

good correlation coefficient with r=0.92 was found with microcolumn test kit. This

colorimetric method was able to meet many criteria of an ideal laboratory test

(Karunanayake and Chandrasekharan, 1985).

Merits: More rapid and reproducible than other methods and correlates well with

standard liquid-chromatographic method. This method is cheap and not laborious. No

specialized equipments and expertise is required and can even be automated, if required.

Demerits: Most manual methods for quantitating HbA1C based on this approach require

very long incubation period (approx. 3-16 h) for the production of HMF. The yield of 5-

HMF is low and depends critically on the protein concentration and results may vary.


19

2.4.7. Fluorometry

Principle: The method is based upon periodate oxidation of the carbohydrate moieties

present on both the - and Ԑ -amino groups of globin. The formaldehyde product is

measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the

condensation of formaldehyde with acetylacetone and ammonia.

A sensitive fluorometric method was based on periodate oxidation and by

measurement of the formaldehyde produced, for the measurement of HbA1C. Thus, it was

necessary to remove all periodate sensitive formaldehyde yielding compounds. This was

achieved by preparing globin with acid tetrahydrofuran (THF), as both the hemin and the

acetone usually used for the preparation of globin were found to interfere with the

fluorometric determination of formaldehyde (Gallop et al., 1981). The formaldehyde

liberated was measured spectrofluorometrically with the DDL assay (Nash, 1953). The

linearity of glycoglobin was found to be in the range, 1 to 5 mg. The method was

rigorously designed to assay HbA1C levels and to give a direct measure of the number of

glycogroups per mg of Hb. It requires only 1 mg of protein and may also be used to

determine the extent of the nonenzymatic glycosylation of other proteins. Coefficient of

variance and correlation coefficient were found to be 1.3-3.8% and 0.95 respectively.

2.4.8. Turbidimetric Immunoassay

Principle: The method utilizes the interaction of antigen and antibody to directly

determine HbA1C in whole blood.

A light-scattering competitive immunoassay for HbA1C was developed and

compared to an automated clinical chemistry analyzer (Vucic et al., 1998). The method is

based on a combination of the latex-enhanced competitive turbidimetric immunoassay for

HbA1C and colorimetric determination of total Hb within the same sample. After

hemolysis combined with enzymatic cleavage, HbA1C and total Hb were determined by

immunoassay and colorimetry, respectively, and final result was calculated as a

percentage from the two measured parameters ratio. The enzymatic cleavage led to

release of -N-terminal fragments for reaction with a fixed amount of latex-bound


20

monoclonal antibodies. Antibody excess was then removed by agglutination with a

synthetic polyvalent complex of -N-terminal fragments. The reaction was monitored by

turbidimetry with light-scattering signal (absorbance at 540 nm) inversely related to the

amount of HbA1C in the sample. Total Hb was measured in the same hemolysate by the

alkaline hematin method (absorbance at 410 nm). The within and between batch

coefficient of variance, correlation coefficient with standard ion-exchange

chromatography (n=117) and stability of assay were found to be <5%, r=0.989 and 2

months respectively.

A turbidimetric immunoassay was developed in which mouse antihuman A1C

monoclonal antibodies were added to whole blood, latex A1C antihuman A1C antibody

complex was formed. Agglutination occured when goat antimouse IgG polyclonal

antibody interacts with the monoclonal antibody. The amount of agglutination measured

as absorbance was proportional to the amount of A1C absorbed onto the surface of latex

particles. Multipoint calibration was used for preparing calibration curves. The method

was compared with standard HPLC method on Biorad variant with r=0.986 (n=15) and

an intraclass correlation value of 0.993 was obtained. Stability of the assay was found to

be 15 days (Lakshmy and Gupta, 2009).

2.4.9. Automated HbA1C immunoassay

In the method, whole blood is hemolyzed using an automatically onboard analyzer. The

results of imprecision studies showed comparable performance of both procedures,

coefficients of variation being slightly higher with the automated procedure (2.2-2.7%

versus 1.7-2.1% in within-run experiments, and 2.4-3.5% versus 2.1-3.0% in between-run

experiments). Comparison of results in 100 fresh samples showed acceptable correlation

between the two procedures (R2=0.94, y=0.98x+0.43). Sedimentation of whole blood in

sample tubes prior to automatic hemolysis did not alter the results (Schneider et al.,

2002).

A heterogeneous affinity-matrix based immunoassay was developed for the

determination of HbA1C. The method is based on an immunoenzymometric assay

(IEMA), where the glycated pentapeptide Val-His-Leu-Thr-Pro (VHLTP), as HbA1C


21

analogon was immobilized either to the surface of a microtiter plate by adsorption or to

an amino-modified cellulose membrane by covalent linkage. The immobilized analogon

competed together with the HbA1C in the sample for the antigen binding sites of the anti-

HbA1C antibodies. Glucose oxidase-labeled antibodies were used to indicate the antigen

antibody reaction indirectly and enzyme activity was detected optically. A linear range

was obtained between 5-50 % HbA1C (Stöllner et al., 2001).

Demerit: The automated procedures developed for the autoanalyzer require a manual

extraction into a suitable solvent and a preliminary purification of the extract, before it is

introduced into the instrument. Thus, the autoanalyzer procedures are only partly

mechanized.

2.4.10. Enzymatic Assays

The method is based on structural difference of HbA1C and HbA0. Lysed blood samples

are subjected to proteolytic digestion. Glycated valines are released and serve as substrate

for fructosyl valine oxidase. The produced hydrogen peroxide is measured using a

horseradish peroxidase-catalyzed reaction with a chromogen.

2.5. Biosensors

A biosensor is a system of two transducers, biochemical and physical, in intimate contact

or in close proximity with each other that relates the concentration of an analyte to a

measurable signal. It uses specific biochemical reactions mediated by isolated enzymes,

immunosystems, tissues, organelles or whole cells to detect chemical compounds usually

by electrical, thermal or optical signals. Biosensors are used for analysis of bio-material

samples to gain an understanding of their bio-composition, structure and function and

also provide selective identification of toxic chemical compounds at ultratrace levels in

industrial products, chemical substances, environmental samples (e.g., air, soil, and

water) or biological systems (e.g., bacteria, virus, or tissue components) for biomedical

diagnosis.

The history of biosensors started in 1962, with the development of enzyme

electrodes by scientist Leland C. Clark (Clark and Lyons, 1962), who is known as the


22

father of Biosensors. He carried out this by using an enzyme transducer. Clark’s oxygen

electrode was the enzyme glucose oxidase, entrapped in dialysis membrane. The method

of detection was based on the decrease in oxygen concentration that is proportional to

glucose concentration. Clark's ideas became commercial reality in 1975 with the

successful re-launch (first launch 1973) of the Yellow Springs Instrument Company

(Ohio) glucose analyser based on the amperometric detection of hydrogen peroxide.

Since then, the designs and applications of biosensors in analytical chemistry have

developed rapidly for the last thirty years and communities from various fields such as

very large scale integration (VLSI), physics, chemistry, and material science have come

together to develop more sophisticated, reliable, and mature biosensing devices.

Applications for these devices are in the fields of medicine, agriculture, biotechnology as

well as the military and bioterrorism detection and prevention.

Fig. 6 List of possible biological and sensor elements in biosensor


23

2.5.1. Basic principle of biosensors

A biosensor is generally defined as an analytical device which converts a biological

response into a quantifiable and processable signal (Lowe, 1984). Fig. 7 shows

schematically the parts comprising a typical biosensor: a) bioreceptors that specifically

bind to the analyte; b) an interface architecture where a specific biological event takes

place and gives rise to a signal picked up by c) the transducer element; the transducer

signal (which could be anything from the in-coupling angle of a laser beam to the current

produced at an electrode) is converted to an electronic signal and amplified by a detector

circuit using the appropriate reference and sent for processing by, e.g., d) computer

software to be converted to a meaningful physical parameter describing the process being

investigated; finally, the resulting quantity has to be presented through e) an interface to

the human operator. Biosensors can be applied to a large variety of samples including

body fluids, food samples, cell cultures and be used to analyze environmental samples.

Fig. 7 Mechanism of action of a biosensor

2.5.2. Classification of biosensor

2.5.2.1. Classification of biosensor based on evolution

A. First generation biosensors are those in which the normal product of the reaction

diffuses to the transducer and causes the electrical response. It works by means of the

direct detection of electroactive species that are enzymatically produced or consumed


24

(Dicks et al., 1989). A good example is the traditional glucose sensor, which can detect

the hydrogen peroxide produced in the oxidation process catalyzed by a glucose oxidase

enzyme.

B. Second generation biosensors involve specific 'mediators' between the reaction and

the transducer in order to generate improved response. It makes use of an artificial

electron carrier, or mediator, in place of oxygen, which shuttles the electron involved in

the redox process from the active center of the enzyme to the electrode or vice versa.

Both mediator and enzymatic substrate must be in the analytical solution.

C. Third generation biosensors are those in which the reaction itself causes the

response and no product or mediator diffusion is directly involved. Interest about third

generation biosensors grew a few years ago when it was found that direct

electrochemistry with redox proteins is possible without using a co-substrate (Armstrong

et al., 1987). The foremost work in this field used mercury electrodes, onto which

enzyme adsorption was strong and lead to molecule denaturation.

2.5.2.2. Classification of biosensor based on transducer element involved

A. Electrochemical transducers: In this type of sensor, sensing molecules are either

coated onto or covalently bonded to a probe surface. The sensing molecules react

specifically with analyte, sparking an electrical signal proportional to its concentration.

The underlying principle for this class of biosensors is that many chemical reactions

produce or consume ions or electrons, causing some change in the electrical properties of

the solution that can be sensed out and used as a measuring parameter. These can be

classified based on the measuring electrical parameters as conductometric,

amperometric, and potentiometric (Fig. 8). Amperometric and potentiometric

transducers are the most commonly used electrochemical transducers.

(i) Amperometric. Amperometric is a high sensitivity biosensor that can detect

electroactive species present in biological test samples. The potential between the two

electrodes is set and the current produced by the oxidation or reduction of electroactive

species is measured. The height of the peak current is directly proportional to the concen-


25

Fig. 8 Types of different biosensors

-tration of the electroactive material. Most electrodes are made of metals like platinum,

gold, sliver, and stainless steel, or carbon-based materials that are inert at the potentials at

which the electrochemical reaction takes place. This mode is also known as voltammetric

(Fig. 9).

(ii) Potentiometric. These involve the measurement of the emf (potential) of an

electrochemical cell at very low current. The emf is proportional to the logarithm of the

concentration of the substance being determined. The working principle relies on the fact

that when a ramp voltage is applied to an electrode in solution, a current flow occurs

because of electrochemical reactions. The voltage at which these reactions occur,

indicates a particular reaction and particular species (Fig. 10).

(iii) Conductometric. Most electrochemical reactions involve a change in the

composition of the solution resulting in production of ions or electrons. This will

normally result in a change in the electrical conductivity of the solution, which can be

measured electrically. Conductance measurements have relatively low sensitivity. The

electric field is generated using a sinusoidal voltage (ac), which helps in minimizing

undesirable effects such as Faradaic processes, double layer charging, and concentration

polarization.


26

(iv) FET-based sensors. Miniaturization can sometimes be achieved by constructing one

of the above types of electrochemical transducers on a silicon chip-based field-effect

transistor (FET). FET is a potentiometric devices based on the measurement of potential

at an insulator-electrolyte interface. The metal gate of a FET can be substituted by an ion

selective membrane to make a pH transducer (pH ISFET). ISFETs having an ion-

sensitive surface. The surface electrical potential changes when the ions and the

semiconductor interact. This change in the potential can be subsequently measured. The

Fig. 9 Schematic representation of an amperometric biosensor

Enzymes

Potential Response

Analyte

Signal

Amplifier

Biocompatible layer

Transducer


27

Fig. 10 Schematic representation of potentiometric biosensor

ISFET can be constructed by covering the sensor electrode with a polymer layer. This

polymer layer is selectively permeable to analyte ions. The ions diffuse through the

polymer layer, causing a change in the FET surface potential. This method has mainly

been used with potentiometric sensors, but could also be used with voltammetric or

conductometric sensors. This type of biosensor is primarily used for pH detection (Fig.

11).

B. Optical Sensors: In optical biosensors, the output transduced signal that is measured

is light. The biosensor can be made based on optical diffraction or electro-

chemiluminescence. Since they are non-electrical, optical biosensors have the advantages

of lending themselves to in vivo applications and allowing multiple analytes to be

detected by using different monitoring wavelengths. The versatility of fiber optics probes

is due to their capacity to transmit signals that reports on changes in wavelength, wave

propagation, time, intensity, distribution of the spectrum, or polarity of the light. In

n source n drain gate

Biocatalytic

membrane Ion selective

membrane

H+ sensitive

membrane

p silicon

Encapsulant

Reference

electrode

+

-


28

general, acquisition of the signal from these devices is accomplished through flexible

cables, which can transmit light to the biological component.

Fig. 11 Schematic outline of biosensors based on ion-selective field effect transistor

C. Piezoelectric Sensors: Guilbault and Montalvo (1969) were the first to detail a

potentiometric enzyme electrode. These devices involve the generation of electric

currents from a vibrating crystal. In this mode, sensing molecules are attached to a

piezoelectric surface-a mass to frequency transducer-in which interactions between the

analyte and the sensing molecules set up mechanical vibrations that can be translated into

an electrical signal proportional to the amount of the analyte. Example of such a sensor is

quartz crystal micro or nano balance (Fig. 12).

D. Acoustic. Electroacoustic devices used in biosensors are based on the detection of a

change of mass density, elastic, viscoelastic, electric, or dielectric properties of a

membrane made of chemically interactive materials in contact with a piezoelectric

material. Bulk acoustic wave (BAW) and surface acoustic wave (SAW) propagation

transducers are commonly used. In the first, a crystal resonator, usually quartz, is

connected to an amplifier to form an oscillator whose resonant frequency is a function of

the properties of two membranes attached to it. The latter is based on the propagation of

n+

n+

P

Sapphire

Au

Immobilized

enzyme

membrane

Inactivated

enzyme

membrane

Si3N4

SiO2

P


29

Fig. 12 Schematic representation of piezoelectric biosensor

SAWs along a layer of a substrate covered by the membrane whose properties affect the

propagation loss and phase velocity of the wave.

E. Thermal Sensors. Thermal detection biosensors are based on absorption or

production of heat that in turn changes the temperature of the medium in which the

reaction takes place. When the analyte comes in contact with the enzyme, the heat

reaction of the enzyme is measured and calibrated against the analyte concentration. The

total heat produced or absorbed is proportional to the molar enthalpy and the total number

of molecules in the reaction.

2.5.2.3. Basic Principles of HbA1C biosensors

HbA1C is formed through the nonenzymatic glycation of the terminal valine of β chain in

Hb. This HbA1C can be digested to small glycated peptide FV that can be further

oxidized by the enzyme FAO. Enzymatic assay of HbA1C is based on the oxidation of FV

(as a model compound). The general strategy used for electrochemical sensing of HbA1C

is by measuring oxygen consumption or the amount of hydrogen peroxide produced by

Oscillator

Piezoelectric crystal

21258967.998 Hz

Frequency counter


30

the enzyme reaction. The enzymatically produced H2O2 can be monitored by an

electrochemical sensor. Therefore, the HbA1C level can then be determined using this

biosensor by measuring the output current changes from the enzymatic oxidation reaction

producing H2O2. The reactions involved were as shown in Fig. 13.

2.6. Classifications of HbA1C biosensors

2.6.1. Biosensors based on Fructosyl Valine (FV)

The current electrochemical measurements of FV need to be improved in terms of the

sensitivity and the repeatability of the detection. Ideally a FV biosensor should operate at

a lower potential, ambient temperature and would require a small sample volume in order

to minimize the potential interference, improving the operational convenience and

patient’s compliance. Various biosensors based on FV detection have been reported and

extensively studied; in order to construct a biosensor, which could lay a successful foun-

dation for future HbA1C biosensing. Principle of FV based biosensor is as given below:

Fru-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-ser… Protease Fructosyl valyl histidine + amino

(N-terminal residue of β-chain in HbA1C) and/or Fructosyl valine acid

(His, Leu, Thr)

Fructosyl valyl histidine and/or + O2 + H2O FAO Valyl histidine + D-glucosone

Fructosyl valine or valine +H2O2

H2O2 High potential 2H+ + O2 + 2e

2e- through nanoparticles/immobilization support working electrode

2.6.1.1. Amperometric FV biosensors

2.6.1.1.1. Platinum (Pt) electrode coated with membrane

A novel amperometric FV sensor was prepared by coating the surface of a Pt electrode

with a membrane. The partially-purified fructosyl amino acid oxidase (FAO) (0.05 units)

was casted onto the membrane and placed onto a 3 mm Pt electrode. This membrane was

successfully employed as an immobilization support for FAO (from marine yeast). This

FAO membrane catalyzed FV producing hydrogen peroxide, which was then detected


31

Fig. 13 Scheme of electrochemical reactions involved in HbA1C biosensor based on FAO

(Where MNPs = Metal nanoparticles)

31

MNPs

FAO

Protease Fru-Val-His-Leu-Thr-Pro-Glu-Glu-Lys-ser…

(N-terminal residue of β-chain in HbA1c)

Fructosyl valyl histidine + amino acid

or Fructosyl valine (His, Leu, Thr)

valyl histidine + D-glucosone +H2O2

2H+ + O2 + 2e

Gold electrode

Amperometric

response


32

electrochemically in a stirred 10 ml testing solution.

The biosensor had following characteristics:

Optimum pH: 7.0-7.5; Optimum temperature: 30-45ºC; Linearity: 0.05-1.8 mM;

Working potential: 0.6 V (vs Ag/AgCl reference electrode); Detection limit: 0.05 mM

Sensitivity: 0.42 µAmM-1

cm-2

(Tsugawa et al., 2000).

2.6.1.1.2. Polyvinylimidazole polymer onto carbon paste electrode

Polyvinylimidazole (PVI), a synthetic polymer was used as a catalyst for fabrication of an

amperometric FV sensor. Molecular imprinting technique was used for oxidative

cleavage of FV. A mixture of PVI and carbon paste was applied on the electrode. The

constructed electrode was then immersed in the phosphate buffer electrolyte containing

1-methoxyphenazinemethosulphate (m-PMS) as mediator.


Linearity: 20-700 mM; Working potential: +0.1 V (vs Ag/AgCl electrode); Detection

limit: 20 mM; Sensitivity: 0.135 µAmM

-1cm

-1 (Sode et. al., 2001b).

Since FV is an expensive reagent, it is the limiting factor for its utilization as the

template for sensor fabrication. Proteolytic digestion of HbA1C for production of FV also

leads to the formation of another fructosylamine compound (fructosyl lysine denoted as

Fru-ε-lys) which is the proteolytic product of digestion of glycated albumin in the blood

and can interfere with the detection of FV. So, Sode et al., 2003 developed a sensor for

better selectivity for FV over fructosyl lysine and used methyl valine (m-val), which is a

cheaper analogue of expensive FV as the template. Also, they used the positively charged

functional monomer allylamine to improve the selectivity of the sensor toward FV. Both

the sensitivity and selectivity (FV/Fru-ε-lys) decreased from 135 nA/mM to 95 nA/mM

and 1.8 to 1.6, respectively. However, with the introduction of alkylamine as the

functional monomer, the selectivity increased to 1.9, while a sensitivity of 95 nA/mM

could be maintained. Thus, with these two modifications, selectivity increased slightly,

while the sensitivity decreased in exchange for a more inexpensive template (m-val).

2.6.1.1.3. Prussian Blue (PB) modified enzyme electrode


33

Low potential amperometric enzyme sensor for fructosyl-valine, a model compound of

HbA1C, was constructed using a FAO and Prussian blue film as the artificial peroxidase.

These results obtained indicated that the PB-based FAO electrode may avoid the inherent

problems of hydrogen peroxide measurement at the high potential and also have

advantage in its simple construct of the system compared with the two enzyme

peroxidase-FAO-ferrocene system. The biosensor had following characteristics:

Linear range: 0.1-0.3 mM; Sensitivity: 0.42 µAmM-1

cm-2

; Applied potential: -0.05 V

(Tsugawa et al., 2001).

2.6.1.1.4. Poly(vinylalcohol)-Stylbazole (PVA–SbQ) membrane on oxygen electrode

An amperometric FV biosensor based on FAO immobilized onto silk fibroin membrane

mounted over an oxygen electrode in flow injection analysis (FIA) was constructed.

Source of FAO: Pichia sp. N1-1 strain, Type of immobilization: Cross-linking


Optimum pH: 7.0; Optimum temperature: 25ºC, Linearity: 0.2-10.0 mM;

Working potential: 0.6 V; Detection limit: 0.2 mM; Sensitivity: 0.0046 µAmM−1

cm−2

;

Application: The biosensor was used for determination of blood HbA1C; Operational

stability: The sensor was capable of detecting 120 consecutive sample injections over 20

h (Ogawa et al., 2002).

This FIA enzyme biosensor was claimed to be the first sensor towards fructosyl

dipeptide, though with insufficient sensitivity.

2.6.1.1.5. Screen-printed iridium modified carbon electrode

A methodology was devised for determination of FV by iridium modified screen-printed

carbon electrodes. It was single-use, disposable FV amperometric biosensor. A single-use

amperometric FV biosensor, incorporating a three-electrode configuration was fabricated

on a polyester substrate using low cost screen printing (thick-film) technology. Both the

working and counter electrodes were prepared by screen-printing commercial carbon ink.

Source of FAO: Recombinant Escherichia coli, Type of immobilization: Adsorption



34

Optimum pH: 7.0; Optimum temperature: 25ºC; Linearity: 0.2-2.0 mM; Working

potential: 0.25 V; Response time: 120s; Detection limit: 0.2 mM; Sensitivity: 21.5

μAmM−1

cm−2

; Application: The biosensor was used for determination of blood-HbA1C

(Fang et al., 2009).

Merits: Cost-effective and disposable, so routine measurement of HbA1C level could be

significantly easier for the diabetic patient management.

Demerits: It is single use, interference by presence of potential interferents, since FV is

an expensive reagent, it is the limiting factor for its utilization as the template for sensor

fabrication.

2.6.1.1.6. Non-enzymatic biosensor based on glassy carbon paste electrode (GCPE)

GCPE serving as the working electrode was obtained by dropping the glassy carbon

microparticle carbon paste onto the indium-doped tin oxide (ITO) substrate using a

baking process. A coiled Pt wire and Ag/AgCl electrode (in saturated KCl) were used as

the counter electrode and the reference electrode, respectively. This GCPE was

characterized and reported to have higher sensitivity on FV and lower background current

compared with conventional glassy carbon electrodes.

Optimum pH: 7.4; Optimum temperature: 35ºC; Linearity: 0-1 mM; Working

potential: 1 V; Response time: 40s; Detection limit: 0.05 mM; Sensitivity: 5.26

AmM-1

cm-2

; Square correlation coefficient (R2): 0.999; Interference: No interference

on current response from D-fructose, D-glucose and L-valine.

Merits: Relatively low oxidation potential required for detection of FV without the need

of an enzyme, low cost and the ease of fabrication of the materials involved. These

HbA1C and glucose sensor array were expected to be used for simultaneous point-of-care

measurements, thereby gathering more accurate patient information (Chien and Chou,

2010).

2.6.1.1.7. Non-enzymatic biosensor based on ferrocene boronic acid (FcBA) bound

glassy carbon paste electrode (GCPE)

A novel non-enzymatic amperometric method using a GCPE for fast monitoring of FV


35

was developed. The method had advantages in terms of: the relatively low oxidation

potential required for detection in 3 mM FcBA, low cost, and the ease of fabrication of

the materials involved. FcBA was employed as useful tool for the capture of Fru-Val

through the interaction of the boronic acid-diols to form a complex and for the signal

transfer by iron ion oxidation. In conclusion, our novel method shows that it is possible to

generate an easily detectable signal, using a very low oxidation potential, to detect Fru-

Val without the need for an enzyme.

Optimum pH: 7.4; Optimum temperature: 25ºC; Linearity: 0.5-4.0 mM; Working

potential: 0.1 V; Response time: 10s; Detection limit: 0.5 mM; Sensitivity: 5.9

AmM-1

cm-2

; Application: The biosensor was used for determination of blood HbA1C

(Chien and Chou, 2011).

Merits: These type of biosensors are amenable to miniaturization and have compatible

instrumental sensitivity.

2.6.1.1.8. Core-shell magnetic bionanoparticles (MNPs) modified gold electrode

An electrochemical biosensor for HbA1C detection by monitoring FV level based on

core-shell MNPs modified gold electrode has been reported by us recently (Fig. 14). The

biosensor had following characteristics:

Working voltage (V): 250 mV; Response time: 4s; Optimum pH: 7.5; Optimum

temperature: 35

oC; Linear range: 0.1-2.0 mM; Detection limit (mM): 0.1 mM;

Stability: 90 days; Reusability: 250 times (Chawla and Pundir, 2011).

2.6.1.2. Potentiometric FV biosensors

2.6.1.2.1. Poly-aminophenylboronic acid (p-APBA) polymer on conductive ITO

electrode

Molecular imprinting technique was used to fabricate a potentiometric FV biosensor.

Molecular imprints of FV in a poly-aminophenylboronic acid (p-APBA) polymer on

conductive ITO electrodes were made. Electrochemical characterization of the fabricated

biosensor was carried out by comparing the open circuit potential (Eoc) of the ITO

carrying the molecular imprinted polymer (MIP) with that measured on a non-imprinted

control in 10 ml of 0.1 M phosphate buffer (pH 7.0) with a standard Ag/AgCl reference


36

Fig. 14 Chemical sequence of electropolymerization of magnetic bionanoparticles onto

gold electrode and chemical reaction of immobilization of FAO on modified Au electrode

electrode to assess the affinity of the FV imprints for FV, D-fructose, D-glucose and L-

valine. The affinity of the imprinted electrode for FV was higher than for the others. The

measurement of open circuit potentials (ΔEoc) showed that imprinted p-APBA was able

to demonstrate selectivity for the carbohydrate component of the Amadori compound that

is characteristically present in diabetic patients.

Optimum pH: 7.0; Optimum temperature: 25oC; Optimum potential: 5 mV;

Response time: 1000s (Chuang et. al., 2009).

2.6.2. Biosensors based on HbA1C

Other types of HbA1c biosensors detect HbA1c directly. Different methods and

techniques have been used for these HbA1C biosensor. One of their potential advantages

was that there was no need for two time-consuming preliminary steps to release FV from

HbA1C by a protease (one of the main drawbacks with FV point-of-care (POC)

instruments).


37

2.6.2.1. Amperometric HbA1C biosensors

2.6.2.1.1. Haptoglobin onto 1,1'-carbonyldiimidazole (CDI)-activated cellulose

membranes

An amperometric immunosensor for HbA1C determination, utilizing CDI-activated

cellulose membrane immobilized haptoglobin as affinity matrix with a Pt-working

electrode was described. The HbA1C assay was carried out in a two-step procedure

including the selective Hb enrichment on the sensor surface and the specific HbA1C

detection by a glucose oxidase (GOx) labeled anti- HbA1C antibody.

Enzyme linked immunosorbent assay (ELISA) studies confirmed the advantage of

a sandwich-type format with haptoglobin as capture molecule for selective Hb binding

over the direct adsorption method. Results by the sandwich immunoassay showed a linear

correlation within the clinically relevant range 5-20% (cofficient of variation < 3). The

immobilization procedure of haptoglobin onto CDI-activated cellulose membranes was

optimized for sensor application.

Linearity: 7.8-39 nM (HbA1C linear range: 0 and 25%); Working potential: 0.6 V;

Precision: Within-batch CV 0.9-3.3% for the haptoglobin based assay and 2.2-6.9% for

the anti-Hb antibody based assay (Stöllner et al., 2002).

2.6.2.1.2. Ferroceneboronic acid (FcBA)/zirconium dioxide nanoparticles

(ZrO2NPs)-modified pyrolytic graphite electrode (PGE)

The principle is based on the electrochemical measurement of FcBA that has been

specifically bound to the glycated N-terminus. Hb was immobilized on a ZrO2NPs

modified PGE in the presence of didodecyl dimethyl ammonium bromide (DDAB). The

incubation of this sensor in FcBA solution leads to the formation of an FcBA-modified

surface due to the affinity interaction between boronate and the glycated sites of the Hb.

The PGE is used for protein (total Hb) immobilization and DDAB accelerates electron

transfer between Hb and the electrode. Purified hemolysed erythrocytes from real human

blood sample were mixed with the suspension of ZrO2NPs in the DDAB solution and

then applied to the electrode surface for total Hb immobilization. Square wave


38

voltammetry (SWV), was used instead of cyclic voltammetry (CV) since the chemically

modified sensor with bound Hb exhibited a relatively large charging current and higher

sensitivity for the Fc label.

Optimum pH: 8.0; Optimum temperature: 22ºC; Working potential: 0.299 V versus

Ag/AgCl (1M KCl); Linearity: HbA1C from 6.8 to 14.0%; Incubation time: 30 min.;

Reproducibility: 12.7% for 10.2% HbA1C samples at the different total Hb

concentrations (n=3) (Liu et al., 2006a).

Demerits: The major drawback of this method was that deviations between the results

obtained with the present two methods range from 10.7 to 31%. The requirement for the

separate determination of the total Hb content also made this an inconvenient aspect of

this method. Further improvement of the method is needed in terms of measuring time

and precision.

2.6.2.1.3. 3-AminoPhenylboronic acid (APBA) modified graphene oxide (GO) onto

glassy carbon electrode

APBA modified GO was synthesized and was applied for electrochemical detection of

HbA1C through modified glassy carbon electrode. The compound (GO-APBA) was

synthesized by forming an amide linkage between the amino group (-NH2) of APBA and

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

spectroscopy. Detection of HbA1C was carried out using Electrochemical Impedance

Spectroscopic (EIS) measurements. The other characteristics of the biosensor were:

Optimum pH: 8.0; Optimum potential: 0.2 V (Krishna et al., 2011).

Table 1 provides a comparison of analytical properties of amperometric FV and HbA1C

biosensors.

2.6.2.2. Potentiometric HbA1C biosensors

2.6.2.2.1. Integrated chip and a micro extended gate electrode array

A micro potentiometric immunosensor based on an integrated chip and a micro extended

gate electrode array was reported, which can detect Hb and HbA1C simultaneously. It is

free labeling, simple and convenient. The integrated chip based on standard complement-


39

Table 1. A comparison of analytical properties of amperometric FV and HbA1C biosensors

39

S.

No.

Type of

biosensor

Support of

immobilization

Working

voltage

(V)

Response

time (s) Optimum

pH Optimum

temp. (

oC)

Linear

range

(mM)

Detection

limit

(mM)

Sensitivity

(µA

mM-1

cm-2

)

Coefficient

of variance

(%)

Reference

1. Amperometric FAO/membrane/Pt

electrode

0.6

ND

7.0-7.5

30-45

0.05-1.8

0.05

0.42

ND

Tsugawa

et al., 2000

2. Amperometric PVI polymer onto

carbon paste

electrode

0.1

ND

ND

ND

20-700

20

0.135

ND

Sode et.

al., 2001

3. Amperometric PB film/Electrode

-0.05

ND

ND

ND

0.1-0.3

0.1

0.42

ND

Tsugawa

et al., 2001

4. Amperometric FAO/PVA-SbQ on

oxygen electrode

0.6

ND

7.0

25

0.2-10.0

0.2

0.0046

ND

Ogawa

et al., 2002

5. Amperometric Haptoglobin/CDI-

activated cellulose

membranes

0.6

ND

ND

ND

0.78x10-5-

3.9 x10-5

ND

ND

0.9-3.3

Stöllner et

al., 2002

6. Amperometric FcBA/ZrO2NPs/PG

E

0.299

1800

8.0

22

6.8 to

14.0%

ND

ND

12.7

Liu et al.,

2006a

7. Amperometric FAO/iridium

modified carbon

electrode

0.25

120

7.0

25

0.2-2.0

0.2

21.5

ND

Fang

et al., 2009

8. Non-

enzymatic

Glassy carbon

microparticle

carbon paste/ITO

1.0

40

7.4

35

0-1.0

0.05

5.26

ND

Chien and

Chou, 2010


40

FAO: Fructosyl amino acid oxidase; PVI: Polyvinylimidazole; PB: Prussian blue; PVA-SbQ: Poly(vinylalcohol)-Stylbazole; CDI: 1,1'-

carbonyldiimidazole; FcBA: Ferroceneboronic acid; GCPE: Glassy carbon paste electrode; MNPs: Magnetic bionanoparticles; APBA: 3-

AminoPhenylboronic acid; GO: Graphene oxide; GCE: Glassy carbon electrode; ND: Not detected; ZnONPs: zinc oxide nanoparticles; PPy:

polypyrrole

9. Non-

enzymatic

FcBA/GCPE

0.1

10

7.4

25

0.5-4.0

0.5

5.90

ND

Chien

and Chou,

2011

10. Amperometric FAO/core shell

MNPs/Au electrode

0.25

4

7.5

35

0.1-2.0

0.1

35.72

2.58-5.63

Chawla and

Pundir, 2011

11. Amperometric APBA/GO/GCE

0.2

ND

8.0

ND

ND

ND

ND

ND

Krishna et

al., 2011

12. Amperometric FAO/ZnONPs/PPy/

Au

0.27 2 7.0 35 0.1-3.0 0.05 38.42 1.58- 2.07 Present

40


41

-ary metal-oxide-semiconductor (CMOS) technology consisted of the ion-sensitive field-

effect transistors (ISFET)/reference field-effect transistor (REFET) sensing device and

signal-processing circuit. The electrode array was fabricated by micro electro mechanical

systems (MEMS) method. A two-layer structured monolayer is formed on the anti-

HbA1C/anti-Hb gold electrode by the mixed self-assembled monolayers (SAMs)

technique. Following characteristics of biosensor were studied:

Optimum pH: 7.4; Optimum temperature: 25oC; HbA1C range: 5-20%; Sensitivity:

0.35 mV/μg ml-1

and 0.13mV/μg ml-1

for HbA1C and Hb, respectively; Response time:

120s; Accuracy: 99.4%; Correlation coefficient: 0.996; RSD: 0.85% (Xue et al., 2010).

2.6.2.2.2. Mixed self assembled monolayers (SAMs) wrapped nano-spheres array

A micro potentiometric immunosensor based on mixed SAMs wrapped nano-spheres

array for the detection of HbA1C level. Nano-spheres array was prepared by wrapping

gold nanoparticle with mixed SAMs on the surface of micro immunosensor. Mixed

SAMs make the nano-gold particles distribute uniformly without aggregation and render

the signal less susceptible to noise. Based on this nano-spheres array, antibody is

covalently immobilized on the immunosensor surface. The micro immunosensor,

consisting of ISFET integrated chip and MEMS electrodes array is applied to measure

HbA1c level by detecting the concentration of HbA1c and Hb simultaneously. The

biosensor had following characteristics:

Linearity: 166.7-570 ng/ml Hb and 50-170.5 ng/ml HbA1C; Recovery: 99.08% and

103.27%; RSD: 5%; Sensitivity: 94.73µV/(ngmL−1

); Interference: No significant

interference by interfering substances like bovine serum albumin, lysis solution and some

potassium ion, chloride ion. Although the HbA1C detected channel and Hb detected

channel were integrated on one electrode array chip, the two channels can get good

results in one drop of simulated blood sample contained Hb and HbA1C, with no

interference each other (Xue et al., 2011a).

2.6.2.2.3. FET sensor chip and a disposable microelectrode-array chip

Field-effect transistor (FET)-based micro potentiometric immunosensor consisting of an


42

integrated FET sensor chip was fabricated with CMOS processes and a disposable micro

electrode array chip fabricated by MEMS technique. This immunosensor was applied for

simultaneous detection of Hb and HbA1C. The integrated FET sensor chip consisted of

ISFET/REFET sensing devices and signal-processing circuit. Three methods were

researched for immobilizing antibody i.e. mixed SAMs method, seed mediated growth

nano-gold method and nano-gold wrapped with mixed SAMs method on the micro

extended gate electrode array chip. The immunosensor fabricated by nano-gold wrapped

with mixed SAMs method showed the highest sensitivity. Whole blood samples were

detected by this immunosensor. Good consistency and high accuracy were achieved. As

shown from the results, the immunosensor is free labeling, simple and has the potential to

become a portable device for diabetes control. The biosensor had following

characteristics:

Sensitivity (V/ng mL−1

): 189.8 for HbA1C and 40.42 for Hb; Recovery: 102.54; RSD:

2.63

Merits: It could detect Hb and HbA1C at the same time (Xue et al., 2011b).

2.6.2.2.4. Polypyrrole (PPy)-gold nanoparticles (AuNPs) composite

A novel micro-potentiometric HbA1C immunosensor based on electrochemically

synthesized PPy-AuNPs composite. PPy-AuNPs film with AuNPs uniformly distributed

in it was deposited on gold electrode surface by a simple and direct procedure, without

the addition of any nanoparticles or reducing agent. This generic method made it possible

to deposit different polymers on miniaturized electrodes. With the existence of AuNPs,

the antibody immobilization onto the electrode surface was facilitated. Based on an

ISFETs integrated chip, a micro-potentiometric immunosensor for Hb and HbA1C has

been constructed.


Linearity: 60-180 µg/ml Hb and 4-18µg/ml HbA1C; Sensitivity: 0.20mV µg−1

ml-1

;

Response time: less than 1 min.; Linear dose-response behavior: 125 and 197 µg/ml;

Storage stability: 35.1% at 4oC under dry conditions for 5 days in 8 µg/ml Hb in

phosphate buffer saline (PBS) (pH 7.4); Reproducibility: The variation coefficients were


43

relatively high. Further optimization is needed to improve the reproducibility for real

sample analysis; Interference: 9.2% decrease in biosensor response by number of

interferents such as immunoglobulin G, α-fetoprotein and BSA (Qu et al., 2009).

2.6.2.2.5. Boronic acid-modified thin film/gold electrode

A boronic acid-modified thin film interface for selective binding of HbA1C followed by

electrochemical biosensing using an enzymatic backfilling assay. A freshly evaporated

gold working electrode for the bottom-up layer formation process. This procedure began

with the formation of an amine-reactive Dithiobis(succinimidyl propionate) (DTSP)

SAM on the gold which was then transferred to a poly(amidoamine) G4 dendrimer

solution. Then 4-formyl-phenylboronic acid (FPBA) was immobilized on the dendrimer

layer selective for HbA1C. FPBA functionalization was confirmed by XPS and cyclic

voltammetry. To carry out the backfilling assay, samples with various ratios of

HbA1C/HbA0 (with normal adult human Hb concentration i.e. 150 mg/ml) in a pH 9.0

bicarbonate buffer were contacted with the functionalized surface to react with FPBA for

1 hour. After rinsing with buffer and PBS, 1 mg/ml activated GOx in PBS was added in

order to bind to the remaining unreacted amine groups on the dendrimer-FPBA layer or

30 min. The response of this electrode sensor was assessed by subjecting it to a

voltammetric scan from 0 to +500 mV vs. Ag/AgCl at a rate of 5 mV/s in PBS in the

presence of 0.1 mM ferrocenemethanol (as mediator) and 10 mM glucose (as substrate).

The anodic current measured at +400 mV was chosen as the sensor signal because of

stable current at this potential in the voltammogram. Although this sensor had the

advantage of signal amplification without the need for pretreatment such as labelling or

use of labeled secondary antibody, if required incubation of the Hb sample and then GOx

solution requires 1 hour and 30 minutes, respectively. In addition, the sensitivity at

HbA1C levels below 5% is not sufficient. The biosensor had following characteristics:

Optimum pH: 7.2; Working potential: 0.4 V versus Ag/AgCl; Potential sweep rate:

50 mV/s; Linearity: HbA1C from 2.5-15% (Song and Yoon, 2009).

Table 2 provides a comparison of analytical properties of potentiometric FV and HbA1C

biosensors.


44

2.6.2.3. Piezoelectric sensor

2.6.2.3.1. Ferroceneboronic acid modified piezoelectric quartz crystal

An electrochemical immunoassay based on the multiple affinity labeling of the indicator

antibody with an electro-active tag is presented. Hb is adsorbed to the surfactant-

modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance

is used to validate the total Hb binding, the HbA1C on the sensor surface is recognized by

an antibody and quantified electrochemically after the sugar moieties of the antibody

have been labeled in-situ with ferroceneboronic acid. The sensitivity of this sensor is

about threefold higher than the sensitivity of a Hb sensor, where the ferroceneboronic

acid is bound directly to HbA1C. The total Hb content was determined using a mass-

sensitive quartz crystal modified with a surfactant, while the FcBA-bound HbA1C on the

surface was measured using square wave voltammetry. The biosensor had following

characteristics:

Optimum pH: 8.0; Optimum temperature: 25oC; Response time: 300s; Optimum

potential (vs. Ag/AgCl): +200mV; RSD: 5.1% (Halamek et al., 2007).

2.6.2.3.2. 3-aminophenylboronic acid (APBA) modified gold electrode

The biosensor employed a combination of the piezoelectric biosensor for HbA1C and the

flow-through photometric sensor for total haemoglobin (Hb). The modification of gold

electrodes with APBA, as a specific ligand was studied; the chemisorbed conjugate of

APBA with a long-chain thiocompound (11- mercaptoundecanoic acid) provided the best

affinity for HbA1C. The total haemoglobin content was analysed from 50 to 1000 µg/ml

as absorbance of the haemoglobin-cyanide derivative at 540 nm. Thus, Interaction

between immobilised boronic acid and glycated haemoglobin was used as the recognition

reaction on the surface of the piezoelectric biosensor. Only one standard (calibrator)

diluted in various ratio was necessary for calibration and 1 µl of blood was sufficient for

analysis. The full range of HbA1C content (4-15%) in blood was analysed. In the

presence of higher concentrations of HbA1C, the surface became saturated and the

response versus concentration dependency was non-linear.


45

Table 2. A comparison of analytical properties of potentiometric FV and HbA1C biosensors

Poly-aminophenylboronic acid: p-APBA; Polypyrrole: PPy; AuNPs: Gold nanoparticles Indium tin oxide: ITO; Self Assembled Monolayers:

SAMs; FET: Field effect transistor; ND: Not detected; RSD: Relative standard oxidation

S.

No.

Support of

immobilization

Linear range

(%)

Applied

potential

(mV)

Response

time (s)

Optimum pH Optimum

temperature

(oC)

Sensitivity

(mVµg−1

mL-1

)

RSD (%) Reference

1. p-APBA

polymer on

ITO electrode

ND +5 1000 7.0 25 ND ND Chuang et al.,

2009

2. PPy/AuNPs

composite

4-18 µg mL-1 ND 60 ND ND 0.20 0.20 Qu et al., 2009

3. Boronic acid-

modified thin

film/Au

electrode

2.5-15.0 +400 ND 7.2 ND ND ND Song and Yoon,

2009

4. Integrated chip

and micro

extended gate

electrode array

5.0-20.0 ND 120 7.4 25 0.35 0.85 Xue et al., 2010

5. Mixed SAMs

wrapped nano-

spheres array

0.05-0.171 g

mL-1

ND 120 7.4 ND 0.095x10-3 5.0 Xue et al.,

2011a

6. FET sensor chip

and a disposable

micro electrode-

array chip

5.0-15.0 ND ND ND ND 0.189 2.63 Xue et al.,

2011b

45


46

Optimum pH: 8.0-9.0; Optimum temperature: 22ºC; Working range: 10-90 µg/ml;

Response time: 15 min/sample with 20 measurements without any user intervention.

The developed method was successfully evaluated on blood samples collected from

diabetics (Pˇribyl and Skl´adal, 2006).

2.6.2.3.3. 3-boronic acid (T3BA) self-assembled monolayer (SAM)/Au electrode

HbA1C was selectively immobilized on a gold electrode modified with T3BA-SAM and

was detected by label-free electrochemical impedance spectroscopy (EIS). The reported

binding mechanism is based on bonding between the sulphur atom of the π-stacked

thiophene SAM and the gold. The binding of T3BA and formation of a SAM on the gold

was confirmed by the use of a quartz crystal microbalance (QCM), atomic force

microscopy (AFM) and EIS experiments. Electrochemical determination was based on

measuring the change in the capability of the gold electrode for electron transfer due to

blocking of the electrode surface by HbA1C after immobilization. The biosensor had

following characteristics:

Optimum pH: 8.5; Concentration range: 0.1-1 µg/mL; Response time: 600s

Drawbacks: The variation of signal with HbA1C concentration also depends on total Hb

concentration. Consequently, the total Hb concentration was also needed to be

determined separately to obtain the HbA1C content. Since this method is not selective for

HbA1C over glycated albumin, glycated albumin must be separated from RBC by centrif-

-ugation (Park et. al., 2008).

2.6.2.4. Biochips for HbA1C detection

2.6.2.4.1. m-amino-phenylboronic acid (m-APBA) modified agarose bead/inter-

digitated array IDA electrodes

A disposable biochip was constructed by using an integrated system consisting of a pair

of interdigitated array (IDA) electrodes, HbA1C binding chamber, blood lysis chamber,

filter, micro-pump and microchannel. Ferricyanide (K3Fe(CN)6) was used as a mediator

so that the electrons released from the oxidation of Fe2+

in Hb were transferred to the

electrode by the ferricyanide/ferrocyanide couple. When Fe2+

in Hb is oxidized to Fe3+

by


47

an electron delivery medium of ferricyanide (K3Fe3+

(CN)6), the ferricyanide is

deoxidized to a ferrocyanide (K4Fe2+

(CN)6), and current flows as a result of the applied

electric potential to the ferrocyanide. After plasma separation (1) and red blood cell

(RBC) lysis (2), the total Hb stream branches off into two separate streams: in the lower

stream HbA1C was immobilized on a packed agarose bead containing m-amino-

phenylboronic acid (m-APBA) in the binding chamber and releases Hb, while total Hb

flows in the upper stream (3). The ratio of the resulting electrochemical signals from the

lower and upper streams after passing through the IDA electrodes yields the % HbA1C.

Due to the nonhomogeneous distribution of Hb, the instantaneous current varies as a

sample flows through the IDA electrodes. Consequently, the integral of the current over

time was used for measurement of % HbA1C (Son et al., 2006).

2.6.2.4.2. Microfluidic system using Au dielectrophoretic (DEP) electrodes

RBCs were successfully separated and lysed in an automatic manner utilizing the Au

dielectrophoretic (DEP) forces in a microfluidic system composed of a circular

micropump and normally closed valves. The chip was fabricated using MEMS technique.

The electrochemical detection module composed of three electrodes on a glass substrate

for sensing the concentration of HbA1C electrochemically. The three electrodes including

a working (carbon paper), a counter (Pt) and a reference (Ag) electrode were designed

and fabricated using thin-film deposition processes.

Optimum pH: 7.4; Optimum potential: 1000 mV; Linear range: 0 to 2 mg/ml;

Response time: 10 min.; Standard deviation: 1.56% (Huang et al., 2011).

Table 3 provides a comparison of analytical properties of piezoelectric biosensors and

biochips for HbA1C detection.

2.7. Conclusion and Future Outlook

During past few years, various methods for HbA1C determination were reported.

Compared to conventional methods, the biosensing methods are very rapid, selective and

sensitive. However, more efforts are needed to tackle technical difficulties in biosensor to

find better alternatives such as: (1) need of blood pretreatment (2) separate determination


48

of total Hb (3) leaching out of enzyme or larger biomolecules from electrode (4)

selectivity and detection limit require to be more selective and wide. There is still need of

more reliable, specific, accurate and portable HbA1C biosensors.

Most of the reports have focused on the use of FAO for detection of HbA1C.

These biosensors have the potential to be converted to point-of-care testing (POCT)

devices, which can potentially play an important role in diabetes diagnosis and

management. Miniaturization is expected to have a marked impact on the development

and applications of biosensors. Miniaturization of a biosensor not only reduces the size of

detection device and sample volume, but also integrates all steps of the analytical process

into a single-sensor device. Thus, it results in reduction of both time and cost of analysis.

Moreover, it is expected to lead to further portability for in vivo sensing and in-field

applications. Trials can be made to integrate the laboratory model with chip designing

companies (VLSI) in order to develop a fully automated chip system in a device, to be

used by any patient on his bedside.


49

Table 3. A comparison of analytical properties of piezoelectric biosensors and biochips for HbA1C detection

FcBA: Ferroceneboronic acid; APBA: 3-aminophenylboronic acid; T3BA: 3-boronic acid; SAM: Self-assembled monolayer; ND: Not detected;

RSD: Relative standard deviation

*******************

S. No. Support of

immobilization

Linear

range (µg

mL-1

)

Applied

potential (mV)

Response time

(s)

Optimum pH Optimum

temperature

(oC)

RSD (%) Reference

1. APBA modified

Au electrode

10-90 ND 900 8.0-9.0 22 ND Pˇribyl and Skl´adal,

2006

2. FcBA modified

piezoelectric

quartz crystal

ND +200 300 8.0 25 5.1 Halamek et al., 2007

3. T3BA/SAM/Au

electrode

0.1-1.0 ND 600 8.5 ND ND Park et. al., 2008

4. Integrated chip

and a micro

extended gate

electrode array

0 to 2000 +1000 600 7.4 ND 1.56 Huang et al., 2011

49


50

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